Combining metabolic engineering and evolutionary adaptation in Klebsiella oxytoca KMS004 to significantly improve optica
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
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 Sokra In 1 & Panwana Khunnonkwao 1 & Nonthaporn Wong 1 & Chutchawan Phosiran 1 & Sirima Suvarnakuta Jantama 2 & Kaemwich Jantama 1 Received: 16 March 2020 / Revised: 15 August 2020 / Accepted: 23 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. Keywords Metabolic engineering . Evolutionary adaptation . K. oxytoca . D-(−)-lactate . Cassava
Introduction * Kaemwich Jantama [email protected] 1
Metabolic Engineering Research Unit, School of Biotechnology, Suranaree University of Technology, 111 University Avenue, Suranaree, Muang, Nakhon Ratchasima 30000, Thailand
2
Department of Bio-pharmacy, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Warinchamrap, Ubon Ratchathani 34190, Thailand
Researchers have been attracted to the sustainable productions of biofuels and biochemicals from renewable biomass to reduce demands for crude oil and petroleum derivatives (Lee et al. 2017) including 2,3-butanediol, ethanol, succinic acid, acetic acid, and lactic acid. Among these, D-(−)- and L-(+)lactic acid isomers have long been of interest as biochemical products and precursors for se
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