| N-acetylneuraminic acid(Neu5Ac)is an important functional monosaccharide.Because of its unique physiological and biochemical properties,it has attracted more and more attention,and has broad application prospects in the field of food and medicine.Market demand urgently requires an economical and efficient Neu5Ac production process.The microbial fermentation method has the advantages of sustainability,low environmental pollution,low energy consumption and emissions,etc.By using engineered microbes,biological processes could be promising in economic feasibility with high titer/yield/productivity.In this study,on the basis of the previously constructed N-acetylglucosamine producing strain E.coli GLA-0 in our laboratory,CRISPR/Cas9 gene editing technology was used to construct and optimize the synthesis pathway of Neu5Ac,so as to achieve the efficient microbial synthesis of Neu5Ac.(1)Firstly,the dephosphorylase gene yqaB was overexpressed,and the production of GlcNAc was increased by 13.92%at shake flask level.Then,the nanATEK gene was knocked out,which blocked the degradation of Neu5Ac.Subsequently,the source of N-acetylglucosamine 2-epimerase(AGE),the key enzyme of Neu5Ac synthesis pathway,was selected by comparing the catalytic efficiency of PT7-bAGE from Anabaena sp.CH1 and FT7-slr1975 from Synechocystis sp.PCC6803.According to shake flask fermentation results,PT7-bAGE from Anabaena sp.CH1 was much better.Finally,the Neu5Ac synthetase gene PT7-neuB from Neisseria meningitidis was introduced to construct the synthesis pathway of Neu5Ac,and the titer of Neu5Ac in the engineered strain(NEA-4)was 3.2 g/L.(2)The expression strength of the two key enzymes AGE and NeuB of the Neu5Ac synthesis pathway was optimized.The fermentation results showed that when the copy numbers of PT7-bAGE and PT7-neuB were 1 and 2,respectively,strain NEA-6 had the highest yield of Neu5Ac,reaching 4.24 g/L.On this basis,the UDP-N-acetylglucosamine 2-epimerase gene from Neisseria meningitidis was introduced to increase the supply of ManNAc.The shake flask results showed that the introduction of UDP-N-acetylglucosamine 2-epimerase increased the accumulation of GlcNAc and ManNAc,but the accumulation of Neu5Ac did not increase,indicating that the supply level of ManNAc was not a factor limiting the further increase in the production of Neu5 Ac of NEA-6 strain.(3)On the basis of strain NEA-6,the supply of precursor PEP was further enhanced.By eliminating the glucose-specific PTS system and introducing the glucose facilitator protein Glf transporter from Zymomonas mobilis,the accumulation of Neu5Ac reached 5.31 g/L,which was 25.23%higher than that of NEA-6,the dry cell weight(DCW)decreased by 13.34%,and the unit cell yield increased by 44.6%;the overexpression of phosphoenolphin synthase gene pps,the accumulation of Neu5Ac reached 5.89 g/L,which was 39.15%higher than that of NEA-6,and DCW decreased 31%,the unit cell yield increased by 99.8%;the pyruvate kinase Ⅰ gene pykF was knocked out and the PEP carboxykinase gene pck was overexpressed,the accumulation of Neu5Ac reached 5.24 g/L,which was 23.58%higher than that of NEA-6,and DCW decreased 26.24%,unit cell yield increased by 67.7%.(4)In order to attenuate the accumulation of acetic acid,the acetyltransferase gene patZ was knocked out with the simultaneous overexpression of acs gene encoding acetyl-CoA synthetase,which could convert acetic acid into acetyl-CoA benefiting the GlcNAc synthesis and cell growth.The shake flask fermentation results showed that △patZ::Ptrc-acs effectively reduced the accumulation of acetic acid in the fermentation broth,but did not have any positive effects on the increase of biomass and the accumulation of Neu5Ac.In this study,rational design was performed on a GlcNAc producing Eschericia coli,resulting an engineered E.coli NEA-10 that efficiently synthesizd Neu5Ac.With glucose as single carbon source,the titer of Neu5Ac in the final fermentation broth reached 5.89 g/L,and the unit cell yield reached 0.5 g/g DCW. |
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