Industrial Agricultural Biotech – Eleftherios T. Papoutsakis, Christopher A. Tomas, Marija Tesic, Jose Y. Santiago, Northwestern University
Abstract for “Increased cell resistance against toxic organic substances”
“Recombinant microorganisms” and other methods to increase tolerance to toxic substances. These microorganisms can also be used to increase solvent production.
Background for “Increased cell resistance against toxic organic substances”
“Metabolic engineering is the manipulation of living organisms in order to produce desired metabolic substrates, products, and/or byproducts. This allows for the production of a wide range of valuable commodity or specialty chemicals. Recombinant DNA technology allows metabolic pathway modification by using targeted genetic modifications in place of or in addition to traditional mutagenesis. Numerous examples of metabolic engineering have been documented since the 1980s. To achieve desired results, variables like temperature and aerobic conditions can be also engineered or modified. One of the main limitations to such bio-production is the toxic effects of many desired substrates, products, and/or byproducts. This problem is both particular and general in that it can be found in the production of chemical commodity chemicals from renewable sources, bioremediation technologies and the use cells in biocatalysis which involves toxic organic molecules.
“The strictly anaerobic and Gram-positive, solventogenic Clostridium (C. acetobutylicum or C. beijerinckii) are great candidates for creating metabolically engineered strains that can be used in a variety of applications. These species can be used to produce butanol and butyric acids, acetones, propanol and 1,3-propanediol as well as polysaccharides and enzymes. Clostridium can be grown under low redox potential. This allows for a wide range of stereospecific reductions that yield chiral products, which are hard to synthesize chemically. These clostridial species, as well as related clostridial species, can also degrade toxic chemicals making them good candidates for bioremediation. Solventogenic clostrides have the advantage of being able to use a wide range of substrates, including mono-, oligo, and poly-saccharides. This includes common pentoses, hexoses, as well as biomass hydrolysates.
“Important potential uses of solventogenic Clostridium include the production of butanol or acetone. Poor process economics can affect such production, for example, a low butanol concentration in the product stream. These low butanol tolerances are caused by the low concentrations of butanol in these organisms. Because of this, separation costs for butanol are high. Economic analysis shows that the separation costs could be reduced by doubling the concentration of final butanol from 12 to 19 grams/l.
“Butanol toxicities are quite severe, as we have already mentioned. The final product stream should not contain more than 12-13 g/l of butanol before it causes cellular destruction. Butanol at high levels inhibits active nutrient transportation, membrane bound ATPase and glucose uptake. It also partially or totally abolishes the membrane pH and?pH. Butanol inhibits active nutrient transport, membrane bound ATPase, glucose uptake, partially or completely abolishes the membrane?pH and???? and lowers intracellular pH. Butanol’s chaotropic effect upon the cell membrane has been the reason for its toxic effects. The membrane model does not provide a complete explanation of butanol toxicity. A Clostridium acetobutylicum kinase activation mutant strain produces 230 mM (17 g/l), butanol, 83 mM (4.75 g/l), acetone, and 69 mM (3.2/l) ethanol. ?, Biotechnol. Bioeng., 67: 1-11 (2000)]. This strain was able to surpass the limit of 12-13 g/l Butanol-toxicity without any adaptation or selection for an increased butanol tolerance.
“Stress-response protein” is a group of specialized proteins that are essential for cellular function. They are found in normal cells under normal growth conditions and play an important role in cell biology as a protective cellular reaction to environmental stress. Due to their high abundance after heat shock, a common family of stress response proteins is called heat shock proteins (HSPs). Stress response proteins are able to recognize hydrophobic surfaces and aid in folding proteins. They can also bind the normative states of proteins, helping them to fold properly. The majority of stress response proteins form noncovalent interactions to the hydrophobic areas of misfolded protein proteins. This helps stabilize them from irreversible multimeric aggregates, misfolding of nascent proteins, unraveling under stress, and eventual degradation. “The properly folded and stabilized proteins can be used to perform their cellular function(s).
“The major heat-shock protein classes include the 90-kDa Heat Shock Protein (HSP90), 60-kDa Heat Shock Protein (HSP60), and 70-kDa Heat shock Protein (HSP70; DnaK for E. coli), as well as the 40-kDa Heat shock Protein (HSP40; or the DnaJ Family). Chaperonin 10, a co-chaperone with HSP60, is another important protein in the heat shock response. GroES in E. coli).”
“DnaK functions by binding to the nascent polypeptide chain on ribosomes. This prevents premature folding, misfolding or aggregation. DnaK consists of two functional domains. The NH2-terminal ATPase Domain and the COOH?terminal Domain. The NH2-terminal ATPase domain binds ADP, ATP and hydrolyzes ATP. While the COOH-terminal domain binds polypeptides. DnaJ acts as a co-chaperone to DnaK. GrpE, another chaperone in DnaKJ’s folding pathway, facilitates the exchange between ADP/ATP. “The dnaK operon is where the genes for DnaK and DnaJ, as well as GrpE, are organized.
GroEL/ES proteins are another class of HSPs. They bind partially folded intermediates to prevent their aggregation and facilitate folding and assembly. It has also been suggested that GroEL may help misfolded structures unfold and refold with the aid of its co-chaperonin GroES. E. coli’s GroEL consists of 14 subunits within two-stacked heptameric rings that each contain a central cavity. This chaperone system can handle proteins up to 50-60 kDa due to the size of its complex cavity. GroEL/ES genes are also organized in an operon (the “groE” operon). B. subtilis’ expression of the dnaK/groE operons can be negatively controlled by a repressor protein via a CIRCE DNA elements (a palindromic sequence that runs between the promoter codon and the initiation codon). In B. subtilis, for example, the inactivation (HrcA?) of the repressor protein (whose activity is modulated through GroEL/ES) results in constitutive expression the two HSP operons. This enhances folding and secretion of proteins that are difficult to fold.
“HSPs were also detected in solventogenic Clostridium. Terracciano, et al. Terracciano et al. showed that C. acetobutylicum can respond to a slight increase in butanol. This is similar to the response that heat shock produces. Using a chemostat, Pich, et al. Pich et al. demonstrated that HSP synthesis rates and solventogenic proteins increased hours before solvents could be detected in the medium. Bahl also observed that certain factors, such as a lower pH, low growth rate and excess carbohydrates, may be involved in the induction stress response in C. Acetobutylicum.
The studies mentioned above only mention the existence of heat/shock stress reaction proteins in solventogenic bacteria. However, there is no conclusive evidence about the role of these proteins or guidance regarding their use in solvent production. The prior art has not resolved the long-standing concern about solventogenic bacteria’s viability. The art still needs to provide an organism or a related method that can enhance the fermentation of such bacteria and increase solvent generation.
The following publications provide more detailed and better understanding of the background information.
“41. Herbort M., U. Schon K. Angermann J. Lang and W. Schumann Cloning and sequencing the dnaK operon for Bacillus stearothermophilus. Gene, 1996. 170(1): p. 81-84.”
“DESCRIPTION DU DRAWINGS”
“FIG. “FIG. Italics are used to indicate genes that have been cloned. Enzyme names indicated in bold and are abbreviated as follows: LDH, lactate dehydrogenase, HYDA, hydrogenase, PTA, phosphotransacetylase, AK, acetate kinase, THL, thiolase, CoAT, CoA transferase, AADC, acetoacetate decarboxylase, BHBD, ?-hydroxybutyryl DH, CRO, crotonase, BCD, butyryl-CoA DH, PTB, phosphotransbutyrylase, BK, butyrate kinase, AAD, alcohol/aldehyde DH, and BDHA & BDHB butanol DH (isozymes A & B).”
“FIGS. “FIGS. Wild type (open circles); 824-pGROE1 (closed squares); 824-pSOS95del (open squares).
“FIG. “FIG. Data are averages of two fermentations. rGLY1, glucose utilization; rTHL, acetyl-CoA utilization; rHYD, hydrogen formation; rPTBBK, butyrate formation; rPTAAK, acetate formation; rBYUP, butyrate uptake; rACUP, acetate uptake; rACETONE, acetone formation; rBUOH, butanol formation.”
“In light the above, the object of the present invention is to provide a method for increasing cell resistance to toxic organic compounds, thereby addressing many issues and concerns in the prior art, such as those mentioned above. The skilled in the art will understand that the invention may have one or more components that meet certain objectives. However, one or more of its aspects may meet other objectives. Each objective might not be applicable equally to all aspects of the invention. The following objects may be considered alternatively with respect to any aspect of the invention.
“It is the object of the present invention that an organism genetically modified to allow increased resistance to toxic organic compounds be made.”
“It is the object of the present invention that an organism genetically modified to enhance expression of solvent tolerance genes or to overexpress stress response/heat proteins in order to increase cellular resistance against toxic organic molecules”
“It is another object in the present invention to attain higher cell densities for bacteria that can withstand increased levels of toxic organic compounds.”
“It is possible to also be an object in the present invention, either alone or in combination with another objective, to provide a method of enhanced fermentation by solventogenic bacteria.”
“It is also possible to use the present invention to provide a method for using protein expression to increase solvent levels from solventogenic organisms. Such protein expression can be provided by the recombinant bacteria discussed herein.”
“Other features, benefits, and advantages of this invention will be evident from this summary and its descriptions. They will also be immediately apparent to those skilled with basic knowledge in gene expression, molecular genetics and biochemistry. These objects, features, advantages and benefits will be obvious when they are viewed in conjunction with the data, figures, and examples that accompany them.
The present invention provides an organism that can be used to make prokaryotes or eukaryotes more resistant to toxic organic compounds. A strain of bacteria can be created by transforming a plasmid that contains genes for a stress response proteins into a strain of bacteria for gene expression. This bacteria can then be used to produce and/or transform toxic organic molecules. The skilled artisan will quickly notice that the plasmid can be episomal or integrative. Plasmid construction can be done using common techniques that are well-known to those with ordinary skill in the field. The plasmid constructed contains genetic code that allows expression of stress response proteins. This material may include, but not be limited to, heat shock proteins GroES/EL and DnaK or a combination thereof.
“A preferred embodiment allows for the expression of stress response proteins to be overexpressed by the constructed plasmid. You can achieve this by using methods that are well-known in the art. For example, you could provide the genetic sequence of a stress response gene or express a gene to induce the endogenous stress reaction protein operon. The genetic sequence of the constructedplasmid could include, for example, C. acetobutylicum’s groE operon. The alternative to the groE operon or in combination with it, the constructedplasmid could contain the dnaK or the genetic sequence of other stress response proteins, which may be from bacteria with similar characteristics. The operons that code for stress response proteins could be controlled by their natural promoters, or other promoters created to increase expression of the operons. The constructed plasmid-manipulated bacteria should exhibit prolonged fermentation and lower cell lysis than wild-type strains.
“In another embodiment, a strain may be created by inactivating or removing genetic material that normally suppresses expression solvent-tolerant genes. These genes confer greater resistance to toxic organic compounds and/or molecules. A negative regulatory element, such as CIRCE, can be removed or inactivated to increase expression of specific solvent-tolerant genes. These genes can increase tolerance to solvents and other toxic organic molecules, as well as produce higher titers during bio-production. Solvent-tolerance genes include those that increase cell resistance to toxic organic compounds and may also include proteins from the multi-drug efflux pump. No matter whether the preferred recombinant species are modified by downregulation, overexpression, or a combination thereof they can still be used. Other recombinant bacteria of the same nature are included in this invention, as described below.
