Part:BBa_K2570018
Production of PLA: TyrB+LDH+rocG
The D-lactic dehydrogenase used in this project was derived from Lactobacillus bulgaricus ATCC 11842 D-ldh BBa_K2570012, and the 52nd D-lactic dehydrogenase was mutated into the hydrophobic amino acid leucine by fusing PCR, resulting in improvement of the enzymatic activity of the D-lactic dehydrogenase. The phenylalanine transaminase Tyrb BBa_K2570002 was derived from Escherichia coli 21B, and it was co-expressed with D-lactic acid dehydrogenase to in the vector （pRB1s), and then the engineered host cells can produce D-PLA using phenylalanine as the substrate. To improve the NADH availablity of the engineered host cells, we introduced a glutamate dehydrogenase rocG BBa_K2570013 into the cells to enhance the D-lactate dehydrogenase-dependent NADH cofactor regeneration system for improvement of the D-PLA production. Then the improved NADH regeneration can be achieved when the co-expression of rocG, D-lactate dehydrogenase D-ldh and phenylalanine transaminase Tyrb were realized. Finally, the conditions of whole-cell transformation were optimized and the D-PLA production by the recombinant engineered E. coli was further improved.
This part solves the problem of co-expression of the rocG, D-lactate dehydrogenase D-ldh and phenylalanine transaminase Tyrb. The expression level of D-lactate dehydrogenase can be effectively reduced by introducing rare codons of arginine into the gene of D-lactate dehydrogenase. In order to verify the effects of bypass pathway of D-PLA synthesis in E. coli, we transformed the recombinant pathways into E.coli with two bypass pathways that had influences on D-PLA synthesis were knocked out. Finally, the improved D-PLA production can be obtained through optimizing the transformation conditions of recombinant E. coli (J23119-Dldh-Tyrb-rocG).
The picture above is a result of the SDS-page protein expressed by the recombinant bacteria（BW/pRB1s-Dldh-Tryb-rocG）. We induced culture of recombinant bacteria（BW/pRB1s-Dldh-Tryb-rocG）and then performed SDS-page to verify its protein expression.
The picture above is a result of time profile for D-PLA concentration under optimum condition by the recombinant bacteria（BW/pRB1s-Dldh-Tryb-rocG）. We try to Inoculated bacterial solution and cultured it, then we used HPLC to measure the content of D-PLA in the conversion solution.We can see the concentration of phenylalanine gradually decreased and the concentration of PLA gradually increased
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2054
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2839
Illegal NgoMIV site found at 3378
Illegal AgeI site found at 1115
Illegal AgeI site found at 2527 - 1000COMPATIBLE WITH RFC[1000]
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