PFK2/FBPase-2 is a potential target for metabolic engineering in the filamentous fungus Myceliophthora thermophila. PFK2/FBPase-2 is a potential target for metabolic engineering in the filamentous fungus Myceliophthora thermophila

The PFK-2/ FBPase-2 are important regulatory enzymes in the glycolytic and are present in all major eukaryotic taxa. However, unlike the well-studied animal and plant PFK-2/ FBPase-2, their functions in filamentous fungi remain little investigated. As a typical filamentous fungus and industrial microorganisms, M. thermophila was choose to investigate the functions of this gene. At the same time, the application of regulating this gene in improving metabolites production was also studied. Overall design: There are three isomers of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase in M. thermophila, named pfk2-a(Mycth_71484), pfk2-b(Mycth_2301421), pfk2-c(Mycth_2312639), respectively.To learn more about the function of PFK-2/FBPase-2 in Myceliophthora thermophila. pfk2-a/b/c, single and triple gene knockouts were performed using the CRISPR/Cas9 system in M. thermophila wild-type strain (WT), respectively.Knockout of the above three genes and named koa, kob, and koc, respectively.While simultaneous knockout three genes from wlid-type was named koabc. Mycelia for RNA isolation were harvested after 2 days and 4 days of cultivation.

PFK-2/FBPase-2是糖酵解途径中的重要调控酶，广泛存在于所有主要的真核生物类群中。然而，相较于已被深入研究的动物和植物来源的PFK-2/FBPase-2，该酶在丝状真菌中的功能仍鲜有探究。作为典型的丝状真菌与工业微生物，嗜热毁丝霉（Myceliophthora thermophila）被选为研究该基因功能的模式菌株，同时本研究还探讨了调控该基因以提升代谢产物产量的应用潜力。实验设计概述：嗜热毁丝霉中存在6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase）的三种同工型，分别命名为pfk2-a (Mycth_71484)、pfk2-b (Mycth_2301421)与pfk2-c (Mycth_2312639)。为深入解析嗜热毁丝霉中PFK-2/FBPase-2的功能，本研究借助CRISPR/Cas9基因编辑系统，分别在嗜热毁丝霉野生型（WT）菌株中构建了pfk2-a、pfk2-b、pfk2-c的单基因敲除株与三基因同时敲除株。其中，单基因敲除株分别命名为koa、kob与koc；从野生型菌株中同时敲除三个基因的菌株则命名为koabc。分别在培养2天与4天后收集菌丝体，用于RNA提取。