“In part, this invention also provides a method for solvent production and/or using protein expression to increase solvent titers. This is done by creating a cell line called solventogenic clostridium and then manipulating the genetic code to overexpress stress response genes. The manipulated cells are able to resist solvents better when given the right conditions and media. Solventogenic clostridium is described in detail herein. However, the methods of this invention are extensible to other types of cells to make solvents, such as butanol and methanol, ethanol, acetone, and so on. These cell lines can be genetically modified in accordance to the teachings and will be easily understood by those who are skilled in the art. Additionally, the conditions for cell growth and production of useful metabolic byproducts can be modified depending on the cell’s particular needs, including temperature, oxygen, and nutrients. This is also known to those who are skilled in the art.
“Partially, the invention also addresses one or more methods for increasing solvent titer levels in solventogenic organisms. Alternate, but similar, methods may also be used to prolong the fermentation stage for solventogenic cells, such as solventogenic Clostridium or other anaerobic Gram-positive bacteria. Solventogenic cells can be added to any number of cultures or fermentation systems, batch or not, whose constitution will be well-known by those who are skilled in the art. Overexpression of heat shock proteins can increase the fermentation stage of solventogenic cell. The groE and dnaK operons are genetic sequences that encode heat shock proteins, as we have already discussed. The genes orfA to grpE are all part of the dnaK operon. OrfA may be similar to HrcA, a repressor protein of CIRCE-regulated heat shock genes of B. subliminis. Its overexpression could also be problematic. The function of the orfC, and orfD genes is unknown. A genetic sequence containing the genes grpE and dnaK can be made into solventogenic cells using a plasmid, or other techniques that are well-known to those who are skilled in the art. These genes can be overexpressed using a functional promoter during both the stationary and exponential phases of culture. Other genetic sequences that encode different stress response proteins can be combined or used alone to achieve the same results. This includes overexpression of the GroE operon (ca. 2.2 kB) and groE, the dnaK and dnaJ genes and grpE genes (thus, total size for all 5 genes of the DnaK operon of ca. 6 kb) simultaneously from a single plasmid with different promoters. The solventogenic cells are able to tolerate the solvent byproducts from fermentation if they have overexpression of stress response proteins. Solventogenic cells can produce higher yields of solvents and other products if they have a greater tolerance.
“Alternatively, stress response protein may be overexpressed by suppressing expression of the gene that codes for a putative regulator using either antisense or gene-knockout RNA techniques. The putative repressor protein, orfA, of C. acetobutylicium atCC 824 works through the CIRCE DNA element. This element is located in front of both the groE (and dnaK) operons. This method is also practical because it allows for increased HSP production by activating a single gene, rather than overexpressing many structural genes.
“While several aspects of this invention have been discussed, in the preceding, with respect to certain bacteria and/or recombinant strains thereof, it will be understood by those skilled in the art made aware of this invention that such aspects can be applied more generally to a wide variety of bacteria including, but not limited, to the following: solventogenic bacteria and/or related clostridia (e.g., C. acetobutylicum, C. beijerickii, C. cellulolyticum, C. thermocellum, C. butyricum, C. saccharoperbutylacetonicum, and others), Escherichia (including E. coli), Zymomonas mobilis, Enterobacter, Serratia, Erwinia, acetic acid bacteria, Bacillus (including B. subtilis), Lactobacilli, Pseudomonas, methylotrophic bacteria, Acetobacter, Lactococcus, Arthrobacter, Ralstonia, Gluconobacter, Klebsiella, Propionibacterium, Eubacterium, Streptomyces, and Rhodococcus.”
“Likewise, the invention and the related methods can be made, as would be understood by those skilled in the art, using all major yeast genera and/or their recombinant varieties, including but not limited to Saccharomyces and Schizosaccharomyces, Kluyveromyces and Candida, Pichia, and methylotropic yeast. Other filamentous fungi can also be used and/or recombinant varieties thereof.
“The present invention may also relate to one or several methods for using heat shock proteins or their expression to increase microorganism tolerance of toxic substances. This method involves (i) creating a recombinant organism that has been transformed with a genetic code to increase the expression of a heat-shock protein; (ii), providing a suitable fermentation system; and (iii), contacting the recombinant organism with the system for a sufficient time to allow the expression of the heat-shock protein. These microorganisms are useful depending on the system used and the desired fermentation effect. For instance, various solventogenic bacteria can be utilized where the toxic substance may be the solvent or a solvent by-product of such a system, such toxins/products/solvents including, but not limited to, butanol, acetone, ethanol and other substances of the sort described herein.”
“Various other microorganisms can also be used and/or recombinant forms thereof depending on the bioremediation system in which this invention is used to increase/enhance tolerance to toxic substances such as alcohols, carboxylic acid, ketones and aldehydes and derivatives, derivatives or carboxylic acid derivatives, ethers. amines. phenols. glycols. epoxides. keto acids. hydroxy acids. heterocyclic compounds. Although Aspergillus is the preferred recombinant microorganism, other bacteria, yeast, or fungi can be used. Similar to this, these microorganisms, or recombinant variants thereof, can be used in a biocatalytic process, where expression of heat shock proteins increases tolerance to the starting substrate, byproduct, resulting product, or any combination of them. Referring to the biomediation discussion above, such substrates/products can include those listed with, whose identity will determine which microorganism is used. These considerations are why preferred recombinant microorganisms are species from the genera Escherichia and Saccharomyces, as well as those of the genus Pichia. Each of these can be provided in accordance with the instructions or using recombinant techniques that are well-known to those who have the necessary skill.
“Preferred embodiments have been described using a combination of recombinant techniques and plasmid. As those who are skilled in the art will know, bacteriophage is also a viable cloning vector. In a non-related context, the feasibility of phage-cloning Vectors in C. Acetobutylicum was also discussed in prior art. Jones and Woods, “Acetone-Butanol Fermentation Rediscovered”,? Microbiological Review, 1986, 50, 484-524. The present invention contemplates the use of a bacteriophage that can increase expression of a heat-shock protein in combination with the methods and/or recombinant organisms described herein.
“While many examples, tables, figures, and supporting data have been provided for the purpose of general illustration in the context solvent production and increased tolerance thereto it will be apparent to those skilled in art that the present invention is easily extended to a variety bioremediation or biocatalytic system. In both cases, it has been shown that the availability of microorganisms for bioremediation//or biocatalysis is limited by toxic effects of the target molecule, substrate, product, and/or byproduct. This is in a manner similar to the one described herein for solvent manufacturing. With regard to biocatalysis and the effect of such toxins on microorganisms, consider, for example, ?Production and Biotransformation of 6-pentyl-alpha-pyrone by Trichoderma harzianum in Two-phase Culture Systems,? Serrano-Carreon, BalderasRuiz K. Galindo E. Rito-Palomares MM Applied Microbiology and Biotechnology 58(2): 170-174 Feb 2002; and?Organic Solvent Toxicity in Photoautotrophic Unicellular Microorganisms. Leon R. Garbayo I. Hernandez R. Vigara J. Vilchez C Enzyme & Microbial Technology 29(2): 173-181 Aug. 72001. For more information on bioremediation and the resulting toxicities, please see,?Detoxification Of Reactive Intermediates During Microbial Metabolism of Halogenated Compounds?. Vlieg JETV. Poelarends J, Mars A. E. Janssen D. Current Opinion In Microbiology 3 (3) June 2000: 257-262; and?In-situ Bioremediation via Mulching of Soil Polluted By a Copper Nickel Smelter. Kiikkila O. Perkiomaki JS, Barnette M. Derome J. Pennanen T. Tulisalo e, Fritze H Journal of Environmental Quality (30 (4): 1134-1143, July-August 2001. Although the prior art recognizes these concerns, there is no solution.
“Accordingly, it will become apparent that increased tolerance to toxic substances can increase the effectiveness of bioremediation and biocatalytic processes. This is possible through a simple extension of the methods and recombinant microorganisms described in this invention. Overexpression of heat shock proteins is shown to increase solvent production. However, it can also be used for other toxic effects. The present invention allows for bioremediation and biocatalysis in conditions that might otherwise prohibit such processes.
“This invention illustrates the design and development for several preferred embodiments. It is important to first understand two common stress responses proteins that are useful for the above-described methods and constructs. GroESL, and DnaKJ both are stress response proteins commonly called heat shock proteins. These operons were cloned using C. acetobutylicum. The groE operon is composed of two genes: groES, and groEL. The dnaK operon is made up of seven genes: orfA to grpE and dnaK. The groE operon can be transcribed as a bicistronic text that is 2.2 kb long. It has a transcription terminator downstream of groEL, and a transcriptional starting site upstream of groES. For the dnaK operon, there were two transcription start sites. One transcriptional start site is located upstream from orfA and the other upstream from grpE. The four transcripts had a size of 5.0, 3.8 and 3.8 kb respectively. Both the groE, dnaK operons had putative ribosome binding sites that were identical to the ones found in E. coli and those found in gram-positive bacteria. The consensus promoter sequences from gram-positive bacteria were also identified and compared. Recently, the DnaK, DnaJ, GrpE, and OrfA of C. acetobutylicum were shown to refold guanidium hydrochloride-denatured firefly luciferase in vitro. The DnaK system of C. Acetobutylicum also prevented the aggregation OrfA from C. Acetobutylicum. orfA codes are used to identify a potential repressor for the dnaK operon via a similar CIRCE regulatory element to that in B. subtilis.”
Because all the genes in FIG. 1 have been cloned, and its physiology has been studied extensively among C. Acetobutylicum strains. The US DOE published the genetic and physical maps of this organism. Solventogenic clostridium such as C. Acetobutylicum ATCC824 has been described in greater detail below. However, it should be obvious that the same principles can be applied to other anaerobic protokaryotes as well as solventogenic clostridium for making solvents like butanol or acetone as well as other chemicals and enzymes used for biotransformations, and bioremediation.
“Further research has revealed that solvent tolerance is influenced by multi-drug efflux proteins. Multi-drug efflux pumps are responsible for detoxifying the intracellular environment by exporting toxic organic components. The acrAB operon in E. coli forms a multidrug efflux pump. It has been shown to be highly expressed by solvent-tolerant mutants. A deletion of acrAB can cause loss of tolerance to n-hexane or cyclohexane. The transcriptional regulators marR, marA and soxR regulate the expression of acrAB as well as other related genes. Overexpression of soxS and marA was shown to increase organic solvent tolerance in several E. coli strains. Two multi-drug efflux proteins in B. subtilis have been shown to confer resistance against various organic compounds (ethidium brmide, fluoroquinolone antibiotics, and acridine colors). Blt and br are the genes that code for these homologous transporters. Their expression is controlled by regulators encoded in bltR or bmrR, respectively. Pseudomonas.aeruginosa has shown antibiotic resistance and solvent tolerance to be linked to several efflux pumps, including MexA-MexB, MexC?MexD, OprJ, as well as MexE?MexF?OprN. These pumps consist of a periplasmic linking protein (MexA-MexC, MexC, MexE), a mexoplasmic protein [MexB, MexD and MexF] and an outer membrane protein (?OprM,? OprJ and OprN). Similar efflux systems were found in P. putida strains. They have been shown to affect solvent tolerance. Below is a table that shows the collective results of several genes. These same constructs and methodologies can be used to create solvent-resistant strains of bacteria using the overexpression or similiar proteins.
