Production Of D-lactic Acid By Microbial Fermentation

D-Lactic acid as an important platform chemical is widely used in the food,pharmaceutical,cosmetic,and other industries.Recent demands for D-lactic acid have surged due to the increasing production of biodegradable polylactic acid(PLA),since a stereocomplex of poly(L-lactic acid)and poly(D-lactic acid)can improve the mechanical performance,thermal resistance,and hydrolysis resistance of PLA-based materials.However,the production technology for D-lactic acid is rather backward compared to that of L-lactic acid,resulting in a 3 to 5-fold higher price of D-lactic acid.This study aims to develop economical and efficient D-lactic acid producers by engineering a cyanobacterium and a thermotolerant Bacillus strain.In addition,methylomes were analyzed to provide mechanism regarding to the thermophilic adaptation of two Bacillus strains.It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO₂.However,cofactor bias of cyanobacteria is different from bacteria that prefer NADH,which hampers cyanobacterial strain engineering.In this study,the key enzyme D-lactate dehydrogenase(Ldh D)from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH.Then,the engineered enzyme was introduced into Synechococcus elongatus PCC7942to construct an efficient light-driven system that produces D-lactic acid from CO₂,the resulting recombinant strain increased D-lactate productivity by over 3.6-fold.We further demonstrated that introduction of a lactic acid transporter and bubbling CO₂-enriched air also enhanced D-lactate productivity.Using this combinational strategy,enhanced D-lactate concentration and productivity were achieved.High-temperature fermentation has multiple advantages,such as minimized the contamination risk,high mass transfer rate and the reductions in equipment requirements and energy consumption.Herein,we hunted and introduced a unique D-lactate dehydrogenase into a thermotolerant 2,3-butanediol-producing strain.A carbon flux trapping effect was observed due to a“trapping point”created by the cooperation between the introduced enzyme and the host-borne efflux pump,which enabled the irreversible transport of D-lactic acid.The overall carbon flux of the engineered strain was significantly enhanced and was dominantly(>90%)redistributed to D-lactic acid.Under optimized conditions,D-lactic acid reached an extremely high titer(226.6 g/L)that was newly recorded to date.In addition,to develop a low-cost process,corn steep powder(CSP)has been evaluated as the sole nitrogen source for D-lactic acid production.After conducting fed-batch fermentation under optimal CSP concentration,a high titer(169.2 g/L)of D-lactic acid was produced.To further reduce the cost,an economical fermentative medium was acquired with the method of response surface analysis.As a result,135.4 g/L of D-lactic acid was achieved.It is reported that thermophilic B.coagulans was also a lactic acid producing strain,however,it was rarely applied to any host cell due to its incompleted genetic manipulation.On the basis of above point and the fact that the role of DNA methylation in regulating bacterial adaptation to high-temperature environment is less understood,this study evaluated the global DNA methylome of two closely related thermotolerant B.coagulans strains using single-molecule real-time(SMRT)sequencing approach.Bioinformatics analysis on distribution of methylated gene function group and hypermethylated regions revealed that wide differences was discovered in the two strains under both high and low growth temperatures.Accordingly,we speculated that m⁴C modification might play a major role in bacterial heat adaptation which attributable to type??DNA methyltransferases(MTases).The results supported the assumption that the formed relaxed-specificity of DNA MTases in hot environments has increased their recognition sites thus led to better DNA methylation protection.Taken together,the present study shed new light on physiological functions of DNA methylation.