Progress 10/02/02 to 09/30/07
Outputs
OUTPUTS: Our focus during this CRIS project period was towards developing microbial biocatalysts that ferment sugars to various chemicals and fuels in a cost-effective manner. Towards this goal we have accomplished the following. 1. Developed microbial biocatalysts that produced optically pure lactic acid at high concentrations from both pentoses and hexoses that can be derived from lignocellulosic biomass. 2. Developed microbial biocatalysts that produced succinic acid at high concentrations by fermentation of sugars. Lactic acid and succinic acid are potential feedstocks for production of various industrial chemicals and serve as replacements for petroleum derived chemicals. Both these organic acids are also used as the raw materials for production of bioplastics that can replace the plastics that are manufactured today using petroleum. 3. Construction of ethanologenic microbial biocatalysts that ferment all the sugars that are present in lignocellulosic biomass is a critical national need for production of cellulosic ethanol as transportation fuel. During this CRIS project period, we have improved on the ethanologenic E. coli for higher rate of ethanol production in mineral salts medium with high concentrations of glucose or xylose. In addition, we also developed ethanologenic E. coli that can tolerate higher concentrations of ethanol for production of higher ethanol broth to reduce the overall cost of cellulosic ethanol. 4. To further reduce the cost cellulosic ethanol we also identified a thermophilic and acid-tolerant Bacillus coagulans as a potential microbial biocatalyst for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol. The ability of this bacterium to grow at high temperature and low pH, conditions that are optimal for fungal cellulases, help reduce the amount of cellulases needed for SSF by about 70% and associated cellulase cost by about 3-4 -fold. This bacterium ferments both hexoses and pentoses to opticlly pure lactic acid. 5. Towards engineering B. coagulans as an ethanol producer, we have identified sources of bacterial pyruvate decarboxylase (PDC). The PDC enzyme is not common in bacteria as compared to fungi. Four different sources of bacterial PDC were identified, the genes cloned, the enzymes purified and biochemcially characterized. These pdc genes were evaluated for expression in B. coagulans. An alternate ethanologenic pathway that utilizes the pyruvate dehydrogenase and alcohol dehydrogenase was also developed in E. coli to engineer B. coagulans for ethanol production using native enzymes. 6. We also identified a Paenibacillus sp. as a xylan utilizer. This bacterium preferentially utilizes xylan as a C-source over glucose. The genome of this bacterium was sequenced by DOE-JGI. The genome sequence helped in identification of several xylanases, xylosidases, glucuronidases, arabinosidases and esterases, enzymes that are essential for depolymerization of xylan in this bacterium. This bacterium will be the source of these genes/enzymes for engineering other ethanologenic bacteria for conversion of xylan to ethanol. Results from these studies are published in the listed publications. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Biotech industries that are specializing in petroleum replacements both for transportation fuel and bioplastics industries. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have developed various E. coli derivatives that produce ethanol, lactic acid, succinic acid, alanine, etc. as the sole fermentation product. These bacterial biocatalysts can ferment all the sugars present in biomass to any of these products. These biocatalysts are currently licensed to industry for production of the various chemicals and ethanol starting from sugars derived from biomass.
Publications
- Hasona, A., S.W. York, L.P. Yomano, L.O. Ingram and K.T. Shanmugam. 2002. Decreasing the level of ethyl acetate in ethanologenic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase. Appl. Environ. Microbiol. 68(6):2651-2659.
- Underwood, S.A., M.L. Buszko, K.T. Shanmugam, and L.O. Ingram. 2002. Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation. Appl. Environ. Microbiol. 68:1071-1081.
- Gonzalez, R. Han Tao, K.T. Shanmugam, S. W. York, and L. O. Ingram. 2002. Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose. Biotechnol. Prog. 18:6-20.
- Preston, J.F., J.C. Hurlbert, J.D. Rice, A. Ragunathan, and F.J. St. John. 2003 Microbial Strategies for the Depolymerization of Glucuronoxylan: Leads to the Biotechnological Applications of Endoxylanases. in Application of Enzymes to Lignocellulosics, eds S.D. Mansfield and J. N. Saddler, ACS Symposium Series No. 855. Ch 12.
