title: Escherichia coli strains engineered for homofermentative production of D-lactic acid from glycerol.
abstract: Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (DeltapflB) and fumarate reductase (DeltafrdA) (strain LA01) and (ii) inactivation of fumarate reductase (DeltafrdA), phosphate acetyltransferase (Deltapta), and alcohol/acetaldehyde dehydrogenase (DeltaadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Deltadld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Deltadld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main St., MS-362, Houston, TX 77005, USA.
authors.forename: Suman
authors.initials: S
authors.lastname: Mazumdar
authors.forename: James M
authors.initials: JM
authors.lastname: Clomburg
authors.forename: Ramon
authors.initials: R
authors.lastname: Gonzalez
mesh_terms: D000693:Anaerobiosis; D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods*; D005990:Glycerol / Q000378:metabolism*; D019344:Lactic Acid / Q000096:biosynthesis*; D053858:Metabolic Networks and Pathways
chemical_list: D003470:Culture Media; D029968:Escherichia coli Proteins; D019344:Lactic Acid; D005990:Glycerol
references.citation: Curr Opin Biotechnol. 2007 Jun;18(3):213-9
references.pmid: 17532205
references.citation: Appl Environ Microbiol. 2007 Jan;73(2):456-64
references.pmid: 17122396
references.citation: J Bacteriol. 1987 Feb;169(2):507-13
references.pmid: 3027031
references.citation: J Bacteriol. 2004 Aug;186(15):4921-30
references.pmid: 15262929
references.citation: Biochemistry. 1982 Apr 27;21(9):2085-91
references.pmid: 7046793
references.citation: J Bacteriol. 1994 Mar;176(6):1796-800
references.pmid: 8132480
references.citation: Appl Environ Microbiol. 2003 Apr;69(4):2237-44
references.pmid: 12676706
references.citation: Appl Environ Microbiol. 2000 May;66(5):1844-50
references.pmid: 10788348
references.citation: Enzyme Microb Technol. 2000 Feb 1;26(2-4):87-107
references.pmid: 10689064
references.citation: J Bacteriol. 1974 Sep;119(3):736-47
references.pmid: 4604283
references.citation: Appl Microbiol Biotechnol. 2007 Nov;77(2):355-66
references.pmid: 17874321
references.citation: Metab Eng. 2008 Nov;10(6):340-51
references.pmid: 18840539
references.citation: Biotechnol Bioeng. 2009 May 1;103(1):148-61
references.pmid: 19189409
references.citation: Appl Environ Microbiol. 2008 Feb;74(4):1124-35
references.pmid: 18156341
references.citation: J Bacteriol. 1993 Feb;175(3):870-8
references.pmid: 8423158
references.citation: Biotechnol Bioeng. 2006 Aug 5;94(5):821-9
references.pmid: 16715533
references.citation: EMBO J. 2005 Jan 26;24(2):283-93
references.pmid: 15616579
references.citation: Appl Microbiol Biotechnol. 2010 Jan;85(3):413-23
references.pmid: 19826806
references.citation: J Bacteriol. 1971 Dec;108(3):1224-34
references.pmid: 4945192
references.citation: Microbiology (Reading). 2001 Sep;147(Pt 9):2437-2446
references.pmid: 11535784
references.citation: Mol Syst Biol. 2006;2:2006.0008
references.pmid: 16738554
references.citation: J Biol Chem. 1968 May 25;243(10):2587-96
references.pmid: 4297266
references.citation: Biochem J. 2002 Jul 15;365(Pt 2):471-9
references.pmid: 11955283
references.citation: Appl Environ Microbiol. 1991 Dec;57(12):3541-6
references.pmid: 1785929
references.citation: Eur J Biochem. 1969 Dec;11(2):316-27
references.pmid: 4902610
references.citation: EcoSal Plus. 2008 Sep;3(1):
references.pmid: 26443736
references.citation: J Biol Chem. 1967 Mar 10;242(5):1030-5
references.pmid: 5335908
references.citation: J Chromatogr A. 2005 Apr 8;1070(1-2):89-101
references.pmid: 15861792
references.citation: Biochem J. 1972 Aug;128(5):1339-44
references.pmid: 4345358
references.citation: Eur J Biochem. 1971 Dec;24(2):376-84
references.pmid: 4945502
references.citation: Biotechnol Bioeng. 2008 Apr 1;99(5):1140-53
references.pmid: 17972330
references.citation: Appl Environ Microbiol. 1999 Apr;65(4):1384-9
references.pmid: 10103226
references.citation: FEBS Lett. 1983 Apr 18;154(2):343-6
references.pmid: 6219895
references.citation: Annu Rev Microbiol. 1976;30:535-78
references.pmid: 825019
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references.pmid: 10829079
references.citation: Metab Eng. 1999 Apr;1(2):141-52
references.pmid: 10935927
references.citation: Metab Eng. 2008 Sep;10(5):234-45
references.pmid: 18632294
references.citation: Appl Environ Microbiol. 2003 Jan;69(1):399-407
references.pmid: 12514021
references.citation: J Ind Microbiol Biotechnol. 2001 Oct;27(4):259-64
references.pmid: 11687940
references.citation: Biochemistry. 1979 Jan 23;18(2):312-6
references.pmid: 369599
references.citation: Biotechnol Lett. 2008 Dec;30(12):2097-103
references.pmid: 18773150
references.citation: Appl Biochem Biotechnol. 2006 Jan;128(1):47-86
references.pmid: 16415480
references.citation: J Bacteriol. 1996 Oct;178(20):6013-8
references.pmid: 8830700