Department of Chemistry, Tallinn University of Technology, Tallinn, Estonia,
Appl Microbiol Biotechnol. 2014 Jun;98(11):5131-43. doi: 10.1007/s00253-014-5613-y. Epub 2014 Mar 15.
Elimination of acetate overflow in aerobic cultivation of Escherichia coli would improve many bioprocesses as acetate accumulation in the growth environment leads to numerous negative effects, e.g. loss of carbon, inhibition of growth, target product synthesis, etc. Despite many years of studies, the mechanism and regulation of acetate overflow are still not completely understood. Therefore, we studied the growth of E. coli K-12 BW25113 and several of its mutant strains affecting acetate-related pathways using the continuous culture method accelerostat (A-stat) at various specific glucose consumption rates with the aim of diminishing acetate overflow. Absolute quantitative exo-metabolome and proteome analyses coupled to metabolic flux analysis enabled us to demonstrate that onset of acetate overflow can be postponed and acetate excretion strongly reduced in E. coli by coordinated activation of phosphotransacetylase-acetyl-CoA synthetase (PTA-ACS) and tricarboxylic acid (TCA) cycles. Fourfold reduction of acetate excretion (2 vs. 8 % from total carbon) at fastest growth compared to wild type was achieved by deleting the genes responsible for inactivation of acetyl-CoA synthetase protein (pka) and TCA cycle regulator arcA. The Δpka ΔarcA strain did not accumulate any other detrimental by-product besides acetate and showed identical μ max and only ~5 % lower biomass yield compared to wild type. We conclude that a fine-tuned coordination between increasing the recycling capabilities of acetate in the PTA-ACS node through a higher concentration of active acetate scavenging Acs protein and downstream metabolism throughput in the TCA cycle is necessary for diminishing overflow metabolism of acetate in E. coli and achieving higher target product production in bioprocesses.
在好氧培养大肠杆菌的过程中消除乙酸盐溢出可以改善许多生物过程，因为在生长环境中积累乙酸盐会导致许多负面影响，例如碳损失、生长抑制、目标产物合成等。尽管经过多年的研究，乙酸盐溢出的机制和调节仍不完全清楚。因此，我们使用连续培养方法 accelerostat (A-stat) 研究了大肠杆菌 K-12 BW25113 及其影响乙酸盐相关途径的几种突变株的生长，在不同的特定葡萄糖消耗率下，目的是减少乙酸盐溢出。绝对定量的外代谢组和蛋白质组分析结合代谢通量分析使我们能够证明，通过协调磷酸转乙酰酶-乙酰辅酶 A 合成酶 (PTA-ACS) 和三羧酸 (TCA) 循环的激活，可以延迟大肠杆菌中乙酸盐溢出的发生，并强烈减少乙酸盐的排泄。与野生型相比，在最快的生长速度下，通过删除负责乙酰辅酶 A 合成酶蛋白 (pka) 和 TCA 循环调节剂 arcA 失活的基因，乙酸盐排泄减少了四倍 (2%对 8%来自总碳)。除了乙酸盐外，Δpka ΔarcA 菌株没有积累任何其他有害副产物，与野生型相比，最大比生长速率仅降低了约 5%，生物量产率相同。我们得出结论，通过增加 PTA-ACS 节点中乙酸盐的回收能力，通过更高浓度的活性乙酸盐清除 Acs 蛋白，并在 TCA 循环中增加下游代谢通量，对乙酸盐溢出代谢进行精细调节，对于减少大肠杆菌中的乙酸盐溢出代谢并在生物过程中实现更高的目标产物产量是必要的。