- Yilei, Qian, L.P. Yomano, J.F. Preston, H.C. Aldrich and L.O. Ingram. 2003. Cloning, characterization and functional expression of the Klebsiella oxytoca xyloside utilization operon (xynTB) in Escherichia coli. Appl Environ Microbiol. 69(10):5957-67.
- Maupin-Furlow, J.A., S.J. Kaczowka, C.J. Reuter, K. Zuobi-Hasona, and M.A. Gil. 2003. Archael proteasomes: potential in metabolic engineering. Metab. Engin. 5:151-163.
- Gonzalez, R., H. Tao, J. E. Purvis, S. W. York, K. T. Shanmugam and L. O. Ingram. 2003. Gene array-based identification of beneficial changes that contribute to increased ethanol tolerance in ethanologenic Escherichia coli LY01. Biotechnol. Prog. 19:612-623.
- Zhou, Shengde, K.T. Shanmugam and L.O. Ingram. 2003. Functional replacement of Escherichia coli D(-)-lactate dehydrogenase (ldhA) with L(+) -lactate dehydrogenase (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol. 60(4): 2237-2244.
- Causey, T. B., S. Zhou, K. T. Shanmugam, and L. O. Ingram. 2003. Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homo-acetate production. Proc. Natl. Acad. Sci. USA. 100:825-832.
- Zhou, S., T.B. Causey, A. Hasona, K.T. Shanmugam, and L.O. Ingram. 2003. Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69(1):399-407.
- Causey, T.B.,K.T. Shanmugam, L.P. Yomano, and L.O. Ingram. 2003. Engineering Escherichia coli for efficient conversion of glucose to pyruvate. PNAS. 101(8): 2235-2240
- Qian, Y., L.P. Yomano, J.F. Preston, H.C. Aldrich and L.O. Ingram. 2003. Cloning, characterization and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli. Appl. Environ.Microbiol. 69: 5957-67.
- Gonzalez, R., H. Tao, J. E. Purvis, S. W. York, K. T. Shanmugam and L. O. Ingram. 2003. Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli LY01. Biotechnol. Prog. 19: 612-623.
- Zhou, S., K. T. Shanmugam, and L. O. Ingram. 2003. Functional replacement of Escherichia coli D(-)-lactate dehydrogenase (ldhA) with L(+)-lactate dehydrogenase (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol. 69: 2237-2244
- Causey, T.B., S. Zhou, K.T. Shanmugam, and L.O. Ingram. 2003. Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production. Proc. Nat. Acad. Sci. 100: 825-832.
- Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam, and L. O. Ingram. 2003. Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69: 399-407.
- Hasona, Adnan, Y. Kim, F.G. Healy, L.O. Ingram, and K.T. Shanmugam. 2004. Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose. J. Bacteriol. 186(22): 7593-7600.
- Patel, M., M. Ou, L.O. Ingram, and K.T. Shanmugam. 2004. Fermentation of sugarcane bagasse hydrolysate to L(+) lactic acid by a thermotolerant acidophilic Bacillus sp. Biotechnology Letters. 26(11):865-8.
- Maupin-Furlow JA, Gil MA, Karadzic IM, Kirkland PA, Reuter CJ. 2004. Proteasomes: perspectives from the Archaea. Front Biosci. 2004 May 1; 9:1743-58.
- Maupin-Furlow JA, Kaczowka SJ, Reuter CJ, Zuobi-Hasona K, Gil MA. Archaeal proteasomes: potential in metabolic engineering. Metab Eng. 2003 Jul;5(3):151-63. Review.
- Underwood, S.A., M.L. Buszko, K.T. Shanmugam, and L.O. Ingram. 2004. Lack of protective osmolytes limits final cell density and volumetic productivity of ethanologenic Escherichia coli KO11 during xylose fermentation. Appl Environ Microbiol. 2004 May;70(5):2734-40.
- Shukla, V.B., S. Zhou, L.P. Yomano, K.T. Shanmugam, J.F. Preston, and L.O.Ingram. 2004. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters. 26: 689-693.
