| 其他摘要 | 3-Hydroxypropionate (3-HP) is an important platform chemical, and its biosynthesis method is mature, however，there are two major problems block industrial application in biosynthesis: 1, 3-HP toxicity suppress the growth of cells, affect cell growth, increase total production cost; 2, some 3-HP derivatives lack artificial metabolic pathways. In order to solve the two problems, the study has focus on the stress process of E.coli to 3-HP. As a result, we got a cluster genes response to 3-HP, and discovered a new acid resistant mechanism in E.coli, at the same time, we synthesized an important 3-HP derivative-acrylic acid by using metabolic engineering in E.coli.
Organic acids have toxicity repress on the growth of bacteria, in order to understand the mechanism of 3-HP resistance in E.coli, this study employed single gene mutant library (keio collection, NBRP, Japan) to monitor the influence of each mutant. when cells after adaption to 3 g/L 3-HP (pH 4), the growth curve of E.coli were tested in M9 medium, The results showed that 18 mutants become more tolerance and 20 mutants had opposite effect when cells were adapted to 3-HP. To test if same effect of above strains in the middle of the logarithmic phase, the screening strains were conducted acid shock (5 g/L 3 - HP) when cells were in the middle of the logarithmic phase( OD600nm ~ 0.6 ). The result of percentage of survival revealed, 4 mutants still show more tolerance and 7 mutants were sensitive when expose 3-HP.
The study revealed that two-component system CpxRA elements cpxA and cpxR mutant was more sensitive when expose to acids shock, separately. Meanwhile, after adaption to pH, a change in the membrane lipid composition of E.coli was observed. We analyzed the promotor region of fabA and fabB, and a similar conserved sequence of CpxR recongnition site was found. fabA and fabB were critical enzymes of unsaturated fatty acids in E.coli. In the study, we demonstrated that the two-component system CpxRA is directly activated by acid shock, and the CpxRA system could up-regulates transcription level of fabA and fabB, and the UFAs biosynthesis was up-regulated in E.coli under acidic conditions. In conclusion, a new acid resistance system was found in E.coli, and the new acid resistance will help for prevention and clinical treatment and promoter the yield of organic acid in biosynthesis.
Acrylic acid is an important target for fermentative production, as an important organic synthetic raw materials and synthetic resin monome. However, there is not artificial production pathway to produce acrylic acid in engineering strain. 3-HP and lactic acid are isomers, in theory, it maybe generate acrylic acid from 3-HP or lactic acid by dehydrogenation. But in fact, the plan proves infeasible because the C3 structure is very stable, it is hard to conduct redox reaction in the carbon chain skeleton. It’s need to ligate a CoA group at the end of C3 skeleton to activate lactic acid structure, only by this way can dehydrogenation reactions are more likely to perform. For the first time, the study designed a new pathway, and glycerol as substrate, through 3-hydroxypropylaldehyde, 3-hydroxypropionyl coenzyme A, acryloyl coenzyme A, at the end acrylate was produced by E.coli fermentation. 3-hydroxypropionyl CoA dehydrogenase is rate-limiting enzyme in the new pathway, so it is necessary to screen efficient 3-hydroxypropionyl CoA dehydrogenase. According to the reactions, 3-hydroxypropionyl CoA dehydrogenase belongs to enoyl coenzyme A hydratase superfamily. In this study, four candidates of enoyl coenzyme A hydratase were picked out via sequence alignment and literature investigation, and there name were: PhaJ1, EcH, HcaD, PcsII. In order to analyze whcih enoyl-CoA hydratase was more appropriate for the production of acrylic acid, Four candidate enzymes were introduced into new metabolic pathways respectively. The results revealed that PcsII is more suitable as 3-hydroxypropionyl CoA dehydrogenase in the pathway. Although acrylate could be detected after PcsII was introdued in the new pathway, the production of shake flask fermentation yield were quited low. In order to improve the product, chromosomal gene integration (CGI) was employed which two genes were integrated into chromosome in engineering strain, and 6.92 mg/L acrylate were detected by shake flask. After CGI was employed, the product was still low, we speculate the production were repressed by low catalytic efficiency of PcsII. Alignment of PcsII with known structure of the enoyl-CoA hydratases，two amino acid residues were found conserved in all other proteins but not in PcsII：Lys and Ala. So, site-directed mutagenesis was performed to construct PcsII single mutants, and the effect of point mutations was verified by shake flask fermentation. Strains with PcsIIR103K and PcsIID186A produced 16.1 mg/L and 20.8 mg/L acrylic acid, respectively. When the double point mutations R103K/D186A were constructed in PcsII, the titer of acrylic acid was increased to 37.7 mg/L. |
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