Part:BBa_K2570012
D-ldh
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.
This sequence encodes for Phenylalanine aminotransferase Tyrb that is one of the two key enzymes in the anabolic pathway of phenyllactic acid biosynthesis. It can express D-PLA using phenylpyruvate produced by transamination of phenylalanine as raw material under the action of lactate dehydrogenase.
However, high level of D-lactate dehydrogenase expression can be obtained , while the expression levels of the other two enzymes transaminase and glutamate dehydrogenase are very low. The possible explanation is that the expression of D-lactate dehydrogenase significantly inhibits the expression of transaminase and glutamate dehydrogenase. In order to achieve higher D-PLA production, we must solve the balance expression of the D-lactate dehydrogenase, transaminase and glutamate dehydrogenase in the metabolic pathway. Therefore, we reduced the expression level of D-lactate dehydrogenase by introducing of the rare arginine codons in the D-lactate dehydrogenase gene, and It has been proved by experiments that the D-PLA has the highest production after 4 Arg rare codons were introduced.
PCR was performed using D-lactate dehydrogenase gene (D-ldh)
The picture above is an electrophoresis of the product of Dldh amplification. We amplified PCR by D-ldh and purified the product. Finally, we used agarose gel electrophoresis to verify whether the recovered product was purified.
We constructed a recombinant plasmid vector for pRB1s and D-ldh and transferred it into E. coli. After that, we carried out colony PCR of the engineering bacteria, and the products obtained were verified by gel electrophoresis.
We extracted plasmids from the recombinant bacterium BW / pRB1s - Dldh - Tyrb - rocG and obtained different numbers of D - lactate dehydrogenase genes(Dldh-2r、Dldh-4r) by PCR. The PCR products were recovered and purified, and the products above were verified by agarose gel electrophoresis.
The picture above is a result of the SDS-page of the Tyrb protein expressed by the engineered bacteria. We induced culture of engineered bacteria（BW/pRB1s-Dldh-Tyrb）, and then performed SDS-page to verify its protein expression.
We extracted plasmid from recombinant bacterium BW / pRB1s - Dldh - Tyrb - rocG and obtained vector fragment (pRB1s - Tyrb - rocG) by enzyme digestion. Then we connected the pRB1s - Tyrb - rocG and the Dldh gene fragment with the rare codon base through the Gibson system, then we transferred it into E.coli to and got recombinant bacteria (BW/pRB1s-2rDldh-Tyrb-rocG、BW/pRB1s-4rDldh-Tyrb-rocG ). The above engineered bacteria were amplified by PCR and validated by gel electrophoresis
The picture above is a result of the SDS-page protein expressed by the recombinant bacteria. We induced culture of engineered bacteria（BW/pRB1s-Dldh-Tyrb-rocG）(BW/pRB1s-2rDldh-Tyrb-rocG) (BW/pRB1s-4rDldh-Tyrb-rocG), and then performed SDS-page to verify its protein expression
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 773
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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