- Self, W.T., A. Hasona and K.T. Shanmugam. 2004. Expression and regulation of a silent operon, hyf, coding for hydrogenase 4 isoenzyme in Escherichia coli. J. Bacteriology. 186:580-58
- Jarboe, L.R., T.B. Grabar, L.P. Yomano, K.T. Shanmugam, L.O. Ingram. 2007. Development of ethanologenic bacteria. Book Chapter. Adv. Biochem Engin/Biotechnol 108:237-261.
- Peterson J.D., and L.O.Ingram. 2008. Anaerobic respiration in engineered Escherichia coli with an internal electron acceptor to produce fuel ethanol. Ann. N.Y. Acad. Sci. 1125:363-372.
- Jantama, K., M.J. Haupt, S.A. Svoronos, X. Zhang, J.C. Moore, K.T. Shanmugam, L.O. Ingram. 2008. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. Biotechnol and Bioengineering. 99(5):1140-1153.
- Shanmugan, K.T. and L.O. Ingram. 2008. Engineering biocatalysts for production of commodity chemicals. Journal of Molecular Microbiology and Biotechnology. 15:8-15.
- Miller, E.N., L.O. Ingram. 2008. Sucrose and overexpression of trehalose biosynthetic genes (otsBA) increase dessication tolerance of recombinant Escherichia coli. Biotechnol Lett. 30: 503-508.
- Bidle, K. A., P. A. Kirkland1, J. Nannen, and J. Maupin-Furlow. 2008. Proteomic analysis of Haloferax volcanii reveals up-regulation of the transcriptional activator PspA in response to salt stress. Microbiology (accepted pending modification).
- Kirkland, P. A.1, M. A. Gil1, I. M. Karadzic2, and J. A. Maupin-Furlow. 2008. Genetic and proteomic analyses of a proteasome-activating nucleotidase A mutant of the haloarchaeon Haloferax volcanii. J. Bacteriol. 190:193-205.
- Itoh, Y., J.D. Rice, C Goller, A. Pannuri, J. Taylor, J. Meisner, T.J. Beveridge, J.F. Preston, III, and T Romeo. 2008. Roles of pgaABCD genes in synthesis, modification and export of the Escherichia coli biofilm adhesion poly-beta-acectyl-d-glucosamine. J. Bacteriology. 3670-3680.
- Schmidt, L., L. Mouton, G. Nong, D. Ebert, and J. F. Preston, III. 2008. Genetic and immunological comparison of the cladoceran parasite Pasteuria ramosa with the nematode parasite Pasteuria penetrans. Appl. & Environ. Microbiol. 259-264.
- Publications that are relevant to this project. Hurlbert, J.C. and J.F. Preston. 2002. Differences in the solution structures of the parallel Beta-helical pectate lyases as determined by limited proteolysis. Biochimica et Biophysica Acta. 1599:9-20.
- Underwood, S.A.,S. Zhou, T. B. Causey, L.P. Yomano, K.T. Shanmugam, and L.O. Ingram. 2002. Genetic changes to optimize carbon partitioning between ethanol and biosynthesis ethanologenic Escherichia coli. Appl. Environ. Microbiol. 68(12):6263-6272.
- Raj, K.C., L.A. Talarico, L.O. Ingram and J.A. Maupin-Furlow. 2002. Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase (pdc): Comparison of bacterial homologoues. Appl. Environ. Microbiol. 68(6):2869-2876.
- Gutierrez, T., M.L. Buszko, L.O. Ingram, and J.F. Preston. 2002. Reduction of furfural to furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose. Appl. Biochem. Biotechnol. 98-100:327-340.
- El-Borai, F.E., L.W. Duncan, and J.F. Preston. 2005. Bionomics of a phoretic association between putative Paenibacillus sp. and the entomopathogenic nematode Steinernema diaprepesi. J Nematol. 35:18-25.
- Itoh, Y., X. Wang, B.J. Hinnebush, J.F. Preston, and T. Romeo. 2005. Depolymerization of poly-β-1,6-N-acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms. J. Bacteriol. 187:382-387.
- Purvis J.E., L.P. Yomano, and L.O. Ingram. 2005. Enhanced trehalose production improves growth of Echerichia coli under osmotic stress. Appl. Envir. Microbiol. 2005 71: 3761-3769.
- Talarico, L.A., M.A. Gil, L.O. Ingram, and J.A. Maupin-Furlow. 2005. Construction and expression of an ethanol production operon in gram-positive bacteria. Microbiology 151: 4023-4031.