“TABLE 1nOrganic solvent tolerability homologs in C. acetobutylicum ATCC824 gnome\nProtein\nOrganism & Size C. acetobutylicum ATCC824 Homolog Percent\nGene (aa) Function/Role of Protein Identity (Similarity) & Position\nE. Coli acrB1049 Transmembrane component a 23% (44%%) over 782 Aa @ 89613-91907nmajor Eflux pumpnE. Coli acrR 215, Regulator for acrAB operon 25% (54% over 159 Aa @ 3525884-3526357nE. Coli marA 129 Transcriptional activator of the 30% (60%) over 98 aa @ 152061-152354\nmar (multiple antibiotic 30% (58%) over 97 aa @ 1131958-1132245\nresistance) operon (responsible 29% (54%) over 91 aa @ 1258122-1257850\nfor environmental stress factors) 28% (51%) over 97 aa @ 2605268-2604981\n28% (43%) over 99 aa @ 3235580-3235287\nE. Coli soxR 125 Repressor of the mar operon 27% (47%) over 106 aa @ 3166960-31667274\n25% (46%) over 96 aa @ 664993-665280\nE. Coli soxR 154 Transcriptional activator of the 30% (46%) over 95 aa 3269119-3269403\nAraC subfamily 24% (52%) over 113 aa @ 664993-665280\nE. Coli soxS107 Transcriptional activator for the 31% (62%) over 98nAraC Subfamily 31% (52%), over 91nAraC (1158122-1257850n27% (46%), over 103nAraC subfamily 2605286-2604981nB. subtilis bltR 27 Regulator for blt expression 28% (53%) over 268 @ 3306158-33306973nB. subtilis blt 400 Multi-drug efflux protein 24% (44%) over 379 aa @ 543493-542330\nB. subtilis bmr 389 Multi-drug efflux protein 19% (39%) over 353 aa @ 543430-542330\nB. subtilis bmR 279. Regulator for bmr expression 25% (55%) over 275 @ 3306164-33306970nP. aeruginosa1046 Cytoplasmic components of 25% (46%) above 765 aa @89730-91973nMexBmulti-drug efflux pumpsnP. aeruginosa1043 Cytoplasmic components of 22% (45%), over 692 aa @89730-91973nMexDmulti-drug efflux pumpsnP. aeruginosa1062 Cytoplasmic components of 20% (40%) above 781 aa @ 89931-91973nMexFmulti-drug efflux pumps
“EXAMPLES FROM THE INVENTION”
The following data and examples are non-limiting and illustrate various features and aspects of the constructs and/or associated methods of the invention. They include organisms and/or the creation of cells that are resistant to toxic organic chemicals and achieve higher solvent titers. The present constructs and the related methods produce results and data that are unexpected, unexpected and contradictory to the prior arts. Although the invention’s utility is demonstrated through several methods and constructs, those who are skilled in the art will understand that similar results can be obtained with other methods and constructs, provided they are within the scope of the invention.
“General Methods.”
“Stoichiometric Flux Analysis Using a Physiological Contraint.”
The assumption that product yields only affect enzymes in product synthesis is too simplistic. A more comprehensive approach to quantitatively understanding cell metabolism may be able to significantly increase product yields. Papoutsakis, who proposed a method called metabolic flux analysis (MFA) that used metabolic pathway balances to create a model for cellular metabolism in 1984, has proven to be a very useful approach. This model is a set of linear equations that are based on species balances as well as in vivo metabolic pathways fluxes. This system of equations is often underdetermined. It must be further modified to be useful. Typically, biological constraints are used to approximate metabolic intermediates in pseudo-steady states. The unresolved singularity prevented the calculation some critical pathway fluxes in the C. acetobutylicum metabolism. A new technique was developed that uses a physiological nonlinear constraint to relate the fluxes around the singularity using a software code that employs a model-independent heuristic global optimization approach. This solves the nonlinear problem. Correlating intracellular pH profiles and calculated butyrate production pathway fluxes has validated the use of the nonlinear constraint. This model was used for analysing fermentation data from various C. acetobutylicum species. Flux analysis revealed previously unknown roles for the acid-forming pathways. The fluxes from the acetate formation pathway were significant throughout the stationary phase, while the butyrate-formation pathway was responsible for the uptake of butyrate.
“AntisenseRNA (asRNA), as a Metabolic Engineering tool.”
AsRNA strategies could offer several advantages over gene inactivation in metabolic engineering. These include rapid implementation and the ability to induce repression of protein production with inducible promoters. AsRNA has been shown to be involved in the regulation mechanisms of many genes, including the Clostridium species’ glutamine synthetase. Strain 824 (pRD4) was created to produce a 102 nucleotide RNA with 87% complementarity. It also exhibited 85%-90% lower BK, AK and PTB specific activities and 45%-50% higher PTB and PTA activities. Strain 824 (pRD4) also showed earlier solventogenesis induction, resulting in 50% and 35% respectively higher final titers for acetone or butanol. Strain 824 (pRD1) was designed to produce a 698 nucleotide-asRNA with 96% complementarity and 700/o lower PTB and K activities, respectively. Strain 824 (pRD1) also had 300% higher levels a lactate-dehydrogenase. Although the levels of acids were not affected by 824(pRD1) fermentationss, butanol and acetone titers decreased by 96% and 75% respectively. The 824(pRD1) reduction in solvent production was offset by a?100-fold higher level of lactate production. It is clear that the levels of butyrate-formation enzymes do not have a significant effect on butyrate formation fluxes. These strategies and other technologies can be used to downregulate genes that inhibit the expression of heat-shock proteins in microorganisms.
“Phenomenological Regulations of Solvent Formation.”
“In batch fermentation the growth phase solventogenic clostridia of solventogenic clostridia (FIG.1) is acidogenic. This means that it produces acetate, butyrate, and H2 to fulfill cellular requirements for ATP. 1). Acetone and alcohol are associated with reduced H2 production in the non-growth as solventogenic phase. There is also a reduction in H2 uptake and often, butyrate and acetate. There are many fermentation conditions that can affect product formation and the initiation or solvent formation. These include low pH, carboxylic acid additions, iron limitations, and inhibitors (such CO) of H2 production. Some conditions that induce solventogenesis’ metabolic switch were also shown to cause stress (heat shock).
“Transformation, Vectors and Host-Plasmid Interactions”
“Electrotransformation has become the method of choice for introducing DNA. The present invention identifies a restriction system (Cac824I in strain ATCC 824) that frequently cuts DNA originating with E. coli or any other G+C rich DNA. It also prevents efficient transformation. In vivo systems have been developed to methylate DNA in E. coli, specifically at Cac824I recognition points. This protects it from restriction. Several E. coli-C. acetobutylicum acetobutylicum vectors with different origins of replication have been reported. They are stable in segregation and have copy numbers between 7-14 per cell. This makes them suitable for ME applications. Different vectors can elicit unusual, but beneficial, host-plasmid interaction results that are both unique and unexpected (but still useful for solvent production).
“Confirmation of successful transformation and quantitation”
To correlate increased butanol production and higher levels of these HSPs, it is necessary to confirm that the groE and dnaK operon genes are expressed (mRNA & protein) by the recombinant 824 strains created in the present invention. The levels of GroESL and DnaKJ proteins will be found in strain 824 as well as the controls for plasmid carriers, 824 (pIMP1), and 824 (pSOS95del). However, the levels could vary between strains. You can determine the concentrations of dnaK and dnaJ, groEL, grpE, and groES by Northern Blot, RTPCR, or microarray analysis.
Quantitative Western blot (immunoblot), analysis can measure the amount of groE or dnaK operon protein levels. These proteins will need monoclonal or polyclonal antibodies. Polyclonals that are used with E.coli proteins have been shown to cross-react with C. Acetobutylicum proteins. This is likely due to the high homologous HSP protein levels in both organisms. StressGen Biotechnologies in Victoria, BC, Canada, offers monoclonal or polyclonal antibodies against all major E.coli HSPs. If you are interested in the relative amounts of proteins, you can use Western analysis to determine their quantity using commercially available E.coli HSP proteins.
“Example 1”
“The following shows a representative cell constructed in accordance to this invention. It has increased resistance against toxic organic molecules, such as butanol. A plasmid (pSR1) was constructed using the common methods of those skilled in the arts and containing the C. acetobutylicum groE operon. It is controlled by its natural promoter. pSR1 was then electrotransformed into ATCC 824. After confirming the integrity of this strain’s plasmid, a series fermentations were performed with strain 824 (pSR1) as well as its control strain 824 (pIMP1) to verify. Plasmid (pIMP1), a plasmid that naturally occurs in C. Acetobutylicum AtCC 824 is the backbone of the groE operon. The strain 824 (pSR1) showed a prolonged solvent formation phase, decreased cell lysis, and reached a butanol titre of 227 mg (16.8 g/l), compared to the 175 mg (13.5 g/l), for the control strain 824 (pIMP1).
“Example 2”
The following illustrates another aspect to this invention: the increased expression of solvent-tolerant genes to increase cell tolerance to toxic organic compounds. One gene (solR) was identified upstream from aad and encodes a repressor for the sol locus genes (aad), ctfA and ctfB and adc). This gene is responsible for butanol formation in ATCC 824. Due to its deleterious effects on the transcription of the sol locus gene genes, overexpression of solR (pCO1) in 824 resulted in solvent-negative phenotype. By homologous recombination, inactivating solR resulted in strains B, H. These strains had a deregulated solvent production, with higher yields and fluxes towards butanol, acetone, and earlier inductions of aad than the wild-type strain. The recombination events were confirmed by Southern and PCR analyses. Both strains B and H were also subject to controlled pH fermentations for product and flux analysis. The final butanol concentrations in Strain B were 32-fold higher than those of the wild-type. The strain H also had high levels of butanol. These increases were even greater in strains with solR activation and overexpression of the plasmid encoded as aad. The strain H(pTAAD) produced 255 mM butanol, 162 mg of acetone and 43 mM ethanol. These are the highest levels of solvents ever recorded in a Clostridial fermentation at 30 g/L. This strain also exceeded the level ca 180mM butanol, which was considered the highest level of toxity for this solvent.
“The methods, data, and results of examples 3-10 show a variety of methodologies according to this invention. The groESL operon is representative of genetic materials, and plasmids, and can be used in recombinant mikroogranisms that can be applied to a variety of solvent production, bioremediation, or biocatalytic system.”
“Example 3a”
“GroESL overexpressing Strain”
“A recombinant C. Acetobutylicum strain was created that overexpresses the groESL operon gene (groES) and groEL under the control of the Clostridial Thiolase Promoter. PCR was used to amplify the groESL operon genes from the chromosome with primers that were designed to exclude the operons regulatory element and natural promoter. CIRCE (Controlling inverted repeat of chaperone expression) is the regulatory element that controls expression of groESL via interaction with a potential regulatory protein, orfA. OrfA, which is also the major chaperone familiy member, is the first gene of the dnaKJ operaon. To allow expression of the groES, groEL and groES genes on the plasmid under control of the orfA protein derived from the chromosomal orfA gene, the CIRCE element was removed. The groESL operon gene was inserted into C. acetobutylicum/E. coli shuttle vector pSOS95del. The shuttle vector was made by removing the clostridial Acetone operon (ace), genes from pSOS95 (unpublished findings). The thiolase promoter was left for expression of cloned gene clones. The resulting groESL-overexpression plasmid (pGROE1) was transformed into C. Acetobutylicum AtCC 824 and used for subsequent characterization experiments.
