| The（R）-citramalate is an important multifunctional organic acid that has significant application prospects not only in food processing,medical and cosmetic fields,but also as a precursor for the chemical synthesis of important polymer intermediates methacrylic acid（MAA）,which is used in industries such as optics and nanotechnology.With the increasing demand for（R）-citramalate,the synthesis and accumulation of citramalate by different ways has gradually become a hot research topic.The problems of toxic contamination,low yield,caused by plant extraction and chemical synthesis methods to produce citramalate do not meet the needs of industrial production.The whole-cell biosynthesis has become an important method for efficient accumulation of citramalate by virtue of its simple operation,intracellular enzymes’stability,high yield and reusability.However,the low tolerance of Escherichia coli to the substrates and products and the possible phage infestation have greatly limited its application.Therefore,in this study,Bacillus licheniformis was used as the chassis cell to construct the citramalate production pathway for the first time successfully.The heterologous expression of citramalate synthase was realized in the recombinant B.licheniformis and the glucose was used as the substrate to achieve the synthesis and accumulation of citramalate.Subsequently,the conditions for the whole-cell biosynthesis were optimized combined with metabolic engineering.The research contents in this study are as follows:（1）B.licheniformis was used as the chassis cell to construct the（R）-citramalate production pathway for the first time successfully.Through heterologous expression of citramalate synthase from different sources and preliminary optimization of fermentation conditions and strains of bacteria,the most favorable enzyme for synthesis of citramalate was identified as citramalate synthase Cim A3.7,which is from Methanococcus jannaschii.And the most optimal strain is B.licheniformis CICIM B1391.The recombinant strain BLA accumulated 1.4 g·L-1citramalate and the conversion rate of it achieved at 7.2 mg·g-1glucose.（2）The recombinant B.licheniformis BLA was used as the whole-cell biocatalyst for synthesis of（R）-citramalate.The conditions for whole-cell biosynthesis were optimized.It was found that the yield of（R）-citramalate was achieved at 8.6 g·L-1and the conversion rate of it achieved at 142.8 mg·g-1glucose after a reaction time of 120 h,by controlling the transformation condition at a p H of 7,temperature of 37℃,rotation speed of 250 rpm,substrate concentration of 100 g·L-1and OD600of 70.The accumulation of citramalate increased 6.1 fold compared to shake flask fermentation.（3）Metabolic engineering was used to regulate the synthesis of citramalate.On the one hand,the strain BLP was constructed by replacing the Pshuttle09promoter with the strong promoter P2through promoter optimization,which regulated the citramalate synthase gene.The results showed that BLP produced 3.4 g·L-1citramalate,which was about twice as productive as BLA;On the other hand,the recombinant B.licheniformis heterologously expressing Cim A3.7 was co-expressed with the 6-phosphogluconate dehydratase（ZMedd）from Zymomonas mobilis or the citrate lyase（ACL）from Chlorobium tepidum respectively.And the obtained strains BLZM,BLAC were verified by fermentation.The yield of citramalate reached 4.5 g·L-1and 3.7 g·L-1,which were 2.4 and 1.9 times higher than the orginating strain BLA respectively.These strains BLP,BLAC and BLZM were used for the whole-cell biosynthesis.After 120 h,they could produce 4.8,9.1 and 11 g·L-1citramalate and the conversion rate of them achieved at 48,91 and 110 mg·g-1glucose respectively.The production intensity of BLZM was up to 91.7 mg·L-1·h-1.（4）The optimal strain BLZM was cultured in fermenter with high density.After 95 h,4.6g·L-1citramalate was produced.The high-density bacteria obtained by fermentation were further used for experimental research on cell reuse,and it was found that the strain BLZM still had the catalytic activity to synthesize citramalate after being reused for four times,the accumulation were 4.5,2.8,2.3 and 1.5 g·L-1respectively.And the production intensity of BLZM was up to 115.3 mg·L-1·h-1. |
