Engineering Escherichia Coli To Synthesize Free Fatty Acids

Global energy demand, rising petroleum prices and environmental concerns have increased the development of replacement of fossil fuels. With the exploitation of microbial pathways based on metabolic engineering and synthetic biology frameworks, microbial synthesis of free fatty acids has a promising solution. This study is about engineering Rhodotorula glutinis and Escherichia coli JM109(DE3) to produce high-yield fatty acids by gene engineering.First, we identified Rhodotorula glutinis based as the sequence of618bp RNA, which is amplified by the ITS region primers. Then through the NCBI Blast, we found that it has an affinity with Rhodotorula dairenensis.Secondly we screened a uracil deficient strain by uracil deficient medium. And Rhodotorula glutinis is sensitive to zeocin resistance when zeocin is about100ug/mL. With the Zeocin resistance, we construct three different promoter’s expression cassette to homologous recombination whose homologous recombination arm is5.8SRNA.Then to amplification Rhodotorula glutinis’ key enzyme gene in the fatty acids pathway by designing degenerate primers. Finally gene ME was amplified in the correct region. Gene ACC and ACL were amplified partly correct. And the gene has an intron sequences.We decided that the lipid content of Rhodotorula glutinis was improved by error-prone amplification of its genomic DNA using random primers and Taq DNA polymerase. The resulting amplification products were transferred into wild-type Rhodotorula glutinis by electroporation, then according homologous recombination to create a library of mutants. And the transformants were performed with culture medium supplemented cerulenin, an inhibitor of fatty acid synthetase (FAS).The high yield strains were isolated by flow cytometric cell sorting. Multi-parameter flow cytometry coupled with the fluorescent dye Nile Red can monitor total microbial lipid content.There is a slightly increasing after a few rounds of transformation and selection。Here we report a genetic engineering approach that regulated the protein expression in the multi-gene fatty acid metabolic pathway to improve the yield of fatty acid in JM109.We combined established genetic engineering strategies including: developed a platform for efficient provision of acetyl-CoA by increasing the glycolytic pathway and acetic acid metabolism; pushed the consumption of acetyl-CoA/malonyl-CoA/ACP to fatty acid pathway by overexpression of fabD gene; and optimized fatty acid metabolic pathway for enabling further production improvement by overexpression of tesA’ and FadR gene, and knockout of fadD fad E in JM109. In this study, five distinct genotypic alterations targeted at improving fatty acids in E.coli JM109(DE3). They included:(a) knocking out the endogenous fadD and fadE to block fatty acid degradation;(b) overexpressing glycolytic pathway enzymes aceE and aceF for efficient provision of acetyl-CoA;(c) pushing acetic acid turn to acetyl-CoA by over-expressing ACS. Co-expression of glycolytic pathway enzymes and acetic acid metabolism pathway enzymes led to a supply of acetyl-CoA;(d) increasing malonyl-CoA flux towards to fatty acid pathway, we overexpress fabD, malonyl-CoA-ACP transacylase;(e) releasing feedback inhibition caused by long-chain fatty acyl-ACPs through overexpression of thioesterase gene tesA’(tesA’; leader sequence deleted);(f)as a transcription factor, overexpression of FadR is expected to slow down fatty acid degradation and enhance unsaturated fatty acid biosynthesis, thus increasing the total fatty acid yield.Finally, we construct the vector:PETDuet-tesA’-fabD-ACS-FadR and pACYC-duet-1-aceE-aceF. Then knocking out fadD and fadE in E.coli JM109.To make a genetically engineered bacteria E.coli JM109(PETDuet-tesA’-fabD-ACS-FadR, pACYC-duet-1-aceE-aceF,△fadDE)Results:1. In the inducing conditions:4mM of IPTG, cultured at30℃for6h. The expression of tesA’ fabD aceE aceF ACS FadR were confirmed by SDS-PAGE.2. To improve the cell biomass, we evaluate different medium like:LB, M9, M9(5%yeast) and then try to culture cells through two-steps. The result is that cells in LB medium are growing better than the M9medium. The data is OD600=2.02in LB medium, while the OD600=1.34in M9medium. But when we add5%yeast into M9medium, the cell biomass is higher with the OD600=3.45. So we choose the M9(5%yeast) as the fermentation medium.To further improve the biomass, we try to use two-steps to culture cells, the specific operation is seed culture is in LB medium while fermentation is in the M9medium. In the last, the engineered bacteria growing on the rise, whose maximum OD600is3.79.3. The acetic acid content of genetically engineering strain in the fermentation process was measured. As show in the result, genetically engineered bacteria JM109(△frdD+△frdE+tesA’+fabD+aceE+aceF+ACS+FadR) compared with the control bacteria with a16%reduction in LB medium, while a24%reduction in M9medium. The data indicate that the gene ACS play a role in the acetic acid metabolic pathway, and pyruvic acid metabolic by-product can be produced by forming acetyl coenzyme A which is further into the fatty acid synthesis pathway.through acetyl coenzyme A synthetase.4. The result of total free acid of engineering strain in shake-flask fermentation. In general, the yield of fatty acids in the engineering strains has an increase compared with the control. We found that the strain which was knocking out gene frdD and frdE has increased36.57%than the control. Further, when the expression of gene tesA’-frbD-aceE-aceF, the engineering strain increased by approximately128.92%. Moreover, we express the gene tesA’+fabD+aceE+aceF+after ACS+FadR, the shake-flask fermentation in M9(5%yeast) show that the total free fatty acids is275mg/L when the OD600=3.04. In addition, with the two-steps fermentation, the engineering strain has a higher yield than the control, which is472.94mg/L and the OD600=3.79.5. The data of fermentation tank:the pH was maintained at6.8by a proportional-integral derivative (PID) controller by feeding5N ammonium hydroxide. The dissolved oxygen (DO) was decreasing from100%to0in6hours. The ability of the cell to use glucose is better. Through passing up the sugar, glucose solubility has been maintained at5g/L. Ultimately through regulation,48h ferment biomass cell OD600=8.71. Relative to the fermentation production, the data in the fermentor has greatly improved, fatty acid production was2052mg/L at24h.6. The compositions of fatty acids were evaluated. Either in LB medium or M9medium, the engineering strain compared with the control:there is a difference in the distribution of fatty acids that is focused on C12:0, C14:0, and C16:1, C18:1which is the short-chain and unsaturated the fatty acids, and the relative amount of engineering strain relative to the control in all of the components to improve one or more times.