“Example 3b”
“Description of E. Coli Transformation used in Construction of GroESL & DnaKJ Plasmids.”
“Example 4”
“Fermentations with 824 (pGROE1) or 824 (pSOS95del).”
“Construction Shuttle Vector pSOS95del. Plasmid pSOS95 (available from S. B. Tummala & E. T. Papoutsakis in 1999. Clostridium Acetobutylicum ATCC824: Development and Characterization of an Ene Expression Reporter System Appl. Environ. Microbiol. 65:3793-3799 was digested using EheI and purified with the GFX DNA Purification Column. It was then digested again with BamHI. The large fragment (5.0 kb), was isolated using GFX Gel Band Purification columns and blunt-ended with Klenow fragment. Re-ligation of this large fragment led to the formation of the pSOS95del vector.
“A series duplicate fermentations were performed with the 824 (pGROE1) and 824 (pSOS95del), control strains, and wild type 824 to study the effects of groES/groEL overexpression on solvent production, metabolic fluxes, transcriptional and protein expression patterns, as well as on the formation of solvents. The effects of the hostplasmid effect (wild-type versus control) were also examined. The fermenters were kept in anaerobic conditions with nitrogen, and the pH of the fermenters was low at 5.0. Ammonium hydroxide was used to maintain the pH. To prevent total glucose depletion, glucose was given once during fermentation. Supernatants were collected to analyze product formation by HPLC. Cell pellets were then harvested for Western Blot analysis. RNA samples were also taken for microarray and RTPCR analysis.
“Example 5”
“Product Formation and Metabolic Fluxes.”
“The presence and activity of the pGROE1 virus had dramatic effects on the formation of solvent/products, especially in the formation acetone, butanol and acetone, when compared with both wild type and control strains” (Table 2, FIG. 2). The 824(pGROE1) strain produced 231 mM (and 107 mM) of acetone, butanol and butanol respectively. This compares to the wild type’s wild type and wild strain’s wild-type (96 mM, 175 mM, and 96 mg, respectively). This is a 66% and 56% increase in final acetone titers, respectively, compared to the 824 (pSOS95del), control strain. It is interesting to observe that solvent production begins later than expected and occurs in two distinct phases for each recombinant strain. This could be due to the frequent observation of host-plasmid interaction. The final ethanol titers of the 824 (pSOS95del), and 824 (pGROE1) strains were slightly lower than those of the wild-type 824 strain (28 mg). There was no statistical difference in acetate levels between strains. Butyrate levels were slightly lower than the wild type (80 mg) in the two recombinant strains (73mM and 70mM, respectively). Although the 824(pGROE1) strain had higher optical densities, it was still lower than the 824 (pSOS95del), control strain. Both recombinant strains grew at a similar rate (2.01 hours and 1.99 hours), with the wild type exhibiting slower exponential growth (1.24 hours).
“Example 6”
“An analysis of in vivo fluxes specific to a variety of key metabolic reactions gives an excellent portrait of carbon flows among different strains while also accounting for cell densities. CompFlux, a metabolic flux analysis program based on Desai and colleagues, 1999, was used to calculate the in vivo metabolic fluxes for wild type and recombinant species. FIG. FIG. 3 displays time course profiles of nine key fluxes. There are significant differences in the specific in-vivo fluxes between the wild type 824 and 824 (pSOS95del) strains. Both recombinant and wild types exhibit distinct phases. The wild type only has one. This pattern has also been observed in other plasmid-carrying strains and may be due to a generalized effect of plasmids. The plasmid 824(pGROE1) showed a higher glucose utilization (rGLY1), and an increased acetylCoA utilization (rTHL), relative to the control strain. Strain 824(pGROE1) also had higher acetate and butyrate uptake rates, which leads to an increase in acetone formation (rACETONE). Acetone can be made from either acetate uptake or butyrate, it seems. Acetate uptake (rACUP), which is the main factor in acetone production, seems to be more important than butyrate. Both the butyrate uptakes (rBYUP), and butyrate formations (rPTBBK), are small compared to the increases of acetate uptake and formation (rPTAAK). The butanol (rBUOH), formation fluxes in Strain 824(pGROE1) were also significantly higher than those in strain 824[pSOS95del]. These differences in in vivo fluxes correspond to the higher final solvent titers observed in strain 824 (pGROE1) and further demonstrate the delay in solvent production in both of these recombinant strains.
“Example 7a”
“Microarray Analysis”
“Large-scale transcriptional analysis” has been a useful tool in understanding the differences between the genetic programming of different cell types and growth conditions. The TIGR protocol was used to print DNA microarries with more than 1000 genes. This represents approximately one-fourth of the C. Acetobutylicum genome. PCR primers are designed to amplify gene fragments of 500 bp in size so that non-specific hybridization can be minimized. The PCR products were then dried and dissolved in a water-DMSO solution. They were then spotted on Corning CMT?GAPS in triplicate. Glass microarray slides using a BioRobotics DNA arrayer. The ability to spot in three copies allows for more thorough statistical analysis of microarray data. After the slides have been hybridized with cDNA, which has been fluorescently labeled using Cy-3 and Cy-5, they are then scanned with a GSI Lumonics ScanArray. Quantarray Microarray analysis software is used to quantify spot intensities. The data are normalized, and genes with significant expression differences are identified using a novel normalization/filtering method (currently not published). The effects of chaperonin (GroESL), overexpression, as also the effects on other common stresses like heat, oxygen, or solvents are all investigated in the context of this invention.
“Example 7b”
“Gene Clustering Analysis and Self-Organizing Map (SOM), Analysis”
“Microarray analysis generates a lot of data. It is difficult to analyze the data to determine the patterns of gene expression. This is because no two genes will likely exhibit the exact same response and the behavior can be very different. There are many mathematical methods that can be used to identify patterns in gene expression. Hierarchical clustering is the most popular method for this purpose. This involves putting data points into a strict hierarchy with nested subsets. Hierarchical clustering can be useful but has its limitations. Hierarchical clustering 1) is better suited to data that is truly hierarchical, such as the evolution and speciation of species; 2) lacks robustness, uniqueness, inversion problems; 3) is deterministic. This means clustering is based on local decisions, without the ability for reevaluate it. Self-Organizing Maps (SOMs) are a better choice for clustering and analysis. SOMs are much more reliable and accurate than hierarchical clustering. They can be implemented quickly, are scalable to large data sets, and are simple to use.
“Example 8”
“SOM analysis was done on the three data sets from the experiments described above [WT824 v.824(pSOS95del); WT824 v.824(pGROE1)] using GENECLUSTER. (Tamayo et. al., 1999). Cluster analysis was limited to genes that were significantly overexpressed at the 95% confidence level at any one or more times. To determine the number of clusters, we decreased the number of clusters so that all clusters were unique and did not overlap. Samples taken from the WT824, 824 (pSOS95del), and 824 (pGROE1) fermentations are labeled A-B, B, and C. These samples were taken during the exponential growth phase at respective optical densities (A600), of 0.40, 0.88 and 1.83 (S.D.=0.06). Due to apparent instability of RNA in the stationary phase, it has been impossible to isolate intact RNA from later times points.
“Example 9”
“Transcriptional Analysis for GroESL Overexpression”
“Overexpression of GroESL operon genes seems to cause a dramatic shift to transcriptional programming when compared with the plasmid-control strain. Three gene clusters were identified by SOM analysis. Each cluster represent a set of genes which have elevated expression in the 824(pGROE1) culture at one of the three time points (B,C,D; time point A has yet to be hybridized/analyzed). The following is a list of genes that belong to each cluster. First, the 824(pGROE1) strain shows an increased expression of several sporulation genes, mainly spoIII and family genes. The transition to stationary phase in Clostridium Acetobutylicum wild type is marked by sporulation. It is also accompanied by shifts to solvent formation, the accumulation of granulose, and the expression heat shock proteins. It is not known how these processes are controlled in the same way. It is possible that the regulation of key sporulation genes may be affected by the overexpression of heat shock proteins groES or groEL. In addition to the presence a plasmid, it is possible that groES or groEL genes are overexpressed. In general, stress is thought to cause sporulation. A number of genes involved in chemotaxis (Che), are upregulated at each time point. Third, the expression of flagellar genes is enhanced at the early stages, especially at time point C (mid to late exponential). It is not common for these genes to be expressed more often than expected. Spo0A has been shown to up regulate many of the genes involved in sporulation that are expressed more frequently in the GroESL-overexpressing strain. Spo0A levels were between 1.1 and 1.4 times higher in GroESL-overexpressing strains (while these expression levels weren’t high enough to be included as part of the cluster analysis), the nine Spo0A spots on DNA-array slides had an intensity correlation (Cy3 against Cy5) value 0.93 Spo0A also has been shown to regulate flaggelar and chemotaxis genes (Fawcett, et al. 2000). Two distinct cell populations could explain the distinct phases of growth seen during a fermentation (see FIG. 1) for this and other plasmid-carrying strains (see?Transcriptional Analysis of the Host-plasmid Effect?). below). AADC (key solvent-formation enzyme), many histidine kinases, signaling pathways, glycolysis genes and electron transport genes are all genes that show significant upregulation. Finally, both the groES (key solvent formation enzyme) and the groEL (gross enzyme) were significantly upregulated at all three times points (1.8 to 14. fold higher).
“Example 10”
“Transcriptional Analysis for the Host-Plasmid Interaction E”
The plasmid control strain 824(pSOS95del) was compared to the wild-type 824 strain. This shows that the transcriptional interaction between host and plasmid is very significant. Five gene clusters were identified by SOM analysis. The SOM analysis identified five clusters. Four clusters are sets of genes that have been elevated in the 824 (pSOS95del), plasmid-control strain. The fifth cluster is a group of genes that has been down-regulated. The plasmid strain is showing many of the same trends as the wild type. Many genes involved in sporulation, flagellar and chemotaxis are expressed early and more frequently. Many heat shock proteins are also upregulated early suggesting that cells react to plasmid exposure as they do to other stressors. Several other key metabolic genes were also upregulated: the acid formation gene, butyrate kinase; solvent formation genes alcohol hydrogenase and acetoacetate dcarboxylase, (AADC); several glycolytic genes, phosphofructokinase and GAPDH; and a transcriptional regulator for sugar metabolism. Numerous transcriptional and response regulators have also been upregulated. The gene cluster that represents the downregulated genes also includes hrcA, a negative transcriptional regulator of chaperonin-expression, and several primary metabolism genes. It would be expected to see an increase in chaperonins, which hrcA negatively regulates, if hrcA is included in the downregulated gene group. This is evident with the presence of the groES, grEL and grpE genes within the upregulated gene clusters. Additional validation can be done by using a priori gene regulatory information.
While the principles of the invention were described with reference to specific embodiments, it is important to understand that these descriptions are only examples and do not limit the invention’s scope. For instance, butanol and acetone have been described as the toxic organic molecules in this invention, but the methods and constructs described herein may be used to increase microorganism/cellular resistance to other toxic molecules or compounds including hexane derivatives and aromatic compounds like toluene as would otherwise be known to those skilled in the art for various solventogenic purposes or for application in other bioprocesses associated with such endeavors as bioremediation and biocatalysis. The claims that will be filed in this section will reveal other advantages, features, and benefits. These claims are limited by the reasonable equivalents of the claims, which would be easily understood by those who are skilled in the art.
