Pfk基因过表达对乳酸乳球菌N8产nisin速率的影响

Effect of 6-phosphofructokinase gene-pfk overexpression on nisin production in Lactococcus lactis N8

摘要:

摘要:【目的】通过基因工程手段增加糖酵解途径中编码限速酶6-磷酸果糖激酶基因Pfk 在乳酸链球菌素(nisin)产生菌Lactococcus lactis N8中的表达，增快nisin的产生，从而提高单位时间内nisin的产量，缩短发酵周期。【方法】将pfk 基因及编码以cAMP为依赖的蛋白激酶催化亚基基因pkaC克隆到表达质粒pMG36e上，将共表达重组质粒转入L. lactis N8中，使Pfk-pkaC基因过量表达，得到重组菌株L．lactis N8-pMG36epfk-pkaC，并比较该重组菌株与野生菌的生长曲线、胞内6-磷酸果糖激酶活性、发酵上清液的抑菌活性及效价，并从转录水平分析两株菌nisA及pfk-pkaC的转录差异，比较野生菌与重组菌在不同葡萄糖含量下培养产nisin的变化。【结果】Pfk基因与pkaC基因的过表达对重组菌的生长速度没有明显的影响，却能提高重组菌产nisin的速度，在发酵10 h时nisin的产量比野生菌提高了20%，使得发酵周期缩短近2 h。野生菌及重组菌在不同葡萄糖含量下培养发酵上清液的nisin效价没有明显的变化。【结论】糖酵解途径中6-磷酸果糖激酶基因Pfk的过表达可以加快乳酸乳球菌N8产nisin的速率，缩短发酵周期。

Abstract:

Abstract:［Objective］ To accelerate the formation of nisin through overexpressing 6-phosphofructokinase gene pfk in nisin-producer Lactococcus lactis N8． ［Methods］The genes of pfk and pkaC encoding the catalytic subunit of cAMPdependent protein kinase were cloned into the vector pMG36e andtransformed into L．lactis N8，resulting in the recombinant strain L．lactis N8-pMG36e-pfk-pkaC．Several biochemical and physological factors，including growth profiles，activity of 6-phosphofructokinase，expression of nisA，antibacterial activity of supernatants and nisin titer，were monitored to investigate the differences between the recombinant strain and the parental strain.［Results］No significant difference was observed with respect to the growth patterns of the recombinant strain and the wild type．As expected，the biological activity of PFK in recombinant strain was increased for all examined samples．Correspondingly，the yield of nisin was increased by 20% in the recombinant strain after fermentation for 10 hours，which could be attributed to the accelerated biosynthesis of nisin．As a result，the fermentation cycle was reduced about 2 hours． Meanwhile，different concentration of glucose did not affect the formation of nisin．［Conclusion］The overexpression of pfk and pkaC genes in the nisin-producer strain can effectively accelerate nisin biosynthesis.