Difference between revisions of "Team:HUST-China/Description"

Strategy 1:

The lactate dehydrogenase gene and the lactate transporter gene are combined in one circuit to achieve lactate production and transportation. For lactate dehydrogenase gene, we chose ldhD, and ldhDc is a codon-optimized version of ldhD, ldhDnARSdR is ldhD with D176A/I177R/F178S/N180R, and ldhDARSdR is the codon-optimized version of ldhDnARSdR. These codon optimizations are aim at increasing the production of lactate. The lldP ​​protein gene is used to transport the lactate out of the cell. The lldP ​​protein has 12 transmembrane alpha-helical segments and generally lack cleaved signal sequences and the lldP protein cotransports lactate with a proton[1]. At the same time, in order to detect the expression of these two genes, we add the Flag and 6 × His sequences respectively after the two.

Strategy 2:

In response to the preference for NADPH in the metabolism of Synechocystis sp. PCC 6803, transhydrogenase gene is used to achieve the goal of producing more NADPH, and the glycerol dehydrogenase gene is used to increase the lactate production. For transhydrogenase gene, we use TH gene, and gldA gene for glycerol dehydrogenase. The lldP ​​protein gene is also used to transport the lactate out of the cell. And the same as strategy 1, the Flag and 6 × His sequences are used to detect the expression of the two genes.

After successfully constructing these gene circuits, the shuttle plasmid PCK306 is used to transform the genes into Synechocystis sp. PCC 6803, and the yellow fluorescent protein gene on the PCK306 plasmid is used to detect the transformation.

2.Rhodopseudomonas palustris

Rhodopseudomonas palustris is a kind of Purple Non-sulfur Bacteria. Under anaerobic conditions, Rhodopseudomonas palustris can utilize hydrogen, sodium thiosulfate, hydrogen sulfide as electron donors for photoautotrophic growth. Furthermore, it can also have heterotrophic growth under microaerobic to aerobic conditions [2]. In addition, Rhodopseudomonas palustris is widely used in sewage treatment, environmentally friendly, easily accessible [3]. Since it is capable of maintaining an anaerobic environment and reusing the acetate generated by Shewanella, Rhodopseudomonas palustris may be a better carbon source provider for Shewanella compared with Synechocystis sp. PCC 6803. Therefore, we decided to modify Rhodopseudomonas palustris so that it could produce lactate under the anaerobic condition and export it out of the cell. According to KEGG database [4], metabolic pathways, which are related to lactate metabolism in Rhodopseudomonas palustris, are as follows: .

To enhance the production of lactate in Rhodopseudomonas palustris, we plan to promote the conversion efficiency of pyruvate to D-lactate and malate to L-lactate. Therefore, we decide to transfer these two genes, mleS [5] and ldhA, [6] into Rhodopseudomonas palustris to generate more lactate intracelluarly. Considering the necessity of transporting lactate out of the cells, we also apply the gene lldP [7] encoding lactate permease. Since the order of these three genes in a polycistronic lead to different expression levels [8], we build a model to optimize the gene order to maximize the gene expression. Also, we use codon optimized tool Jcat [9] to optimize the codons of these genes.

mleS：malate dehydrogenase, the conversion of malic acid to L-lactate.

ldhA：fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate.

lldP：L-lactate permease, the lactate is transported out of the cell.

To validate our modeling results, we continue to build three different gene circuits which can determine the highest lactate production efficiency of our total circuit:

The amount of electricity produced by Shewanella is closely related to the bacteria’s metabolism.

①. Glycolysis: Glycolysis is the metabolic pathway that converts glucose C6H12O6 into pyruvate. As glyceraldehyde-3-phosphate turns into 1,3-Bisphosphoglycerate, NADH is generated, which would be used to transfer electron.

②. TCA cycle: TCA cycle is a series of chemical reactions happened in mitochondrion. The reactions of the cycle are carried out by eight enzymes and three of them including malate dehydrogenase could help to produce NADH.

③. Pyruvate fermentation: Pyruvate fermentation is a common metabolic pathway in bacteria. Several steps of pyruvate fermentation could also produce NADH and the related enzymes are pyruvate formate-lyase, lactate dehydrogenase and formate dehydrogenase.

Shewanella oneidensis MR-1 prefers to use lactate as its carbon source since the amount of lactate-based biomass is more than acetate-based biomass or pyruvate-based biomass. Dld and lldEFG are D- and L-lactate dehydrogenase enzymes, which is the first step of utilizing lactate. To make the use of lactate more efficiently, we overexpress four genes: dld, lldE, lldF, lldG.[10]

①. dld: dld refers to FAD-dependent D-lactate dehydrogenase which could catalyze D-lactate’s transformation into pyruvate.

②. lldEFG: They could encode a L-lactate dehydrogenase complex which could catalyze D-lactate’s transformation into pyruvate.

To ensure that the genes would be expressed efficiently, we add a promoter before lldEFG:

NADH is a significant part of extracellular electron transfer(EET) as it could carry electron. Strenghthening the regeneration of NADH would make EET more efficiently.

To achieve this goal, we overexpress these four genes: gapA2, mdh, pflB, fdh. [11]

①. gapA: It encodes glyceraldehyde-3-phosphate dehydrogenase which could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate.

②. mdh: It encodes NAD dependent malate dehydrogenase which transforms malate into pyruvate

③. pflB: It encodes pyruvate formate-lyase to transform pyruvate into Acetyl-CoA.

④. fdh: It encodes formate dehydrogenase to transform formate into CO2..

Also, to ensure that the genes would be expressed efficiently, we add an promoter before pflB and fdh: