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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

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Abstract

In this study, K. oxytoca KMS004 (ΔadhE Δpta-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.

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Acknowledgments

This research was financially supported under One Research One Graduation (OROG) scholarship, Suranaree University of Technology, Thailand. Many thanks are also expressed to staff of the Office of International Relations at Ubon Ratchathani University and Dr. Bob Tremayne for their assistance with English.

Funding

This study was funded by Suranaree University of Technology, Thailand under One Research One Graduation (OROG-2559) scholarship.

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KJ conceived and designed research. SI, NW, and CP conducted experiments. PK contributed new reagents or analytical tools. KJ and SSJ analyzed data. SI and KJ wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Kaemwich Jantama.

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In, S., Khunnonkwao, P., Wong, N. et al. 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. Appl Microbiol Biotechnol 104, 9565–9579 (2020). https://doi.org/10.1007/s00253-020-10933-0

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  • DOI: https://doi.org/10.1007/s00253-020-10933-0

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