Vasiliy A. Portnoy1, Markus J. Herrgård2, and Bernhard Ø. Palsson1. (1) Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, (2) Synthetic Genomics, Inc., 11149 N. Torrey Pines Rd., La Jolla, CA 92037
Fermentation of glucose to D-lactic acid by an evolved E. coli mutant deficient in three terminal oxidases under aerobic growth condition is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, cbdAB) were removed from the E. coli K12 MG1655 genome resulting in the ECOM3 (E. coli Cytochrome Oxidase Mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knock-out strain by nearly 85%. Moreover, the knock-out strain was initially incapable of growing on M9 minimal media. After subjecting the ECOM3 strain to adaptive evolution on glucose M9 medium for 60 days, the growth rate equivalent to anaerobic wild type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 strains acquired different phenotypes: one strain produced lactate as a sole fermentation product while the other two strains exhibited a mixed acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed the D-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analysis was employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN (quinol monooxygenase) and sodAB (superoxide dismutase) explain the remaining oxygen uptake that was observed in evolved ECOM3 strains. Phenotypic assessment of these adaptively evolved deletion strains also demonstrated that they exhibit physiological similarities to the lactate producing bacterium Lactoccocus lactis. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose as well as undergo mixed-acid fermentation under oxic conditions.
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