Metabolic Engineering Of Klebsiella Oxytoca For The Production Of D/L-Lactic Acid And Alanine

As one of the three kinds of organic acids in the biosphere,lactic acid has been ranked as one of the most promising platform compounds by the US Department of Energy.In particular,the lactic acid optically pure monomeric polymer(poly-lactic acid,PLA)is biodegradable and environmental friendly,can replace plastics derived from petrochemical sources in daily life.In addition,lactic acid also has a wide range of applications in the food industry,pharmaceutical industry,cosmetics,agriculture and other fields.Therefore,with the increasing demand for lactic acid production,how to produce optically pure lactic acid with high productivity and low cost has become a hot topic of research.The production methods of lactic acid include chemical synthesis,enzyme conversion,and microbial fermentation.The requirements for the first two methods are relatively complex,and microbial fermentation production has several advantages such as simple source,high repeatability,small environmental pollution,and high product purity.Bio-production methods receive more attention.Alanine is a kind of non-essential amino acid and is widely usefed in medicine,food and industry.At present,the production methods of alanine include enzyme catalysis and microbial fermentation.The production of alanine by microbial fermentation has been widely used for its advantages of low cost and low pollution.All work in this study was based on the K.oxycota.To construct the efficient lactic acid producer and alanine producer and obtain high concentration of optical pure lactic acid and alanine,the genetic engineering and microbial fermentation using glucose as the carbon resource were carried out.In order to reduce the inhibition of high concentration of glucose substrate and increase the productivity of D-lactic acid,we implemented genetic engineering in K.oxycota PDL-6 in the second chapter of this thesis.Methylglyoxal synthase gene mgs was knocked out in a new D-lactic acid production strain K.oxycota PDL-6 Amgs.The analysis of the fermentation conditions was performed by a chenostat.Compared to K.oxycota PDL-6,K.oxycota PDL-6 mgs produced D-lactic acid 59.8 g/L from glucose in 30 h and the yield increased by 14.5%.The productivity was 1.99 g/(L·h)and the production productivity increased by 14.4%.The D-lactic acid conversion rate reached 96.7%.On the basis of Chapter 2,we optimized the relevant factors of microbial fermentation including the fermentation medium,rotation speed,ventilation and pH adjustment in order to improve the ability of producing D-lactic acid by K.oxycota PDL-6 Amgs in the third chapter.Four aspects of the reagents were used to optimize the fermentation conditions for K.oxycota PDL-6 ?mgs.Based on the analysis of fermentation conditions and cost situtaion,the optimum fermentation conditions were defined.Specifically,we used No.8 medium as the best fermentation medium,and performed cultivation at the best rotation speed 100 rpm with best ventilation volume of 0 vvm,as well as 6 M NaOH as the pH regulator.Through optimizing the fermentation conditions,the concentrarion of D-lactic acid reached 59.7 g/L,the production intensity reached 0.99 g/(L·h),and the conversion rate reached 99.50%.In order to increase the production intensity of L-lactic acid and alanine,we constructed the PDL-7 ?pflB and PDL-7 ?pflB?mgs based on the original L-lactic acid producing strain PDL-7 which was constructed before in our research group in the fourth chapter.And we use the gene alaD from Geobacillus stearothermophilus XL-65-6 to replace the gene ldhD in PDL-6 ?mgs and constructed the strain PDL-6?mgs?ldhD::alaD for the production of alanine.The fermentation conditions of these two L-lactic production strains and the origin strain PDL-7 were compared and analyzed by chemostat device.The performance of PDL-7 ApflB was best.Specifically,the production of L-lactic acid was 30.6 g/L,the productivity and substrate conversion rate was 1.02 g/(L-h)and 90.0%,respectively.Afterwards,we tried to optimize the fermentation conditions in the fermenter.The conditions of fermentation were same wtih Chapter 3.However,the titer of L-lactic acid obtained was very small because of the high accumulation of pyrvatic acid in fermentation broth.Then,we tested L-lactate dehydrogenase activity and the enzyme activity was low.This result indicated that the lower activity of the enzyme affected the expandability of the strain PDL-7 ?pflB severely.After that,we studied the production capacity of PDL-6 AmgsAldhD::alaD to produce alanine from glucose.Finally,a total of 64 g/L glucose was consumed,and the output of alanine was 43.92 g/L.The conversion rate of glucose to alanine was 68.625%,which realized the high conversion efficiency of glucose to alanine.