Summary for “Increased cell resistance against toxic organic substances”
“Metabolic engineering is the manipulation of living organisms in order to produce desired metabolic substrates, products, and/or byproducts. This allows for the production of a wide range of valuable commodity or specialty chemicals. Recombinant DNA technology allows metabolic pathway modification by using targeted genetic modifications in place of or in addition to traditional mutagenesis. Numerous examples of metabolic engineering have been documented since the 1980s. To achieve desired results, variables like temperature and aerobic conditions can be also engineered or modified. One of the main limitations to such bio-production is the toxic effects of many desired substrates, products, and/or byproducts. This problem is both particular and general in that it can be found in the production of chemical commodity chemicals from renewable sources, bioremediation technologies and the use cells in biocatalysis which involves toxic organic molecules.
“The strictly anaerobic and Gram-positive, solventogenic Clostridium (C. acetobutylicum or C. beijerinckii) are great candidates for creating metabolically engineered strains that can be used in a variety of applications. These species can be used to produce butanol and butyric acids, acetones, propanol and 1,3-propanediol as well as polysaccharides and enzymes. Clostridium can be grown under low redox potential. This allows for a wide range of stereospecific reductions that yield chiral products, which are hard to synthesize chemically. These clostridial species, as well as related clostridial species, can also degrade toxic chemicals making them good candidates for bioremediation. Solventogenic clostrides have the advantage of being able to use a wide range of substrates, including mono-, oligo, and poly-saccharides. This includes common pentoses, hexoses, as well as biomass hydrolysates.
“Important potential uses of solventogenic Clostridium include the production of butanol or acetone. Poor process economics can affect such production, for example, a low butanol concentration in the product stream. These low butanol tolerances are caused by the low concentrations of butanol in these organisms. Because of this, separation costs for butanol are high. Economic analysis shows that the separation costs could be reduced by doubling the concentration of final butanol from 12 to 19 grams/l.
“Butanol toxicities are quite severe, as we have already mentioned. The final product stream should not contain more than 12-13 g/l of butanol before it causes cellular destruction. Butanol at high levels inhibits active nutrient transportation, membrane bound ATPase and glucose uptake. It also partially or totally abolishes the membrane pH and?pH. Butanol inhibits active nutrient transport, membrane bound ATPase, glucose uptake, partially or completely abolishes the membrane?pH and???? and lowers intracellular pH. Butanol’s chaotropic effect upon the cell membrane has been the reason for its toxic effects. The membrane model does not provide a complete explanation of butanol toxicity. A Clostridium acetobutylicum kinase activation mutant strain produces 230 mM (17 g/l), butanol, 83 mM (4.75 g/l), acetone, and 69 mM (3.2/l) ethanol. ?, Biotechnol. Bioeng., 67: 1-11 (2000)]. This strain was able to surpass the limit of 12-13 g/l Butanol-toxicity without any adaptation or selection for an increased butanol tolerance.
“Stress-response protein” is a group of specialized proteins that are essential for cellular function. They are found in normal cells under normal growth conditions and play an important role in cell biology as a protective cellular reaction to environmental stress. Due to their high abundance after heat shock, a common family of stress response proteins is called heat shock proteins (HSPs). Stress response proteins are able to recognize hydrophobic surfaces and aid in folding proteins. They can also bind the normative states of proteins, helping them to fold properly. The majority of stress response proteins form noncovalent interactions to the hydrophobic areas of misfolded protein proteins. This helps stabilize them from irreversible multimeric aggregates, misfolding of nascent proteins, unraveling under stress, and eventual degradation. “The properly folded and stabilized proteins can be used to perform their cellular function(s).
“The major heat-shock protein classes include the 90-kDa Heat Shock Protein (HSP90), 60-kDa Heat Shock Protein (HSP60), and 70-kDa Heat shock Protein (HSP70; DnaK for E. coli), as well as the 40-kDa Heat shock Protein (HSP40; or the DnaJ Family). Chaperonin 10, a co-chaperone with HSP60, is another important protein in the heat shock response. GroES in E. coli).”
“DnaK functions by binding to the nascent polypeptide chain on ribosomes. This prevents premature folding, misfolding or aggregation. DnaK consists of two functional domains. The NH2-terminal ATPase Domain and the COOH?terminal Domain. The NH2-terminal ATPase domain binds ADP, ATP and hydrolyzes ATP. While the COOH-terminal domain binds polypeptides. DnaJ acts as a co-chaperone to DnaK. GrpE, another chaperone in DnaKJ’s folding pathway, facilitates the exchange between ADP/ATP. “The dnaK operon is where the genes for DnaK and DnaJ, as well as GrpE, are organized.
GroEL/ES proteins are another class of HSPs. They bind partially folded intermediates to prevent their aggregation and facilitate folding and assembly. It has also been suggested that GroEL may help misfolded structures unfold and refold with the aid of its co-chaperonin GroES. E. coli’s GroEL consists of 14 subunits within two-stacked heptameric rings that each contain a central cavity. This chaperone system can handle proteins up to 50-60 kDa due to the size of its complex cavity. GroEL/ES genes are also organized in an operon (the “groE” operon). B. subtilis’ expression of the dnaK/groE operons can be negatively controlled by a repressor protein via a CIRCE DNA elements (a palindromic sequence that runs between the promoter codon and the initiation codon). In B. subtilis, for example, the inactivation (HrcA?) of the repressor protein (whose activity is modulated through GroEL/ES) results in constitutive expression the two HSP operons. This enhances folding and secretion of proteins that are difficult to fold.
“HSPs were also detected in solventogenic Clostridium. Terracciano, et al. Terracciano et al. showed that C. acetobutylicum can respond to a slight increase in butanol. This is similar to the response that heat shock produces. Using a chemostat, Pich, et al. Pich et al. demonstrated that HSP synthesis rates and solventogenic proteins increased hours before solvents could be detected in the medium. Bahl also observed that certain factors, such as a lower pH, low growth rate and excess carbohydrates, may be involved in the induction stress response in C. Acetobutylicum.
The studies mentioned above only mention the existence of heat/shock stress reaction proteins in solventogenic bacteria. However, there is no conclusive evidence about the role of these proteins or guidance regarding their use in solvent production. The prior art has not resolved the long-standing concern about solventogenic bacteria’s viability. The art still needs to provide an organism or a related method that can enhance the fermentation of such bacteria and increase solvent generation.
The following publications provide more detailed and better understanding of the background information.
“41. Herbort M., U. Schon K. Angermann J. Lang and W. Schumann Cloning and sequencing the dnaK operon for Bacillus stearothermophilus. Gene, 1996. 170(1): p. 81-84.”
“DESCRIPTION DU DRAWINGS”
“FIG. “FIG. Italics are used to indicate genes that have been cloned. Enzyme names indicated in bold and are abbreviated as follows: LDH, lactate dehydrogenase, HYDA, hydrogenase, PTA, phosphotransacetylase, AK, acetate kinase, THL, thiolase, CoAT, CoA transferase, AADC, acetoacetate decarboxylase, BHBD, ?-hydroxybutyryl DH, CRO, crotonase, BCD, butyryl-CoA DH, PTB, phosphotransbutyrylase, BK, butyrate kinase, AAD, alcohol/aldehyde DH, and BDHA & BDHB butanol DH (isozymes A & B).”
“FIGS. “FIGS. Wild type (open circles); 824-pGROE1 (closed squares); 824-pSOS95del (open squares).
“FIG. “FIG. Data are averages of two fermentations. rGLY1, glucose utilization; rTHL, acetyl-CoA utilization; rHYD, hydrogen formation; rPTBBK, butyrate formation; rPTAAK, acetate formation; rBYUP, butyrate uptake; rACUP, acetate uptake; rACETONE, acetone formation; rBUOH, butanol formation.”
“In light the above, the object of the present invention is to provide a method for increasing cell resistance to toxic organic compounds, thereby addressing many issues and concerns in the prior art, such as those mentioned above. The skilled in the art will understand that the invention may have one or more components that meet certain objectives. However, one or more of its aspects may meet other objectives. Each objective might not be applicable equally to all aspects of the invention. The following objects may be considered alternatively with respect to any aspect of the invention.
“It is the object of the present invention that an organism genetically modified to allow increased resistance to toxic organic compounds be made.”
“It is the object of the present invention that an organism genetically modified to enhance expression of solvent tolerance genes or to overexpress stress response/heat proteins in order to increase cellular resistance against toxic organic molecules”
“It is another object in the present invention to attain higher cell densities for bacteria that can withstand increased levels of toxic organic compounds.”
“It is possible to also be an object in the present invention, either alone or in combination with another objective, to provide a method of enhanced fermentation by solventogenic bacteria.”
“It is also possible to use the present invention to provide a method for using protein expression to increase solvent levels from solventogenic organisms. Such protein expression can be provided by the recombinant bacteria discussed herein.”
“Other features, benefits, and advantages of this invention will be evident from this summary and its descriptions. They will also be immediately apparent to those skilled with basic knowledge in gene expression, molecular genetics and biochemistry. These objects, features, advantages and benefits will be obvious when they are viewed in conjunction with the data, figures, and examples that accompany them.
The present invention provides an organism that can be used to make prokaryotes or eukaryotes more resistant to toxic organic compounds. A strain of bacteria can be created by transforming a plasmid that contains genes for a stress response proteins into a strain of bacteria for gene expression. This bacteria can then be used to produce and/or transform toxic organic molecules. The skilled artisan will quickly notice that the plasmid can be episomal or integrative. Plasmid construction can be done using common techniques that are well-known to those with ordinary skill in the field. The plasmid constructed contains genetic code that allows expression of stress response proteins. This material may include, but not be limited to, heat shock proteins GroES/EL and DnaK or a combination thereof.
“A preferred embodiment allows for the expression of stress response proteins to be overexpressed by the constructed plasmid. You can achieve this by using methods that are well-known in the art. For example, you could provide the genetic sequence of a stress response gene or express a gene to induce the endogenous stress reaction protein operon. The genetic sequence of the constructedplasmid could include, for example, C. acetobutylicum’s groE operon. The alternative to the groE operon or in combination with it, the constructedplasmid could contain the dnaK or the genetic sequence of other stress response proteins, which may be from bacteria with similar characteristics. The operons that code for stress response proteins could be controlled by their natural promoters, or other promoters created to increase expression of the operons. The constructed plasmid-manipulated bacteria should exhibit prolonged fermentation and lower cell lysis than wild-type strains.
“In another embodiment, a strain may be created by inactivating or removing genetic material that normally suppresses expression solvent-tolerant genes. These genes confer greater resistance to toxic organic compounds and/or molecules. A negative regulatory element, such as CIRCE, can be removed or inactivated to increase expression of specific solvent-tolerant genes. These genes can increase tolerance to solvents and other toxic organic molecules, as well as produce higher titers during bio-production. Solvent-tolerance genes include those that increase cell resistance to toxic organic compounds and may also include proteins from the multi-drug efflux pump. No matter whether the preferred recombinant species are modified by downregulation, overexpression, or a combination thereof they can still be used. Other recombinant bacteria of the same nature are included in this invention, as described below.
