Table2_Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.docx

Introduction:Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology.Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2’-O-fucosyllactose producing strain under industrially relevant conditions.Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode.Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown as a very effective strategy. Interestingly, overexpression of gltA was the most efficient way to reduce acetate accumulation in non-limited cultures, whereas disruption of the poxB and pta genes was more effective for carbon-limited cultures exposed to a sudden glucose shock. Strains from both strategies showed increased tolerance towards a glucose pulse during carbon-limited growth indicating feasible ways to engineer industrial E. coli strains with enhanced robustness.

引言：大肠杆菌，作为一种在生物技术领域中被广泛研究的典型工作马，已被广泛应用于生产多种重组蛋白质和代谢产物。然而，其易发生溢流代谢的倾向成为其主要缺点。该现象于有氧条件下，过量糖分诱导下主要生成乙酸，这种有害的副产物会降低碳利用效率，增加细胞维护成本，并最终抑制细胞生长。尽管通过控制糖碳源供应以限制其可用性可以预防这一现象，但在商业规模的生物反应器中，碳限制的细胞仍可能诱导其发生。尽管已经对这一现象的潜在机制进行了广泛的研究，但这些研究主要基于非限制性培养。相比之下，工业生产通常采用碳限制过程，这导致细胞生理学产生显著差异。目标：本研究的目标是评估和比较不同的代谢工程策略的效率，旨在降低溢流代谢，并提高在工业相关条件下2’-O-岩藻糖乳糖产生菌株的鲁棒性。方法：比较了三种不同的代谢工程策略：i) 改变通向和来自乙酸的途径，ii) 增加三羧酸（TCA）循环的流量，以及iii) 降低葡萄糖摄取速率。在非限制性批次条件、碳限制的连续培养条件以及在连续培养模式下葡萄糖脉冲后，对工程菌株的生长、乙酸形成和产物产量进行了评估。结果与讨论：研究发现，通过删除pta和poxB基因或通过过表达gltA和删除iclR基因增加碳通量进入TCA循环来阻断主要的乙酸生产途径是降低乙酸积累的有效方法。令人惊讶的是，尽管先前研究表明降低葡萄糖摄取速率是一种非常有效的策略，但降低葡萄糖摄取速率并没有降低乙酸形成。有趣的是，gltA的过表达是非限制性培养中降低乙酸积累的最有效方法，而破坏poxB和pta基因对受到突发葡萄糖冲击的碳限制性培养更为有效。两种策略的菌株在碳限制生长过程中对葡萄糖脉冲的耐受性均有所提高，这表明可以通过工程改造工业大肠杆菌菌株，使其具有更高的鲁棒性。