当前位置: X-MOL 学术Metab. Eng. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products.
Metabolic Engineering ( IF 6.8 ) Pub Date : 2020-09-08 , DOI: 10.1016/j.ymben.2020.09.003
John Hellgren 1 , Alexei Godina 2 , Jens Nielsen 3 , Verena Siewers 1
Affiliation  

Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only.



中文翻译:

混杂的磷酸酮醇酶和代谢重组使酵母中的新型非氧化糖酵解能够高产地生产乙酰辅酶 A 衍生产品。

碳节约途径具有提高生物技术过程中产品产量的潜力。该项目的目的是研究一种新的碳保存途径的功能,该途径在酵母酿酒酵母中从 6-磷酸果糖产生 3 mol 乙酰辅酶 A 而没有碳损失。这种循环途径依赖于通用磷酸酮醇酶 (Xfspk),它可以将 5-磷酸木酮糖、6-磷酸果糖和 7-磷酸景天酮糖 (S7P) 转化为乙酰磷酸。该循环旨在克服先前报道的非氧化糖酵解 (NOG) 循环的瓶颈。在这里,在硅模拟显示 S7P 在 NOG 循环中的积累,这可以通过阻断非氧化戊糖磷酸途径并引入 Xfspk 和部分核糖异生途径来解决。为了实现这一点,转酮酶和转醛缺陷的酿酒酵母中产生和循环途径中,ģ lycolysisŤ ernative ħ IGH Ç阿尔邦ý ield Ç ycle(GATHCYC),通过启用xfspk表达和景天庚酮糖二磷酸酶(SHB17)过表达. 通过 GATHCYC 的通量在体外得到证实对无细胞粗提取物进行磷酸酮醇酶测定,并在体内构建依赖于功能途径生存的菌株。最后,我们表明,与仅磷酸酮醇酶途径相比,引入 GATHCYC 作为 3-羟基丙酸 (3-HP) 生产的碳节约途径导致当葡萄糖耗尽时 3-HP 滴度增加 109%。

更新日期:2020-09-08
down
wechat
bug