Liang Jinghui, van Kranenburg Richard, Bolhuis Albert, Leak David J
Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom.
Centre for Sustainable and Circular Technologies (CSCT), University of Bath, Bath, United Kingdom.
Front Microbiol. 2022 Oct 20;13:985465. doi: 10.3389/fmicb.2022.985465. eCollection 2022.
is a thermophilic bacterium of interest for lignocellulosic biomass fermentation. However, carbon catabolite repression (CCR) hinders co-utilization of pentoses and hexoses in the biomass substrate. Hence, to optimize the fermentation process, it is critical to remove CCR in the fermentation strains with minimal fitness cost. In this study, we investigated whether CCR could be removed from DSM 2542 by mutating the Ser46 regulatory sites on HPr and Crh to a non-reactive alanine residue. It was found that neither the (HPr-S46A) nor the (Crh-S46A) mutation individually eliminated CCR in DSM 2542. However, it was not possible to generate a double mutant. While the Crh-S46A mutation had no obvious fitness effect in DSM 2542, the mutation had a negative impact on cell growth and sugar utilization under fermentative conditions. Under these conditions, the mutation was associated with the production of a brown pigment, believed to arise from methylglyoxal production, which is harmful to cells. Subsequently, a less directed adaptive evolution approach was employed, in which DSM 2542 was grown in a mixture of 2-deoxy-D-glucose(2-DG) and xylose. This successfully removed CCR from DSM 2542. Two selection strategies were applied to optimize the phenotypes of evolved strains. Genome sequencing identified key mutations affecting the PTS components PtsI and PtsG, the ribose operon repressor RbsR and adenine phosphoribosyltransferase APRT. Genetic complementation and bioinformatics analysis revealed that the presence of wild type and inhibited xylose uptake or utilization, while and might play a role in the regulation of CCR in DSM 2542.
是一种对木质纤维素生物质发酵有研究价值的嗜热细菌。然而，碳分解代谢物阻遏（CCR）阻碍了生物质底物中戊糖和己糖的共同利用。因此，为了优化发酵过程，以最小的适应性成本去除发酵菌株中的CCR至关重要。在本研究中，我们研究了通过将HPr和Crh上的Ser46调控位点突变为无反应性的丙氨酸残基，是否可以从DSM 2542中去除CCR。结果发现，单独的（HPr-S46A）或（Crh-S46A）突变均未消除DSM 2542中的CCR。然而，无法产生双突变体。虽然Crh-S46A突变在DSM 2542中没有明显的适应性影响，但该突变在发酵条件下对细胞生长和糖利用有负面影响。在这些条件下，该突变与一种棕色色素的产生有关，据信该色素源于甲基乙二醛的产生，对细胞有害。随后，采用了一种较少定向的适应性进化方法，其中DSM 2542在2-脱氧-D-葡萄糖（2-DG）和木糖的混合物中生长。这成功地从DSM 2542中去除了CCR。应用了两种选择策略来优化进化菌株的表型。基因组测序确定了影响磷酸转移酶系统组分PtsI和PtsG、核糖操纵子阻遏物RbsR和腺嘌呤磷酸核糖转移酶APRT的关键突变。遗传互补和生物信息学分析表明，野生型和的存在抑制了木糖的摄取或利用，而和可能在DSM 2542中CCR的调节中起作用。
