Systematic Engineering of Escherichia coli for d-Lactate Production from Crude Glycerol.

Crude glycerol resulting from biodiesel production is an abundant and renewable resource. However, the impurities in crude glycerol usually make microbial fermentation problematic. This issue was addressed by systematic engineering of Escherichia coli for the production of d-lactate from crude glycerol. First, mgsA and the synthetic pathways of undesired products were eliminated in E. coli, rendering the strain capable of homofermentative production of optically pure d-lactate. To direct carbon flux toward d-lactate, the resulting strain was endowed with an enhanced expression of glpD-glpK in the glycerol catabolism and of a heterologous gene encoding d-lactate dehydrogenase. Moreover, the strain was evolved to improve its utilization of cruder glycerol and subsequently equipped with the FocA channel to export intracellular d-lactate. Finally, the fed-batch fermentation with two-phase culturing was carried out with a bioreactor. As a result, the engineered strain enabled production of 105 g/L d-lactate (99.9% optical purity) from 121 g/L crude glycerol at 40 h. The result indicates the feasibility of our approach to engineering E. coli for the crude glycerol-based fermentation.

[1]  K. Shanmugam,et al.  Methylglyoxal Bypass Identified as Source of Chiral Contamination in l(+) and d(−)-lactate Fermentations by Recombinant Escherichia coli , 2006, Biotechnology Letters.

[2]  J. Contiero,et al.  Glycerol: a promising and abundant carbon source for industrial microbiology. , 2009, Biotechnology advances.

[3]  James M Clomburg,et al.  Escherichia coli Strains Engineered for Homofermentative Production of d-Lactic Acid from Glycerol , 2010, Applied and Environmental Microbiology.

[4]  M. Eiteman,et al.  Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains , 2006, Applied and Environmental Microbiology.

[5]  Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites , 2012 .

[6]  Y. Chao,et al.  Systematic approach to engineer Escherichia coli pathways for co-utilization of a glucose-xylose mixture. , 2013, Journal of agricultural and food chemistry.

[7]  Hofvendahl,et al.  Factors affecting the fermentative lactic acid production from renewable resources(1). , 2000, Enzyme and microbial technology.

[8]  Y. Chao,et al.  Metabolic engineering of Escherichia coli for production of butyric acid. , 2014, Journal of agricultural and food chemistry.

[9]  Fangxia Yang,et al.  Value-added uses for crude glycerol--a byproduct of biodiesel production , 2012, Biotechnology for Biofuels.

[10]  R. Gonzalez,et al.  A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. , 2008, Metabolic Engineering.

[11]  James M Clomburg,et al.  Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli , 2013, Microbial Cell Factories.

[12]  Y. Tashiro,et al.  Recent advances in lactic acid production by microbial fermentation processes. , 2013, Biotechnology advances.

[13]  R. Gonzalez,et al.  Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. , 2007, Current opinion in biotechnology.

[14]  Chaoying Yu,et al.  Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols , 2011, Applied Microbiology and Biotechnology.

[15]  P. Gruber,et al.  Polylactic Acid Technology , 2000 .

[16]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[17]  K. Shanmugam,et al.  Production of Optically Pure d-Lactic Acid in Mineral Salts Medium by Metabolically Engineered Escherichia coli W3110 , 2003, Applied and Environmental Microbiology.

[18]  Y. Chao,et al.  Replicon‐free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli , 2008, Biotechnology and bioengineering.

[19]  James M Clomburg,et al.  Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology , 2010, Applied Microbiology and Biotechnology.

[20]  K. Shimizu,et al.  The effect of pfl gene knockout on the metabolism for optically pure d-lactate production by Escherichia coli , 2004, Applied Microbiology and Biotechnology.

[21]  Wei Shen,et al.  Efficient bioconversion of crude glycerol from biodiesel to optically pure D-lactate by metabolically engineered Escherichia coli , 2014 .

[22]  James M Clomburg,et al.  Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli , 2009, Biotechnology and bioengineering.

[23]  Wei Shen,et al.  Genetically switched D-lactate production in Escherichia coli. , 2012, Metabolic engineering.

[24]  S. Andrade,et al.  The formate channel FocA exports the products of mixed-acid fermentation , 2012, Proceedings of the National Academy of Sciences.

[25]  S. Papanikolaou,et al.  Effect of impurities in biodiesel-derived waste glycerol on the performance and feasibility of biotechnological processes , 2012, Applied Microbiology and Biotechnology.

[26]  Y. Chao,et al.  Potential production platform of n-butanol in Escherichia coli. , 2015, Metabolic engineering.

[27]  K. Shanmugam,et al.  Low salt medium for lactate and ethanol production by recombinant Escherichia coli B , 2007, Biotechnology Letters.