- Zhou, S., L.P. Yomano, K.T. Shanmugam, and L.O. Ingram. 2005. Fermentation of 10% (w/v) sugar to D(-)-lactate by engineered Escherichia coli B. Biotechnology Letters. 27: 1891-1896.
- Wood, B.E., L.P. Yomano, S.W. York, and L.O. Ingram. 2005. Development of industrial-medium-required elimination of the 2,3 butanediol fermentation pathway to maintain ethanol yield in an ethanologenic strain if Klebsiella oxytoca. Biotech. Prog.21: 1366-1372.
- Grabar, T. B. S. Zhou, K. T. Shanmugam, L. P. Yomano, & L. O. Ingram. 2006. Methylglyoxal Bypass identified as Source of Chiral Contamination in L(+) and D(-) Lactate Fermentations by Recombinant Escherichia coli. Bioytechnology Letters 28:1527-1535.
- Zhou, S., T.B. Grabar, K.T. Shanmugam and L.O. Ingram. 2006. Betaine tripled the volumetric productivity of D(-)-lactate by Escherichia coli strain SZ132 in mineral salts medium. Biotechnology Letters. 28(9): 671-676
- Zhou, S., K. T. Shanmugam, L. P. Yomano, T. B. Grabar and L. O. Ingram. 2006. Fermentation of 12% (w/v) Glucose to 1.2 m Lactate by Escherichia coli Strain SZ194 using Mineral Salts Medium. Biotechnology Letters. 28(9): 663-670
- St. John, F. J., J.D. Rice, J. F. Preston. 2006. Paenibacillus sp. JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization. Appl. Environ. Microbiol. 72:1496-1506.
- Gutierrez, T., L.O. Ingram, J.F. Preston. 2006. Purification and characterization of a furfural reductase (FFR) form Escherichia coli stain LYO1 - An enzyme important in the detoxification of furfural during ethanol production. J. Of Biotechnology. 121(2): 154-164.
- Patel, M., M. Ou, R. Harbucker, H.C. Aldrich, M.L. Buszko, L.O. Ingram, and K.T. Shanmugam. 2006. Isolation and charactrerization of acid-tolerant, thermophilic bacterial biocatalysts for effective fermentation of biomass-derived sugars to lactic acid. Appl and Environ. Microbiol. 72(5): 3228-3235.
- St. John, F. J., J.D. Rice, J. F. Preston. 2006. Characterization of XynC from Bacillus subtilis subspecies subtilis strain 168 and Analysis of Its Role in Depolymerization of Glucuronoxylan. J. Bacteriol. 24:8617-8626.
- Rhee, M.S., J.-W. Kim, Y. Qian, L.O. Ingram, K.T. Shanmugam. 2007. Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans. Plasmid 58:3.
- Martinez, A., T.B. Grabar, K.T. Shanmugam, L.P. Yomano, S.W. York, and L.O. Ingram. 2007. Low salt medium for lactate and ethanol production by recombinant Esherichia coli B. Biotechnol. Letters. 29:397-404.
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Progress 10/01/05 to 09/30/06
Outputs
Metabolic Engineering of Biocatalysts for lactic acid production Osmotic stress restricts glycolytic flux, growth (rate and yield), D-lactate productivity, and D-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mM betaine doubled cell yield, increased specific productivity of D-lactate and glycolytic flux by 50%, and tripled volumetric productivity. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol D-lactate (90 g) per 100 g sugar (glucose or sucrose). Further metabolic evolution of Escherichia coli B led to strain SZ194 that produced over 1 M D-lactate from glucose (or sucrose) in 72 h using mineral salts medium supplemented with 1 mM betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Chiral purity of D-lactate was estimated to be 95%. Two new strains of Escherichia coli B were engineered for the production of
lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both L (+)- and D (-)-lactate. Strain TG113 contains only native genes and produced optically pure D (-)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only L (+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield. Thermophilic biocatalysts for SSF of cellulose Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current
industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50 degrees C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [(13)C]lactate accounted for more than 90% of the total (13)C-labeled products from [(13)C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to
reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.
