Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 41296, Gothenburg, Sweden.
Microb Cell Fact. 2019 Feb 1;18(1):25. doi: 10.1186/s12934-019-1072-6.
Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized.
Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses.
Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
磷酸酮酶（Xfpk）是酵母中一组非天然的酶，通过减少以 CO 的形式损失的碳，可以与其他代谢酶结合表达，从而积极影响源自乙酰辅酶 A 的产物的产量。在这项研究中，我们对先前发现生长缺陷并增加乙酸产量的短双歧杆菌来源的 Xfpk 进行了特征描述。
在分批和恒化培养中，发现 Xfpk 的表达会增加葡萄糖消耗过程中的呼吸作用并降低生物质产量。通过在不同 pH 值的生物反应器中培养有无 Xfpk 的酵母，我们表明，与 Xfpk 表达相关的某些负生长效应可能部分由质子解耦来解释。在低 pH 值下，这表现为乙醇阶段生物量产量和生长速率的降低。其次，我们表明，Xfpk 表达菌株的细胞内糖磷酸盐池发生了显著变化。特别是检测到木酮糖-5-磷酸和果糖-6-磷酸的底物减少（分别为对照水平的 26%和 74%），同时检测到甘油醛-3-磷酸和赤藓糖-4-磷酸的产物增加（分别为对照水平的 208%和 542%），这清楚地验证了体内 Xfpk 的酶活性。最后，RNAseq 分析表明，Xfpk 表达增加了与乙醛酸循环、三羧酸循环和呼吸作用相关的基因的转录，而与乙醇和乙酸形成相关的基因表达减少。生理和转录变化清楚地表明，异源磷酸酮酶通量与内源性乙酰磷酸水解生成乙酸的组合增加了细胞对乙酸同化和呼吸 ATP 生成的需求，导致碳损失。
我们的研究表明，酵母中 Xfpk 的表达将其糖酵解通量的一小部分转移到乙酸的形成上，这对细胞内糖磷酸盐水平和细胞能量学有重大影响。增加的乙酸通量增加了离子平衡和呼吸吸收的 ATP 需求，导致 CO 产量增加。与 Xfpk 表达相关的大多数负生长效应可能通过直接将乙酰磷酸定向乙酰辅酶 A 以防止乙酸积累来抵消。