“In part, this invention also provides a method for solvent production and/or using protein expression to increase solvent titers. This is done by creating a cell line called solventogenic clostridium and then manipulating the genetic code to overexpress stress response genes. The manipulated cells are able to resist solvents better when given the right conditions and media. Solventogenic clostridium is described in detail herein. However, the methods of this invention are extensible to other types of cells to make solvents, such as butanol and methanol, ethanol, acetone, and so on. These cell lines can be genetically modified in accordance to the teachings and will be easily understood by those who are skilled in the art. Additionally, the conditions for cell growth and production of useful metabolic byproducts can be modified depending on the cell’s particular needs, including temperature, oxygen, and nutrients. This is also known to those who are skilled in the art.
“Partially, the invention also addresses one or more methods for increasing solvent titer levels in solventogenic organisms. Alternate, but similar, methods may also be used to prolong the fermentation stage for solventogenic cells, such as solventogenic Clostridium or other anaerobic Gram-positive bacteria. Solventogenic cells can be added to any number of cultures or fermentation systems, batch or not, whose constitution will be well-known by those who are skilled in the art. Overexpression of heat shock proteins can increase the fermentation stage of solventogenic cell. The groE and dnaK operons are genetic sequences that encode heat shock proteins, as we have already discussed. The genes orfA to grpE are all part of the dnaK operon. OrfA may be similar to HrcA, a repressor protein of CIRCE-regulated heat shock genes of B. subliminis. Its overexpression could also be problematic. The function of the orfC, and orfD genes is unknown. A genetic sequence containing the genes grpE and dnaK can be made into solventogenic cells using a plasmid, or other techniques that are well-known to those who are skilled in the art. These genes can be overexpressed using a functional promoter during both the stationary and exponential phases of culture. Other genetic sequences that encode different stress response proteins can be combined or used alone to achieve the same results. This includes overexpression of the GroE operon (ca. 2.2 kB) and groE, the dnaK and dnaJ genes and grpE genes (thus, total size for all 5 genes of the DnaK operon of ca. 6 kb) simultaneously from a single plasmid with different promoters. The solventogenic cells are able to tolerate the solvent byproducts from fermentation if they have overexpression of stress response proteins. Solventogenic cells can produce higher yields of solvents and other products if they have a greater tolerance.
“Alternatively, stress response protein may be overexpressed by suppressing expression of the gene that codes for a putative regulator using either antisense or gene-knockout RNA techniques. The putative repressor protein, orfA, of C. acetobutylicium atCC 824 works through the CIRCE DNA element. This element is located in front of both the groE (and dnaK) operons. This method is also practical because it allows for increased HSP production by activating a single gene, rather than overexpressing many structural genes.
“While several aspects of this invention have been discussed, in the preceding, with respect to certain bacteria and/or recombinant strains thereof, it will be understood by those skilled in the art made aware of this invention that such aspects can be applied more generally to a wide variety of bacteria including, but not limited, to the following: solventogenic bacteria and/or related clostridia (e.g., C. acetobutylicum, C. beijerickii, C. cellulolyticum, C. thermocellum, C. butyricum, C. saccharoperbutylacetonicum, and others), Escherichia (including E. coli), Zymomonas mobilis, Enterobacter, Serratia, Erwinia, acetic acid bacteria, Bacillus (including B. subtilis), Lactobacilli, Pseudomonas, methylotrophic bacteria, Acetobacter, Lactococcus, Arthrobacter, Ralstonia, Gluconobacter, Klebsiella, Propionibacterium, Eubacterium, Streptomyces, and Rhodococcus.”
“Likewise, the invention and the related methods can be made, as would be understood by those skilled in the art, using all major yeast genera and/or their recombinant varieties, including but not limited to Saccharomyces and Schizosaccharomyces, Kluyveromyces and Candida, Pichia, and methylotropic yeast. Other filamentous fungi can also be used and/or recombinant varieties thereof.
“The present invention may also relate to one or several methods for using heat shock proteins or their expression to increase microorganism tolerance of toxic substances. This method involves (i) creating a recombinant organism that has been transformed with a genetic code to increase the expression of a heat-shock protein; (ii), providing a suitable fermentation system; and (iii), contacting the recombinant organism with the system for a sufficient time to allow the expression of the heat-shock protein. These microorganisms are useful depending on the system used and the desired fermentation effect. For instance, various solventogenic bacteria can be utilized where the toxic substance may be the solvent or a solvent by-product of such a system, such toxins/products/solvents including, but not limited to, butanol, acetone, ethanol and other substances of the sort described herein.”
“Various other microorganisms can also be used and/or recombinant forms thereof depending on the bioremediation system in which this invention is used to increase/enhance tolerance to toxic substances such as alcohols, carboxylic acid, ketones and aldehydes and derivatives, derivatives or carboxylic acid derivatives, ethers. amines. phenols. glycols. epoxides. keto acids. hydroxy acids. heterocyclic compounds. Although Aspergillus is the preferred recombinant microorganism, other bacteria, yeast, or fungi can be used. Similar to this, these microorganisms, or recombinant variants thereof, can be used in a biocatalytic process, where expression of heat shock proteins increases tolerance to the starting substrate, byproduct, resulting product, or any combination of them. Referring to the biomediation discussion above, such substrates/products can include those listed with, whose identity will determine which microorganism is used. These considerations are why preferred recombinant microorganisms are species from the genera Escherichia and Saccharomyces, as well as those of the genus Pichia. Each of these can be provided in accordance with the instructions or using recombinant techniques that are well-known to those who have the necessary skill.
“Preferred embodiments have been described using a combination of recombinant techniques and plasmid. As those who are skilled in the art will know, bacteriophage is also a viable cloning vector. In a non-related context, the feasibility of phage-cloning Vectors in C. Acetobutylicum was also discussed in prior art. Jones and Woods, “Acetone-Butanol Fermentation Rediscovered”,? Microbiological Review, 1986, 50, 484-524. The present invention contemplates the use of a bacteriophage that can increase expression of a heat-shock protein in combination with the methods and/or recombinant organisms described herein.
“While many examples, tables, figures, and supporting data have been provided for the purpose of general illustration in the context solvent production and increased tolerance thereto it will be apparent to those skilled in art that the present invention is easily extended to a variety bioremediation or biocatalytic system. In both cases, it has been shown that the availability of microorganisms for bioremediation//or biocatalysis is limited by toxic effects of the target molecule, substrate, product, and/or byproduct. This is in a manner similar to the one described herein for solvent manufacturing. With regard to biocatalysis and the effect of such toxins on microorganisms, consider, for example, ?Production and Biotransformation of 6-pentyl-alpha-pyrone by Trichoderma harzianum in Two-phase Culture Systems,? Serrano-Carreon, BalderasRuiz K. Galindo E. Rito-Palomares MM Applied Microbiology and Biotechnology 58(2): 170-174 Feb 2002; and?Organic Solvent Toxicity in Photoautotrophic Unicellular Microorganisms. Leon R. Garbayo I. Hernandez R. Vigara J. Vilchez C Enzyme & Microbial Technology 29(2): 173-181 Aug. 72001. For more information on bioremediation and the resulting toxicities, please see,?Detoxification Of Reactive Intermediates During Microbial Metabolism of Halogenated Compounds?. Vlieg JETV. Poelarends J, Mars A. E. Janssen D. Current Opinion In Microbiology 3 (3) June 2000: 257-262; and?In-situ Bioremediation via Mulching of Soil Polluted By a Copper Nickel Smelter. Kiikkila O. Perkiomaki JS, Barnette M. Derome J. Pennanen T. Tulisalo e, Fritze H Journal of Environmental Quality (30 (4): 1134-1143, July-August 2001. Although the prior art recognizes these concerns, there is no solution.
“Accordingly, it will become apparent that increased tolerance to toxic substances can increase the effectiveness of bioremediation and biocatalytic processes. This is possible through a simple extension of the methods and recombinant microorganisms described in this invention. Overexpression of heat shock proteins is shown to increase solvent production. However, it can also be used for other toxic effects. The present invention allows for bioremediation and biocatalysis in conditions that might otherwise prohibit such processes.
“This invention illustrates the design and development for several preferred embodiments. It is important to first understand two common stress responses proteins that are useful for the above-described methods and constructs. GroESL, and DnaKJ both are stress response proteins commonly called heat shock proteins. These operons were cloned using C. acetobutylicum. The groE operon is composed of two genes: groES, and groEL. The dnaK operon is made up of seven genes: orfA to grpE and dnaK. The groE operon can be transcribed as a bicistronic text that is 2.2 kb long. It has a transcription terminator downstream of groEL, and a transcriptional starting site upstream of groES. For the dnaK operon, there were two transcription start sites. One transcriptional start site is located upstream from orfA and the other upstream from grpE. The four transcripts had a size of 5.0, 3.8 and 3.8 kb respectively. Both the groE, dnaK operons had putative ribosome binding sites that were identical to the ones found in E. coli and those found in gram-positive bacteria. The consensus promoter sequences from gram-positive bacteria were also identified and compared. Recently, the DnaK, DnaJ, GrpE, and OrfA of C. acetobutylicum were shown to refold guanidium hydrochloride-denatured firefly luciferase in vitro. The DnaK system of C. Acetobutylicum also prevented the aggregation OrfA from C. Acetobutylicum. orfA codes are used to identify a potential repressor for the dnaK operon via a similar CIRCE regulatory element to that in B. subtilis.”
Because all the genes in FIG. 1 have been cloned, and its physiology has been studied extensively among C. Acetobutylicum strains. The US DOE published the genetic and physical maps of this organism. Solventogenic clostridium such as C. Acetobutylicum ATCC824 has been described in greater detail below. However, it should be obvious that the same principles can be applied to other anaerobic protokaryotes as well as solventogenic clostridium for making solvents like butanol or acetone as well as other chemicals and enzymes used for biotransformations, and bioremediation.
“Further research has revealed that solvent tolerance is influenced by multi-drug efflux proteins. Multi-drug efflux pumps are responsible for detoxifying the intracellular environment by exporting toxic organic components. The acrAB operon in E. coli forms a multidrug efflux pump. It has been shown to be highly expressed by solvent-tolerant mutants. A deletion of acrAB can cause loss of tolerance to n-hexane or cyclohexane. The transcriptional regulators marR, marA and soxR regulate the expression of acrAB as well as other related genes. Overexpression of soxS and marA was shown to increase organic solvent tolerance in several E. coli strains. Two multi-drug efflux proteins in B. subtilis have been shown to confer resistance against various organic compounds (ethidium brmide, fluoroquinolone antibiotics, and acridine colors). Blt and br are the genes that code for these homologous transporters. Their expression is controlled by regulators encoded in bltR or bmrR, respectively. Pseudomonas.aeruginosa has shown antibiotic resistance and solvent tolerance to be linked to several efflux pumps, including MexA-MexB, MexC?MexD, OprJ, as well as MexE?MexF?OprN. These pumps consist of a periplasmic linking protein (MexA-MexC, MexC, MexE), a mexoplasmic protein [MexB, MexD and MexF] and an outer membrane protein (?OprM,? OprJ and OprN). Similar efflux systems were found in P. putida strains. They have been shown to affect solvent tolerance. Below is a table that shows the collective results of several genes. These same constructs and methodologies can be used to create solvent-resistant strains of bacteria using the overexpression or similiar proteins.
