Sun Qing, Liu Dehua, Chen Zhen
Key Laboratory of Industrial Biocatalysis (Ministry of Education), Department of Chemical Engineering, Tsinghua University, Beijing, China.
Tsinghua Innovation Center in Dongguan, Dongguan, China.
Front Bioeng Biotechnol. 2023 Jan 10;10:1089639. doi: 10.3389/fbioe.2022.1089639. eCollection 2022.
Engineering for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into and reprogrammed metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1 g/L yeast extract as co-substrate. C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of , and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of based on the hybrid methanol assimilation pathway with Mdh and Das.
工程化实现高效甲醇同化对于将甲醇开发成为工业生物技术的新兴下一代原料至关重要。虽然最近将[具体生物]工程改造为合成甲基营养菌的尝试取得了巨大成功，但这些工作大多基于原核核糖单磷酸（RuMP）途径的工程改造。在本研究中，我们将由原核甲醇脱氢酶（Mdh）和真核木酮糖单磷酸（XuMP）途径酶二羟基丙酮合酶（Das）组成的混合甲醇同化途径引入[具体生物]，并通过结合理性设计和适应性实验室进化对其代谢进行重新编程，以提高甲醇同化能力。通过删除和下调三羧酸循环（TCA循环）和糖酵解中的关键基因，以增加通向循环XuMP途径的通量，甲醇消耗以及甲醇向生物量的同化显著提高。通过适应性实验室进化实现了甲醇利用和细胞生长的进一步改善，最终进化菌株可以仅以0.1 g/L酵母提取物作为共底物在甲醇上生长。碳-甲醇标记分析表明，糖酵解、TCA循环、磷酸戊糖途径和氨基酸中的细胞内代谢物标记显著更高。转录组学分析表明，[相关基因]、[相关基因]和部分磷酸戊糖途径基因的表达高度上调，表明理性工程策略和适应性进化对于激活循环XuMP途径是有效的。本研究证明了基于具有Mdh和Das的混合甲醇同化途径构建[具体生物]合成甲基营养型的可行性，并提供了新的策略。
