过表达L-赖氨酸合成途径关键基因和ε-聚赖氨酸合成酶基因对ε-聚赖氨酸合成的影响

Effect of overexpression of key genes in L-lysine biosynthesis pathway andε-poly-L-lysine synthase gene onε-poly-L-lysine production

ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是小白链霉菌(Streptomyces albulus)产生的一种具有广谱抑菌活性的次级代谢产物,作为一种新型天然食品防腐剂在食品工业领域获得了广泛应用。通过代谢工程手段提高ε-PL生产菌的产物合成能力是降低ε-PL生产成本的有效手段,但目前关于ε-PL生物合成的关键代谢节点和调控机制还不清晰,因此还缺乏有效的靶基因。该研究基于文献挖掘了5个L-赖氨酸合成途径关键基因pyc(丙酮酸羧化酶基因)、ppc(磷酸烯醇式丙酮酸羧化酶基因)、zwf(6-磷酸葡萄糖脱氢酶基因)、dapA(二氢吡啶二羧酸合成酶基因)、lysA(二氨基庚二酸脱羧酶基因)和1个ε-PL合成酶基因(pls),并借助基因过表达手段,寻找影响ε-PL生物合成的关键靶基因。研究结果发现,过表达ppc、pyc和pls能够有效促进S.albulus WG608合成ε-PL。在补料分批发酵方式下,过表达菌株OE-ppc、OE-pyc和OE-pls的ε-PL产量较出发菌株S.albulus WG608分别提高2.8%、9.9%和16.3%,单位菌体合成能力分别提高12.7%、21.8%和56.4%,葡萄糖转化率分别提升了12.3%、10.2%和24.1%。同时,还发现过表达pls结合流加L-赖氨酸是一种有效提高S.albulus发酵生产ε-PL的策略。该策略在5 L罐上补料分批发酵192 h后的ε-PL产量达到61.3 g/L。该研究结果可为后续利用代谢工程手段改造S.albulus提高ε-PL产量提供靶基因。

ε-Poly-L-lysine(ε-PL)is a secondary metabolite produced by Streptomyces albulus.It has a wide range of antibacterial activity,which makes it a higher safe new natural food preservative widely used in the food industry.However,there are some problems in the field ofε-PL production,such as difficult breeding of excellent strains.Metabolic engineering is an effective strategy to improve the yield ofε-PL and eventually enhancing its production.However,there were few studies on the key metabolic nodes and regulatory mechanisms ofε-PL biosynthesis.As a result,there is a lack of effective target genes used for metabolic engineering at present.In this study,five key genes involved in L-lysine biosynthesis pathway and theε-PL synthase gene were picked out based on literatures mining,i.e.pyruvate carboxylase gene(pyc),phosphoenolpyruvate carboxylase gene(ppc),glucose-6-phosphate dehydrogenase gene(zwf),dihydropyridine dicarboxylate synthase gene(dapA),diaminopelate decarboxylase gene(lysA)andε-PL synthase gene(pls).Overexpressing of these genes in S.albulus WG608 individually,and these parameters such asε-PL production,theε-PL production per unit cell and carbon source conversion ratios to were used to evaluate the validity of these genes.The effects of overexpressed strains with target genes were comprehensively investigated onε-PL production at different levels such as shake flask fermentation,1 L batch fermentation,and 5 L fed-batch fermentation.The results showed that overexpression of ppc,pyc and pls could effectively promoted the synthesis ofε-PL in S.albulus WG608.In the fed-batch fermentation,the yields ofε-PL in overexpressed strains OE-ppc,OE-pyc and OE-pls were increased by 2.8%,9.9%,and 16.3%,respectively,compared with the original strain S.albulus WG608.Meanwhile,theε-PL production per unit cell were increased by 12.7%,21.8%,and 56.4%,respectively.In addition,glucose conversion ratios were also increased by 12.5%,9.4%,and 23.0%,respectively,compared with the original strain S.albulus WG608.In order to evaluate the L-lysine utilization capacity of OE-pls,it was found that the yield ofε-PL increased by 81.9%when 4 g/L L-lysine was added at one time during batch fermentation in a 1 L fermenter.Then,the fed-batch fermentation of OE-pls strain was carried out in a 5 L fermenter for 192 h by continuous addition of L-lysine,the yield ofε-PL eventually reached 61.3 g/L.Meanwhile,the results showed that this strategy can not only improve the yield ofε-PL,but also significantly improve the conversion rate of carbon source.It was found that overexpression of pls combined with exogenous addition of L-lysine was an effective strategy to improve the production ofε-PL by S.albulus.The present research is helpful for further genetic manipulation on S.albulus to improveε-PL production.