Combining metabolic engineering and evolutionary adaptation in Klebsiella oxytoca KMS004 to significantly improve optically pure D-(−)-lactic acid yield and specific productivity in low nutrient medium

In this study, K. oxytoca KMS004 (Δ adhE Δ pta-ackA ) was further reengineered by the deletion of frdABCD and pflB genes to divert carbon flux through D-(−)-lactate production. During fermentation of high glucose concentration, the resulted strain named K. oxytoca KIS004 showed poor in growth and glucose consumption due to its insufficient capacity to generate acetyl-CoA for biosynthesis. Evolutionary adaptation was thus employed with the strain to overcome impaired growth and acetate auxotroph. The evolved K. oxytoca KIS004-91T strain exhibited significantly higher glucose-utilizing rate and D-(−)-lactate production as a primary route to regenerate NAD + . D-(−)-lactate at concentration of 133 g/L (1.48 M), with yield and productivity of 0.98 g/g and 2.22 g/L/h, respectively, was obtained by the strain. To the best of our knowledge, this strain provided a relatively high specific productivity of 1.91 g/gCDW/h among those of other previous works. Cassava starch was also used to demonstrate a potential low-cost renewable substrate for D-(−)-lactate production. Production cost of D-(−)-lactate was estimated at $3.72/kg. Therefore, it is possible for the KIS004-91T strain to be an alternative biocatalyst offering a more economically competitive D-(−)-lactate production on an industrial scale. Key Points • KIS004-91T produced optically pure D-(−)-lactate up to 1.48 M in a low salts medium. • It possessed the highest specific D-(−)-lactate productivity than other reported strains. • Cassava starch as a cheap and renewable substrate was used for D-(−)-lactate production. • Costs related to media, fermentation, purification, and waste disposal were reduced.