Usage and biology

It encodes NAD dependent malate dehydrogenase which transforms malate into pyruvate. With the overexpression of mdh, Shewanella could produce NADH more efficiently, which brings more electricity to be produced.

Characterization

This is one section for NADH production part.

Figure1. RBS-mdh

DNA Gel Electrophoretic

To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result.

Figure2. Verification of successful transformation of pYYDT-mdh

Our target gene is 1145bp, and as the marker is DS5000, we could be sure that the bright band in this picture is our target gene.

Real-Time Quantitative PCR

To demonstrate that mdh could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR.

Figure3. Relative expression level of mdh in engineered Shewanella Oneidensis MR-1.

As we can see from this figure, mdh could be overexpressed by engineered Shewanella.

Contribution From NJXDF-CHN 2022

Group: NJXDF-CHN

Author: Yang Gu

Summary: Identification of the mdh gene that supplies NADH in Escherichia coli and demulcent Y.lipolytica to enhance production

Characterization from iGEM22-NJXDF-CHN

Malate dehydrogenase (MDH) is one of the key enzymes in biological metabolism, which can catalyze the reversible conversion between malate and oxaloacetate. Based on different coenzyme specificities, MDHs are classified into NAD-dependent and NADP-independent. RBS-mdh was first registered in 2021. With the overexpression of mdh, Shewanella could produce NADH more efficiently, which brings more electricity to be produced. To verify whether this gene could be used in other microorganisms to provide sufficient NADH for enhanced production, we first introduced the part in E. coli BL21(DE3). It was found that overexpression of this gene was effective in increasing the yield of various hard-to-express proteins, including heterologous toxic protein (GDH,BBa_K2239014) and endogenous membrane proteins (Oxa1 family membrane protein insertase (YidC, BBa_K4297069), F-ATPase subunit b (Ecb,BBa_K4297070), and cytosine transporter protein (CodB, BBa_K4297071) (Fig. 2A).
Further, phenylethylaldehyde reduction is a NADH-dependent reduction reaction. Therefore, we hope to be able to offer additional cofactors to enhance the 2-PE yield to the maximum. To ensure that the gene can be expressed in Y. lipolytica, we replaced the promoter and terminator elements of the original part and constructed TEF-mdh-CYC1t (BBa_K4297066). this part was introduced into the genome of Y. lipolytica polf and tested it in shake flask fermentation with 4 g/L of L-phe. The results showed that NADH supplementation was effective in increasing the yield of 2-PE up to 1.08 g/L (Fig. 2B). These results provide effective guide for future iGEM team to improve the production by NADH supplementation.

Fig. 2. A. Overexpression of MDH enhances the yield of various recombinant proteins in E.coli. B. Overexpression of MDH enhances the 2-PE yield in Y.lipolytica.