Engineering A High Efficient Xylose-utilization Escherichia Coli For D-lactate Production And Research On Its Fermentation Conditions

D-lactic acid and its derivatives, precursors for the synthesis of a variety of chiral substances, are widely used in pharmaceutical, pesticide and chemical industry. Using renewable resources with lower price and extensive sources as the raw material to yield high optical purity of D-lactic acid by microbial fermentation is thought to be an effective way to achieve the large-scale production of D-lactic acid. But few microorganisms could use xylose in these materials for fermentation. Therefore, we constructed a Escherichia coli strain for the production of D-lactic acid using technique of genetic engineering.First of all, using xylose-utilizing E.coli WL204(Δfrd BC Δldh A::ldh L Δack A Δfoc A-pfl B Δadh E Δpdh R::pfl Bp6-pfl Brbs-ace EF-lpd) as the starting strain, we replaced the ldh L gene with adh A gene by RED gene replacement technology to obtained a mutant E.coli LHY010. Then LHY010 was domesticated in anaerobic tube, and the resulted well-grown strain in anaerobic condition was named E. coli LHY02.Secondly, based on the single factor experiment, Reponse Surface approach was applied to further optimize the fermentation conditions of D-lactic acid production for the recombinant strains LHY02. Herein the optimum fermentation conditions as: inoculum 2%, neutralizing agent Ca CO3 at concentration of 64 g/L, rotation speed of 190 r/min, Mg2 + at concentration of 0.08 g/L, 37℃, fermentation 72 h, resulting in production of 38.178±1.846 g/L D-lactic acid. Compared to production in the single factor experiment, the optimized D-lactic acid production was increased by 9.93%.Thirdly, the usage of xylose and mixed sugars in LHY02 were also studied. E.coli LHY02 finished D- lactic acid fermentation at 22 h and 28 h using 5% and 10% glucose as carbon resource respectively. Small amount of acetic acid was generated,and the D-lactic acid production was 48.18g/L and 85.6 g/L while the productivity were 2.19 g/L/h and 3.06 g/L/h respectively. When 10% xylose was used for fermentation, D-lactic acid production reached 84.4 g/L with productivity of 0.93 g/L/h. On contrast in mixed-sugar(5% xylose and 5% glucose) condition, xylose would not be used until glucose substantially depleted, and the ultimate D-lactic acid production was 66.12 g/L with yield of 80.51%, productivity of 0.19 g/L/h,D-lactic acid optical purity of 99.5%.Finally, the software 1stopt was applied in parameter estimation and nonlinear optimal fit for xylose fermentation kinetics model of LHY02. Model predictions fitted well with the experimental data with relative error less than 10%. It could be refered that D- lactic acid fermentation was growth-related type. Providing a theoretical basis for the simulation enlargement of experimental and optimization in fermentation process.