【摘要】 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。更多还原

【Abstract】 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 of ε-PL production, such as difficult breeding of excellent strains. Metabolic engineering is an effective strategy to improve the yield of ε-PL and eventually enhancing its production. However, there were few studies on the key metabolic nodes and regulatory mechanisms of ε-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 the ε-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)and ε-PL synthase gene(pls). Overexpressing of these genes in S. albulus WG608 individually, and these parameters such as ε-PL production, the ε-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 on ε-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 of ε-PL in S. albulus WG608. In the fed-batch fermentation, the yields of ε-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, the ε-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 of ε-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 of ε-PL eventually reached 61.3 g/L. Meanwhile, the results showed that this strategy can not only improve the yield of ε-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 of ε-PL by S. albulus. The present research is helpful for further genetic manipulation on S. albulus to improve ε-PL production.更多还原