| In this study, a new artificial biosynthetic pathway of L-malate was constructed and optimized to produce L-malate in the modle strain Escherichia coli w3110(Fig.1). Firstly, the pool of pyruvate was construed to provide precursor for L-malate synthesis, and then the NADP-ME2 from Arabidopsis thaliana was selected and engineered to directly convert pyruvate to L-malate. The genes of frd BC, fum B and fum AC were deleted to block L-malate consumption. Lastly, cofactor recycling system was used to optimize the L-malate synthesis by regulating cofactor balance and supply NADPH, which lead to great improve in the titer of L-malate.The main results were described as follows:(1) Pyruvate was designed as direct precursor of L-malate in our study, in order to supply the enough pyruvate for L-malate synthesis, multiple gene combination knockout strategy was applied into wild-type strain E coli w3110. Those metabolites were catalyzed by lactate dehydrogenase( ldh A), pyruvate oxidase(pox B) and pyruvate formate lyase(pfl B) from pyruvate. The successive deletion of gene ldh A, pox B and pfl B lead to pyruvate increase to 12.35 g·L-1, 15.16 g·L-1 and 15.3 g·L-1, the titer of acetate and formic acid was 3.48 g·L-1 and 0.56 g·L-1 in final strain E. coli F0401, was lower 26.3% and 76.1% than the corresponding val ue in E. coli w3110, and no lactate was detected. The strain E. coli F0501 with deletion of pta-ack A lead to pyruvate increase to 20.9 g·L-1, and acetate was 0.22 g·L-1, was lower 93.5% than the corresponding val ue in E. coli F0401.(2) Five kinds of malic enzyme were chosen and overexpressed, and gained the optimal malic enzyme with the highest ability to convert pyr uvate to L-malate(reverse reaction) by in vitro conversion. NADP-ME2 from A.thaliana was the only enzyme that could convert pyruvate to L-malate, and the L-malate titer was 0.044 g·L-1. For further to enhance the reverse reaction capacity of NADP-ME2, NADP-ME2 was modified by protein engineering and gained the best m utant contained C490 S, which showed a 56% reverse activity improvement reach at 0.039 U·mg-1protein and the Km of HCO3-was 10.92 m M with 48.8% decrease compared to NADP-ME2. For constructing L-malate synthesis pathway, the m utant C490 S was introduced into E. coli F0501 and gained the strain E. coli F0511, with introduction of the mutant C490 S, the titer of L-malate in E. coli F0511 was achieved at 1.46 g·L-1, which was 87.2% higher than that of E. coli F0501, and pyruvate was correspondingly decreased 12.9%. In addition, the titer of s uccinate was 3.25 g·L-1, increased 31.6% compare to that of the strain E. coli F0501.(3)In order to inhibit the conversion of L-malate to succinate, the genes frd BC(fumarate reductase), fum B and fum AC(fumarase) were sequentially knocked out in the E. coli F0511, and gain the strain E. coli F0711, E. coli F0811 and E. coli F0911. With the deletion of frd BC, fum B and fum AC lead to L-malate gradually increase to 2.54 g·L-1, 3.03 g·L-1 and 3.27 g·L-1, the titer of succinate is 1.02 g·L-1, 0.55 g·L-1 and 0.32 g·L-1, was lower 68.6%, 41.7% and 41.8% than the corresponding val ue in E. coli F0711, E. coli F0811 and E. coli F0911. Finally, the L-malate titer and production capacity of strain E. coli F0911 increased 123.9% and 110.8% compare to E. coli F0511.(4) The basic cause of the low glucose consumption rate was thought to be due to NADH could not rapidly be oxidized under low oxygen level for the gene of pfl B and ldh A defected, and high NADH/NAD+ ratio inhibited the glycolytic pathway. To alleviate this cofactor imbalance causing decreased glucose consumption rate and supply NADPH, two strategies were attempted:(i) achieving the NADH reoxidation by overexpressing ldh A gene in E. coli F0911 and gained E. coli F0921;(ii) converting NADH to NADPH by overexpressing NADH kinase(pos5) from S.cerevisiae in E. coli F0911 and gained E. coli F0931. With overexpression of pos5, the NADH/NAD+ ratio and the concentration of NADPH is 0.38 and 1.43 μmol·g-1 DCW, were lower 56.2% and higher 45.9% when compare the corresponding value in E. coli F0911.The strain E. coli F0931 could produce 9.34 g·L-1 L-malate, was higher 203.8% compare to E. coli F0911, and the concentration of cell was also increased 54.2%.(5) Optimize the fermentation condition to improve L-malate production. The cell concentration was multiply increased to evaluate the L-malate productivity at the time of 12 h, 12 g·L-1 DCW was found to be best concentration use for producing L-malate, which lead to 16.56 g·L-1 L-malate produced with 128.9% higher than the control(2 g·L-1DCW). With recovery of cell viability, the synthesis rate of L-malate was increased 28% reach to 0.25 g·L-1·h-1 in the stage of 36~72 h. As a result, the titer of L-malate was increased by 30% reach to 21.65 g·L-1. These results indicated that the cell concentration and cell viability play an important role in L-malate production. |
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