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.

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 lycolysis甲升Ť 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%。