Impacts
Lactic acid is an important commodity chemical that is increasingly used for biodegradable and green plastics produciton. We have developed E. coli derivatives that produce high levels of either of the two lactic acid optical isomers in pure form in mineral salts medium. We also established that a thermophilic biocatalyst, Bacillus coagulans whose growth adn fermentation characteristics match that of fungal cellulase activity optimum. this match is expected to reduce the amount and cost of cellulase required for SSF of cellulose.
Publications
- Patel, M. A., M. Ou, L. O. Ingram and K. T. Shanmugam. 2005. Simultaneous saccharification and fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp. Biotechnol. Prog. 21:1453-1460.
- Tao, H., A. Hasona, P. M. Do, L. O. Ingram and K. T. Shanmugam. 2005. Global gene expression analysis revealed an unsuspected deo operon under the control of molybdate sensor, ModE protein, in Escherichia coli. Arch. Microbiol.184:225-233.
- Zhou, S., L. P. Yomano, K. T. Shanmugam and L. O. Ingram. 2005. Fermentation of 10
- Gutierrez, T., L.O. Ingram, and J.F. Preston. 2006. Purification and characterization of a furfural reductase (FFR) from Escherichia coli strain LY01: an enzyme important in the detoxification of furfural during ethanol production. J. Biotechnol. 121: 154-164.
- St. John, F. J., J.D. Rice, J. F. Preston. 2006. Paenibacillus sp. JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization. Appl. Environ. Microbiol. 72:1496-1506.
- Zhou, S., K. T. Shanmugam, L. P. Yomano, T. B. Grabar and L. O. Ingram. 2006. Fermentation of 12
- Zhou, S., T. B. Grabar, K. T. Shanmugam and L. O. Ingram. 2006. Betaine tripled the volumetric productivity of D(-)-lactate by Escherichia coli strain SZ132 in mineral salts medium. Biotechnol. Lett. 28:671-676.
- Patel, M. A., M. S. Ou, R. Harbrucker, H. C. Aldrich, M. L. Buszko, L. O. Ingram and K. T. Shanmugam. 2006. Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic Acid. Appl. Environ. Microbiol. 72:3228-3235.
- Grabar, T. B., S. Zhou, K. T. Shanmugam, L. P. Yomano and L. O. Ingram. 2006. Methylglyoxal bypass identified as source of chiral contamination in L(+) and D(-)-lactate fermentations by recombinant Escherichia coli. Biotechnol. Lett.28:1527-1535.
- Humbard, M. A., S. Stevens Jr., and J. A. Maupin-Furlow. 2006. Post-translational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii. J. Bacteriol. 188:7521-30.
- Dinitz-Bab, E., H. Shmuely, J. Maupin-Furlow, J. Eichler and B. Shaanan. 2006. Haloferax volcanii PitA: An example of functional interaction between the Pfam chlorite dismutase and antibiotic biosynthesis monooxygenase families? Bioinformatics 22:671-675.
- Kirkland, P. A., J. Busby, S. Stevens, and J. A. Maupin-Furlow. 2006. Trizol-based method for sample preparation and isoelectric focusing of halophilic proteins. Anal. Biochem. 351: 254-259.
- Maupin-Furlow, J. A., M. A. Humbard, P. A. Kirkland, W. Li, C. J. Reuter, A. J. Wright, and G. Zhou. 2006. Proteasomes from structure to function: perspectives from archaea. Curr. Top. Dev. Biol. 75: 125-169.
- Maupin-Furlow, J. A., M. A. Gil, M. A. Humbard, P. A. Kirkland, W. Li, C. J. Reuter, and A. J. Wright. 2006. Proteasomes and other nanocompartmentalized proteases in archaea. J. M. Shively (ed.) Springer-Verlag. Complex Intracellular Structures in Prokaryotes pp. 23-46.
- De Castro, R. E., J. A. Maupin-Furlow, M. I. Gimenez, M. K. Herrera Seitz, and J. J. Sanchez. 2006. Haloarchaeal proteases and proteolytic systems. FEMS Microbiol. Rev. 30:17-35.
