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Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli
Author(s) -
Durnin Guyton,
Clomburg James,
Yeates Zeno,
Alvarez Pedro J.J.,
Zygourakis Kyriacos,
Campbell Paul,
Gonzalez Ramon
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22246
Subject(s) - glycerol , acetate kinase , biochemistry , formate , fermentation , mixed acid fermentation , metabolic engineering , glycerol kinase , chemistry , formate dehydrogenase , escherichia coli , biology , enzyme , bacteria , lactic acid , lactic acid fermentation , catalysis , genetics , gene
Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed ∼95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK‐GlpD/GlpABC) and fermentative (GldA‐DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate‐lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl‐CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co‐production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by‐products succinate, acetate, and lactate. Co‐production of hydrogen required the use of acidic pH while formate co‐production was facilitated by inactivation of the enzyme formate‐hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements. Biotechnol. Bioeng. 2009;103: 148–161. © 2008 Wiley Periodicals, Inc.