毕赤酵母(Komagataella phaffii)是一种天然的甲基营养真核微生物,能以甲醇为唯一碳源进行生长代谢,常用于生产蛋白和各种代谢产物,但其内源代谢途径的甲醇利用效率有待进一步提高。该研究过表达磷酸戊糖途径非氧化支路的转酮醇酶(transketolase, tkt)、核糖-5-磷酸-3-差向异构酶(ribulose-5-phosphate-3-epimerase, rpe)和果糖二磷酸酶(fructose-1,6-bisphosphatase, fba)的编码基因,以促进甲醇的同化代谢。结果表明,tkt-GS115生物量OD600值提升约1.7%,其最大比生长速率(μ)达到0.23 h-1;与对照菌株ΔFLDH相比,fba-ΔFLDH以最大比生长速率为0.23 h-1提前18 h进入生长阶段;rpe-ΔFDH和fba-ΔFDH逆转了其亲本菌株ΔFDH不能在甲醇基本培养基中生长的表型,其中rpe-ΔFDH展现出巨大的生长潜力,最高生物量OD600值达到6.97,而fba-ΔFDH的最大比生长速率高达0.36 h-1。各重组菌株的甲醇利用率因出发菌不同而存在差异:tkt-GS115、tkt-ΔFLDH、fba-ΔFLDH的甲醇利用效率分别提高了0.017、0.007、0.034。该研究通过加强磷酸戊糖途径非氧化支路的3个关键基因,有效提升了K.phaffii重组菌的甲醇代谢能力。
The methylotrophic yeast Komagataella phaffii is a eukaryotic microorganism capable of utilizing methanol as sole carbon source, having been widely employed for protein and metabolite production.However, its endogenous methanol utilization efficiency requires further improvement.This study overexpressed encoding genes of key enzymes in the non-oxidative branch of the pentose phosphate pathway (PPP), including transketolase (tkt), ribulose-5-phosphate-3-epimerase (rpe), and fructose-1,6-bisphosphatase (fba), to enhance methanol assimilation.Results showed that tkt-GS115 exhibited a 1.7% increase in OD600, with a maximum specific growth rate (μ) of 0.23 h-1.Compared to the control strain ΔFLDH, fba-ΔFLDH entered the growth phase 18 h earlier, achieving a similar μ of 0.23 h-1.Notably, rpe-ΔFDH and fba-ΔFDH rescued the growth defect of the parental strain ΔFDH in methanol minimal medium.Specifically, rpe-ΔFDH displayed significant growth potential, reaching a maximum OD600of 6.97, while fba-ΔFDH achieved a remarkably high μ of 0.36 h-1.Methanol utilization efficiency varied among the recombinant strains, including tkt-GS115, tkt-ΔFLDH, and fba-ΔFLDH showed improvements of 0.017, 0.007, and 0.034, respectively.This study demonstrates that reinforcing three key genes in the PPP non-oxidative branch effectively enhances methanol utilization efficiency in K.phaffii recombinant strains.
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