Part:BBa_K2570013

rocG

Phenyllactic acid（PLA）is widely found in kimchi, honey and other foods. It is a new type of natural antibacterial substance and preservative, which can inhibit a series of gram-negative, gram-positive bacteria and fungi. There are two isomers of phenyllactic acid, and D-phenyllactic acid (D-PLA) has higher antibacterial activity. In addition, D-PLA has obvious improvement in protection of the cardiovascular system and has been widely used in the food and pharmaceutical industries. In order to enhance the production of D-PLA, we expressed D-lactate dehydrogenase BBa_K2570012, phenylalanine aminotransferase BBa_K2570002 and rocG BBa_K2570013 which is used to introduce the cofactor circulatory system and optimize the transformation system conditions to express D-PLA effectively with the phenylalanine as a substrate.

Fig.1 Schematic diagram and equation of D-PLA production by introducing a self-sufficient system.

Since D - lactate dehydrogenase is a NADH - dependent oxidoreductase in the metabolic pathway, theoretically the cofactor NADH produced by E. coli cells cannot meet the redox reaction requirements of the over-expressed lactate dehydrogenase. In order to avoid the high cost of adding exogenous NADH, we designed the regeneration system of NADH that was regulated by glutamate dehydrogenase to produce additional NADH to meet demand of the catalytic reaction  of D-lactic acid dehydrogenase, resulting in the more efficient conversion of phenylpyruvic acid into D-PLA.

Fig.2 PCR amplification for the rocG Note: M: marker; rocG: PCR product of rocG

The picture above is an electrophoresis of the product of rocG amplification. We amplified PCR by rocG and purified the product. Finally, we used agarose gel electrophoresis to verify whether the recovered product was purified.

Fig.3 PCR verification of Bacterial colony Note：M: DL5000; 1: rocG; 2: PCR products of Prb1s-Dldh-Tyrb

The above picture is the gel electrophoresis of recombinant bacteria. We linked the rocG fragment with the vector fragment pRB1s-Dldh-Tyrb by Gibson method and transferred the product into E. coli.The recombinant strain（BW/pRB1s-Dldh-Tyrb-rocG）was amplified by PCR, and the product was verified by gel electrophoresis.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1054
  
• 1000
  COMPATIBLE WITH RFC[1000]