Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China.
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Molecules. 2022 May 8;27(9):3022. doi: 10.3390/molecules27093022.
Acetate is a promising economical and sustainable carbon source for bioproduction, but it is also a known cell-growth inhibitor. In this study, adaptive laboratory evolution (ALE) with acetate as selective pressure was applied to TD1.0, a fast-growing and contamination-resistant halophilic bacterium that naturally accumulates poly(3-hydroxybutyrate) (PHB). After 71 transfers, the evolved strain, B71, was isolated, which not only showed better fitness (in terms of tolerance and utilization rate) to high concentrations of acetate but also produced a higher PHB titer compared with the parental strain TD1.0. Subsequently, overexpression of acetyl-CoA synthetase (ACS) in B71 resulted in a further increase in acetate utilization but a decrease in PHB production. Through whole-genome resequencing, it was speculated that genetic mutations (single-nucleotide variation (SNV) in , and the upstream of and insertion of ) in B71 might contribute to its improved acetate adaptability and PHB production. Finally, in a 5 L bioreactor with intermittent feeding of acetic acid, B71 was able to produce 49.79 g/L PHB and 70.01 g/L dry cell mass, which were 147.2% and 82.32% higher than those of TD1.0, respectively. These results highlight that ALE provides a reliable method to harness to metabolize acetate for the production of PHB or other high-value chemicals more efficiently.
醋酸盐是一种有前途的经济且可持续的生物生产碳源，但它也是一种已知的细胞生长抑制剂。在这项研究中，以醋酸盐为选择压力的适应性实验室进化（ALE）被应用于 TD1.0，一种快速生长且抗污染的嗜盐菌，它自然积累聚（3-羟基丁酸酯）（PHB）。经过 71 次传代，分离出的进化菌株 B71 不仅对高浓度醋酸盐表现出更好的适应性（在耐受性和利用率方面），而且与亲本菌株 TD1.0 相比，产生了更高的 PHB 效价。随后，在 B71 中过表达乙酰辅酶 A 合成酶（ACS）导致醋酸盐利用率进一步提高，但 PHB 产量下降。通过全基因组重测序，推测 B71 中的遗传突变（ 中的单核苷酸变异（SNV）和 以及 上游的插入）可能有助于其提高醋酸盐适应性和 PHB 产量。最后，在 5 L 生物反应器中进行间歇添加醋酸的实验中，B71 能够生产 49.79 g/L PHB 和 70.01 g/L 干细胞质量，分别比 TD1.0 提高了 147.2%和 82.32%。这些结果表明，ALE 提供了一种可靠的方法，可以利用 来代谢醋酸盐，更有效地生产 PHB 或其他高价值化学品。