“TABLE 1nOrganic solvent tolerability homologs in C. acetobutylicum ATCC824 gnome\nProtein\nOrganism & Size C. acetobutylicum ATCC824 Homolog Percent\nGene (aa) Function/Role of Protein Identity (Similarity) & Position\nE. Coli acrB1049 Transmembrane component a 23% (44%%) over 782 Aa @ 89613-91907nmajor Eflux pumpnE. Coli acrR 215, Regulator for acrAB operon 25% (54% over 159 Aa @ 3525884-3526357nE. Coli marA 129 Transcriptional activator of the 30% (60%) over 98 aa @ 152061-152354\nmar (multiple antibiotic 30% (58%) over 97 aa @ 1131958-1132245\nresistance) operon (responsible 29% (54%) over 91 aa @ 1258122-1257850\nfor environmental stress factors) 28% (51%) over 97 aa @ 2605268-2604981\n28% (43%) over 99 aa @ 3235580-3235287\nE. Coli soxR 125 Repressor of the mar operon 27% (47%) over 106 aa @ 3166960-31667274\n25% (46%) over 96 aa @ 664993-665280\nE. Coli soxR 154 Transcriptional activator of the 30% (46%) over 95 aa 3269119-3269403\nAraC subfamily 24% (52%) over 113 aa @ 664993-665280\nE. Coli soxS107 Transcriptional activator for the 31% (62%) over 98nAraC Subfamily 31% (52%), over 91nAraC (1158122-1257850n27% (46%), over 103nAraC subfamily 2605286-2604981nB. subtilis bltR 27 Regulator for blt expression 28% (53%) over 268 @ 3306158-33306973nB. subtilis blt 400 Multi-drug efflux protein 24% (44%) over 379 aa @ 543493-542330\nB. subtilis bmr 389 Multi-drug efflux protein 19% (39%) over 353 aa @ 543430-542330\nB. subtilis bmR 279. Regulator for bmr expression 25% (55%) over 275 @ 3306164-33306970nP. aeruginosa1046 Cytoplasmic components of 25% (46%) above 765 aa @89730-91973nMexBmulti-drug efflux pumpsnP. aeruginosa1043 Cytoplasmic components of 22% (45%), over 692 aa @89730-91973nMexDmulti-drug efflux pumpsnP. aeruginosa1062 Cytoplasmic components of 20% (40%) above 781 aa @ 89931-91973nMexFmulti-drug efflux pumps
“EXAMPLES FROM THE INVENTION”
The following data and examples are non-limiting and illustrate various features and aspects of the constructs and/or associated methods of the invention. They include organisms and/or the creation of cells that are resistant to toxic organic chemicals and achieve higher solvent titers. The present constructs and the related methods produce results and data that are unexpected, unexpected and contradictory to the prior arts. Although the invention’s utility is demonstrated through several methods and constructs, those who are skilled in the art will understand that similar results can be obtained with other methods and constructs, provided they are within the scope of the invention.
“General Methods.”
“Stoichiometric Flux Analysis Using a Physiological Contraint.”
The assumption that product yields only affect enzymes in product synthesis is too simplistic. A more comprehensive approach to quantitatively understanding cell metabolism may be able to significantly increase product yields. Papoutsakis, who proposed a method called metabolic flux analysis (MFA) that used metabolic pathway balances to create a model for cellular metabolism in 1984, has proven to be a very useful approach. This model is a set of linear equations that are based on species balances as well as in vivo metabolic pathways fluxes. This system of equations is often underdetermined. It must be further modified to be useful. Typically, biological constraints are used to approximate metabolic intermediates in pseudo-steady states. The unresolved singularity prevented the calculation some critical pathway fluxes in the C. acetobutylicum metabolism. A new technique was developed that uses a physiological nonlinear constraint to relate the fluxes around the singularity using a software code that employs a model-independent heuristic global optimization approach. This solves the nonlinear problem. Correlating intracellular pH profiles and calculated butyrate production pathway fluxes has validated the use of the nonlinear constraint. This model was used for analysing fermentation data from various C. acetobutylicum species. Flux analysis revealed previously unknown roles for the acid-forming pathways. The fluxes from the acetate formation pathway were significant throughout the stationary phase, while the butyrate-formation pathway was responsible for the uptake of butyrate.
“AntisenseRNA (asRNA), as a Metabolic Engineering tool.”
AsRNA strategies could offer several advantages over gene inactivation in metabolic engineering. These include rapid implementation and the ability to induce repression of protein production with inducible promoters. AsRNA has been shown to be involved in the regulation mechanisms of many genes, including the Clostridium species’ glutamine synthetase. Strain 824 (pRD4) was created to produce a 102 nucleotide RNA with 87% complementarity. It also exhibited 85%-90% lower BK, AK and PTB specific activities and 45%-50% higher PTB and PTA activities. Strain 824 (pRD4) also showed earlier solventogenesis induction, resulting in 50% and 35% respectively higher final titers for acetone or butanol. Strain 824 (pRD1) was designed to produce a 698 nucleotide-asRNA with 96% complementarity and 700/o lower PTB and K activities, respectively. Strain 824 (pRD1) also had 300% higher levels a lactate-dehydrogenase. Although the levels of acids were not affected by 824(pRD1) fermentationss, butanol and acetone titers decreased by 96% and 75% respectively. The 824(pRD1) reduction in solvent production was offset by a?100-fold higher level of lactate production. It is clear that the levels of butyrate-formation enzymes do not have a significant effect on butyrate formation fluxes. These strategies and other technologies can be used to downregulate genes that inhibit the expression of heat-shock proteins in microorganisms.
“Phenomenological Regulations of Solvent Formation.”
“In batch fermentation the growth phase solventogenic clostridia of solventogenic clostridia (FIG.1) is acidogenic. This means that it produces acetate, butyrate, and H2 to fulfill cellular requirements for ATP. 1). Acetone and alcohol are associated with reduced H2 production in the non-growth as solventogenic phase. There is also a reduction in H2 uptake and often, butyrate and acetate. There are many fermentation conditions that can affect product formation and the initiation or solvent formation. These include low pH, carboxylic acid additions, iron limitations, and inhibitors (such CO) of H2 production. Some conditions that induce solventogenesis’ metabolic switch were also shown to cause stress (heat shock).
“Transformation, Vectors and Host-Plasmid Interactions”
“Electrotransformation has become the method of choice for introducing DNA. The present invention identifies a restriction system (Cac824I in strain ATCC 824) that frequently cuts DNA originating with E. coli or any other G+C rich DNA. It also prevents efficient transformation. In vivo systems have been developed to methylate DNA in E. coli, specifically at Cac824I recognition points. This protects it from restriction. Several E. coli-C. acetobutylicum acetobutylicum vectors with different origins of replication have been reported. They are stable in segregation and have copy numbers between 7-14 per cell. This makes them suitable for ME applications. Different vectors can elicit unusual, but beneficial, host-plasmid interaction results that are both unique and unexpected (but still useful for solvent production).
“Confirmation of successful transformation and quantitation”
To correlate increased butanol production and higher levels of these HSPs, it is necessary to confirm that the groE and dnaK operon genes are expressed (mRNA & protein) by the recombinant 824 strains created in the present invention. The levels of GroESL and DnaKJ proteins will be found in strain 824 as well as the controls for plasmid carriers, 824 (pIMP1), and 824 (pSOS95del). However, the levels could vary between strains. You can determine the concentrations of dnaK and dnaJ, groEL, grpE, and groES by Northern Blot, RTPCR, or microarray analysis.
Quantitative Western blot (immunoblot), analysis can measure the amount of groE or dnaK operon protein levels. These proteins will need monoclonal or polyclonal antibodies. Polyclonals that are used with E.coli proteins have been shown to cross-react with C. Acetobutylicum proteins. This is likely due to the high homologous HSP protein levels in both organisms. StressGen Biotechnologies in Victoria, BC, Canada, offers monoclonal or polyclonal antibodies against all major E.coli HSPs. If you are interested in the relative amounts of proteins, you can use Western analysis to determine their quantity using commercially available E.coli HSP proteins.
“Example 1”
“The following shows a representative cell constructed in accordance to this invention. It has increased resistance against toxic organic molecules, such as butanol. A plasmid (pSR1) was constructed using the common methods of those skilled in the arts and containing the C. acetobutylicum groE operon. It is controlled by its natural promoter. pSR1 was then electrotransformed into ATCC 824. After confirming the integrity of this strain’s plasmid, a series fermentations were performed with strain 824 (pSR1) as well as its control strain 824 (pIMP1) to verify. Plasmid (pIMP1), a plasmid that naturally occurs in C. Acetobutylicum AtCC 824 is the backbone of the groE operon. The strain 824 (pSR1) showed a prolonged solvent formation phase, decreased cell lysis, and reached a butanol titre of 227 mg (16.8 g/l), compared to the 175 mg (13.5 g/l), for the control strain 824 (pIMP1).
“Example 2”
The following illustrates another aspect to this invention: the increased expression of solvent-tolerant genes to increase cell tolerance to toxic organic compounds. One gene (solR) was identified upstream from aad and encodes a repressor for the sol locus genes (aad), ctfA and ctfB and adc). This gene is responsible for butanol formation in ATCC 824. Due to its deleterious effects on the transcription of the sol locus gene genes, overexpression of solR (pCO1) in 824 resulted in solvent-negative phenotype. By homologous recombination, inactivating solR resulted in strains B, H. These strains had a deregulated solvent production, with higher yields and fluxes towards butanol, acetone, and earlier inductions of aad than the wild-type strain. The recombination events were confirmed by Southern and PCR analyses. Both strains B and H were also subject to controlled pH fermentations for product and flux analysis. The final butanol concentrations in Strain B were 32-fold higher than those of the wild-type. The strain H also had high levels of butanol. These increases were even greater in strains with solR activation and overexpression of the plasmid encoded as aad. The strain H(pTAAD) produced 255 mM butanol, 162 mg of acetone and 43 mM ethanol. These are the highest levels of solvents ever recorded in a Clostridial fermentation at 30 g/L. This strain also exceeded the level ca 180mM butanol, which was considered the highest level of toxity for this solvent.
“The methods, data, and results of examples 3-10 show a variety of methodologies according to this invention. The groESL operon is representative of genetic materials, and plasmids, and can be used in recombinant mikroogranisms that can be applied to a variety of solvent production, bioremediation, or biocatalytic system.”
“Example 3a”
“GroESL overexpressing Strain”
“A recombinant C. Acetobutylicum strain was created that overexpresses the groESL operon gene (groES) and groEL under the control of the Clostridial Thiolase Promoter. PCR was used to amplify the groESL operon genes from the chromosome with primers that were designed to exclude the operons regulatory element and natural promoter. CIRCE (Controlling inverted repeat of chaperone expression) is the regulatory element that controls expression of groESL via interaction with a potential regulatory protein, orfA. OrfA, which is also the major chaperone familiy member, is the first gene of the dnaKJ operaon. To allow expression of the groES, groEL and groES genes on the plasmid under control of the orfA protein derived from the chromosomal orfA gene, the CIRCE element was removed. The groESL operon gene was inserted into C. acetobutylicum/E. coli shuttle vector pSOS95del. The shuttle vector was made by removing the clostridial Acetone operon (ace), genes from pSOS95 (unpublished findings). The thiolase promoter was left for expression of cloned gene clones. The resulting groESL-overexpression plasmid (pGROE1) was transformed into C. Acetobutylicum AtCC 824 and used for subsequent characterization experiments.