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Progress 10/01/02 to 10/01/03
Outputs
Current NRI funding has been focused on reducing the high cost of enzymes for cellulose depolymerization. To accomplish this, we have engineered new strains of klebsiella and E. coli that ferment al of the sugar constituents of hemicellulose and also produce some of the enzymes for cellulose depolymerization. We have successfully expressed and integrated genes into E. coli for cellobiose uptake (casA from Klebsiella), phosphocellobiose hydrolysis (casB from Klebsiella), two endoglucanases (celY and celZ from Erwinia), and about 14 additional Erwinia genes that are needed for secretion of CelZ. The two endoglucanases (celY and celZ) and the out genes from Erwinia were functionally integrated into Klebsiella (native casAB cellobiose utilization system)and into E. coli (also including Klebsiella casAB). Fermentations have been compared with hemicellulose hydrolysates, pure sugars, amorphous cellulose, in addition to simultaneous saccharification of cellulose (in
progress). Gene array technology has been developed and improved for the study of microbial metabolism and physiology in engineered strains of E. coli that produce fuel ethanol from all of the sugar constituents of lignocellulose. With the now improved precision and statistical analysis, gene array technology can be used topredict relative levels of key enzymes involved in bioconversion proceses and to identify potential sites where changes in expression levels will promote more rapid processing. Additional analysis revealed molecular adaptations related to ethanol tolerance and stress tolerance. These have led to a new fundamental discovery of the importance of protective osmolytes in fermentations and highlighted the problem of their biosynthesis under anaerobic conditions. These developments have led to substantial improvements in the fermentation of sugars to ethanol. Genetic and inexpensive physiological approaches were demonstrated that circumvent this problem and now allow
effective fermentations in medium in which the requirement for complex nutrients were reduced by 80%. Additional advances have been made related to this work which are documented in numerous publications and by training of graduate students and post-doctoral fellows.
Impacts
Continuing improvements in this USDA-funded technology have fostered the development of a biocatalyst that can efficiently convert the sugar constituents from agricultural and forestry residues into fuel ethanol. With this biocatalyst, a new industry is being built and demonstrated with the potential to significantly relieve the dependence on imported petroleum in many nations.
Publications
- Underwood, S.A., S. Zhou, T. B. Causey, L.P. Yomano, K. T. Shanmugam, and L. O. Ingram. 2002. Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli. Appl. Environ. Microbiol. 68: 6263-6272.
- Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam, and L. O. Ingram. 2003. Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69: 399-407
- Causey, T. B., S. Zhou, K. T. Shanmugam, and L. O. Ingram. 2003. Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homo-acetate production. Proc. Natl. Acad. Sci. USA. 100:825-832.
- Gonzalez, R., H. Tao, J. E. Purvis, S. W. York, K. T. Shanmugam and L. O. Ingram. 2003. Gene array-based identification of changes that contribute to increased ethanol tolerance in ethanologenic Escherichia coli : Comparison of KO11 (parent) to LY01(resistant mutant). Biotechnol. Prog. 19:612-623.
- Zhou, S., K. T. Shanmugam, and L. O. Ingram. 2003. Functional replacement of Escherichia coli D(-)-lactate dehydrogenase (ldhA) with L(+)-lactate dehydrogenase (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol. 60(4): 2237-2244.
- Yilei, Qian, L.P. Yomano, J.F. Preston, H.C. Aldrich and L.O. Ingram. 2003. Cloning, characterization and functional expression of the Klebsiella oxytoca xyloside utilization operon (xynTB) in Escherichia coli. Appl Environ Microbiol. 69(10):5957-67.
- Gonzalez, R., H. Tao, K.T. Shanmugam, S. W. York, and L. O. Ingram. 2002. Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose. Biotechnol. Prog. 18: 6-20.
- Underwood, S.A., M.L. Buszko, K.T. Shanmugam, and L.O. Ingram. 2002. Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation. Appl. Environ. Microbiol. 68: 1071-1081.
- Hasona, A., S.W. York, L.P. Yomano, L.O. Ingram and K.T. Shanmugam. 2002. Decreasing the level of ethyl acetate in ethanologenic broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase. Appl. Environ. Microbiol. 68:2651-2659
- Raj, K.C., L.A. Talarico, L.O. Ingram and J.A. Maupin-Furlow. 2002. Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase (pdc): Comparison of bacterial homologoues. Appl. Environ. Microbiol. 68:2869-2876.
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