“Example 3b”
“Description of E. Coli Transformation used in Construction of GroESL & DnaKJ Plasmids.”
“Example 4”
“Fermentations with 824 (pGROE1) or 824 (pSOS95del).”
“Construction Shuttle Vector pSOS95del. Plasmid pSOS95 (available from S. B. Tummala & E. T. Papoutsakis in 1999. Clostridium Acetobutylicum ATCC824: Development and Characterization of an Ene Expression Reporter System Appl. Environ. Microbiol. 65:3793-3799 was digested using EheI and purified with the GFX DNA Purification Column. It was then digested again with BamHI. The large fragment (5.0 kb), was isolated using GFX Gel Band Purification columns and blunt-ended with Klenow fragment. Re-ligation of this large fragment led to the formation of the pSOS95del vector.
“A series duplicate fermentations were performed with the 824 (pGROE1) and 824 (pSOS95del), control strains, and wild type 824 to study the effects of groES/groEL overexpression on solvent production, metabolic fluxes, transcriptional and protein expression patterns, as well as on the formation of solvents. The effects of the hostplasmid effect (wild-type versus control) were also examined. The fermenters were kept in anaerobic conditions with nitrogen, and the pH of the fermenters was low at 5.0. Ammonium hydroxide was used to maintain the pH. To prevent total glucose depletion, glucose was given once during fermentation. Supernatants were collected to analyze product formation by HPLC. Cell pellets were then harvested for Western Blot analysis. RNA samples were also taken for microarray and RTPCR analysis.
“Example 5”
“Product Formation and Metabolic Fluxes.”
“The presence and activity of the pGROE1 virus had dramatic effects on the formation of solvent/products, especially in the formation acetone, butanol and acetone, when compared with both wild type and control strains” (Table 2, FIG. 2). The 824(pGROE1) strain produced 231 mM (and 107 mM) of acetone, butanol and butanol respectively. This compares to the wild type’s wild type and wild strain’s wild-type (96 mM, 175 mM, and 96 mg, respectively). This is a 66% and 56% increase in final acetone titers, respectively, compared to the 824 (pSOS95del), control strain. It is interesting to observe that solvent production begins later than expected and occurs in two distinct phases for each recombinant strain. This could be due to the frequent observation of host-plasmid interaction. The final ethanol titers of the 824 (pSOS95del), and 824 (pGROE1) strains were slightly lower than those of the wild-type 824 strain (28 mg). There was no statistical difference in acetate levels between strains. Butyrate levels were slightly lower than the wild type (80 mg) in the two recombinant strains (73mM and 70mM, respectively). Although the 824(pGROE1) strain had higher optical densities, it was still lower than the 824 (pSOS95del), control strain. Both recombinant strains grew at a similar rate (2.01 hours and 1.99 hours), with the wild type exhibiting slower exponential growth (1.24 hours).
“Example 6”
“An analysis of in vivo fluxes specific to a variety of key metabolic reactions gives an excellent portrait of carbon flows among different strains while also accounting for cell densities. CompFlux, a metabolic flux analysis program based on Desai and colleagues, 1999, was used to calculate the in vivo metabolic fluxes for wild type and recombinant species. FIG. FIG. 3 displays time course profiles of nine key fluxes. There are significant differences in the specific in-vivo fluxes between the wild type 824 and 824 (pSOS95del) strains. Both recombinant and wild types exhibit distinct phases. The wild type only has one. This pattern has also been observed in other plasmid-carrying strains and may be due to a generalized effect of plasmids. The plasmid 824(pGROE1) showed a higher glucose utilization (rGLY1), and an increased acetylCoA utilization (rTHL), relative to the control strain. Strain 824(pGROE1) also had higher acetate and butyrate uptake rates, which leads to an increase in acetone formation (rACETONE). Acetone can be made from either acetate uptake or butyrate, it seems. Acetate uptake (rACUP), which is the main factor in acetone production, seems to be more important than butyrate. Both the butyrate uptakes (rBYUP), and butyrate formations (rPTBBK), are small compared to the increases of acetate uptake and formation (rPTAAK). The butanol (rBUOH), formation fluxes in Strain 824(pGROE1) were also significantly higher than those in strain 824[pSOS95del]. These differences in in vivo fluxes correspond to the higher final solvent titers observed in strain 824 (pGROE1) and further demonstrate the delay in solvent production in both of these recombinant strains.
“Example 7a”
“Microarray Analysis”
“Large-scale transcriptional analysis” has been a useful tool in understanding the differences between the genetic programming of different cell types and growth conditions. The TIGR protocol was used to print DNA microarries with more than 1000 genes. This represents approximately one-fourth of the C. Acetobutylicum genome. PCR primers are designed to amplify gene fragments of 500 bp in size so that non-specific hybridization can be minimized. The PCR products were then dried and dissolved in a water-DMSO solution. They were then spotted on Corning CMT?GAPS in triplicate. Glass microarray slides using a BioRobotics DNA arrayer. The ability to spot in three copies allows for more thorough statistical analysis of microarray data. After the slides have been hybridized with cDNA, which has been fluorescently labeled using Cy-3 and Cy-5, they are then scanned with a GSI Lumonics ScanArray. Quantarray Microarray analysis software is used to quantify spot intensities. The data are normalized, and genes with significant expression differences are identified using a novel normalization/filtering method (currently not published). The effects of chaperonin (GroESL), overexpression, as also the effects on other common stresses like heat, oxygen, or solvents are all investigated in the context of this invention.
“Example 7b”
“Gene Clustering Analysis and Self-Organizing Map (SOM), Analysis”
“Microarray analysis generates a lot of data. It is difficult to analyze the data to determine the patterns of gene expression. This is because no two genes will likely exhibit the exact same response and the behavior can be very different. There are many mathematical methods that can be used to identify patterns in gene expression. Hierarchical clustering is the most popular method for this purpose. This involves putting data points into a strict hierarchy with nested subsets. Hierarchical clustering can be useful but has its limitations. Hierarchical clustering 1) is better suited to data that is truly hierarchical, such as the evolution and speciation of species; 2) lacks robustness, uniqueness, inversion problems; 3) is deterministic. This means clustering is based on local decisions, without the ability for reevaluate it. Self-Organizing Maps (SOMs) are a better choice for clustering and analysis. SOMs are much more reliable and accurate than hierarchical clustering. They can be implemented quickly, are scalable to large data sets, and are simple to use.
“Example 8”
“SOM analysis was done on the three data sets from the experiments described above [WT824 v.824(pSOS95del); WT824 v.824(pGROE1)] using GENECLUSTER. (Tamayo et. al., 1999). Cluster analysis was limited to genes that were significantly overexpressed at the 95% confidence level at any one or more times. To determine the number of clusters, we decreased the number of clusters so that all clusters were unique and did not overlap. Samples taken from the WT824, 824 (pSOS95del), and 824 (pGROE1) fermentations are labeled A-B, B, and C. These samples were taken during the exponential growth phase at respective optical densities (A600), of 0.40, 0.88 and 1.83 (S.D.=0.06). Due to apparent instability of RNA in the stationary phase, it has been impossible to isolate intact RNA from later times points.
“Example 9”
“Transcriptional Analysis for GroESL Overexpression”
“Overexpression of GroESL operon genes seems to cause a dramatic shift to transcriptional programming when compared with the plasmid-control strain. Three gene clusters were identified by SOM analysis. Each cluster represent a set of genes which have elevated expression in the 824(pGROE1) culture at one of the three time points (B,C,D; time point A has yet to be hybridized/analyzed). The following is a list of genes that belong to each cluster. First, the 824(pGROE1) strain shows an increased expression of several sporulation genes, mainly spoIII and family genes. The transition to stationary phase in Clostridium Acetobutylicum wild type is marked by sporulation. It is also accompanied by shifts to solvent formation, the accumulation of granulose, and the expression heat shock proteins. It is not known how these processes are controlled in the same way. It is possible that the regulation of key sporulation genes may be affected by the overexpression of heat shock proteins groES or groEL. In addition to the presence a plasmid, it is possible that groES or groEL genes are overexpressed. In general, stress is thought to cause sporulation. A number of genes involved in chemotaxis (Che), are upregulated at each time point. Third, the expression of flagellar genes is enhanced at the early stages, especially at time point C (mid to late exponential). It is not common for these genes to be expressed more often than expected. Spo0A has been shown to up regulate many of the genes involved in sporulation that are expressed more frequently in the GroESL-overexpressing strain. Spo0A levels were between 1.1 and 1.4 times higher in GroESL-overexpressing strains (while these expression levels weren’t high enough to be included as part of the cluster analysis), the nine Spo0A spots on DNA-array slides had an intensity correlation (Cy3 against Cy5) value 0.93 Spo0A also has been shown to regulate flaggelar and chemotaxis genes (Fawcett, et al. 2000). Two distinct cell populations could explain the distinct phases of growth seen during a fermentation (see FIG. 1) for this and other plasmid-carrying strains (see?Transcriptional Analysis of the Host-plasmid Effect?). below). AADC (key solvent-formation enzyme), many histidine kinases, signaling pathways, glycolysis genes and electron transport genes are all genes that show significant upregulation. Finally, both the groES (key solvent formation enzyme) and the groEL (gross enzyme) were significantly upregulated at all three times points (1.8 to 14. fold higher).
“Example 10”
“Transcriptional Analysis for the Host-Plasmid Interaction E”
The plasmid control strain 824(pSOS95del) was compared to the wild-type 824 strain. This shows that the transcriptional interaction between host and plasmid is very significant. Five gene clusters were identified by SOM analysis. The SOM analysis identified five clusters. Four clusters are sets of genes that have been elevated in the 824 (pSOS95del), plasmid-control strain. The fifth cluster is a group of genes that has been down-regulated. The plasmid strain is showing many of the same trends as the wild type. Many genes involved in sporulation, flagellar and chemotaxis are expressed early and more frequently. Many heat shock proteins are also upregulated early suggesting that cells react to plasmid exposure as they do to other stressors. Several other key metabolic genes were also upregulated: the acid formation gene, butyrate kinase; solvent formation genes alcohol hydrogenase and acetoacetate dcarboxylase, (AADC); several glycolytic genes, phosphofructokinase and GAPDH; and a transcriptional regulator for sugar metabolism. Numerous transcriptional and response regulators have also been upregulated. The gene cluster that represents the downregulated genes also includes hrcA, a negative transcriptional regulator of chaperonin-expression, and several primary metabolism genes. It would be expected to see an increase in chaperonins, which hrcA negatively regulates, if hrcA is included in the downregulated gene group. This is evident with the presence of the groES, grEL and grpE genes within the upregulated gene clusters. Additional validation can be done by using a priori gene regulatory information.
While the principles of the invention were described with reference to specific embodiments, it is important to understand that these descriptions are only examples and do not limit the invention’s scope. For instance, butanol and acetone have been described as the toxic organic molecules in this invention, but the methods and constructs described herein may be used to increase microorganism/cellular resistance to other toxic molecules or compounds including hexane derivatives and aromatic compounds like toluene as would otherwise be known to those skilled in the art for various solventogenic purposes or for application in other bioprocesses associated with such endeavors as bioremediation and biocatalysis. The claims that will be filed in this section will reveal other advantages, features, and benefits. These claims are limited by the reasonable equivalents of the claims, which would be easily understood by those who are skilled in the art.
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