使用糖化梭菌的代谢工程菌株生产丙酸盐,Applied Microbiology and Biotechnology

当前位置： X-MOL 学术 › Appl. Microbiol. Biotechnol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

使用糖化梭菌的代谢工程菌株生产丙酸盐
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2022-10-25 , DOI: 10.1007/s00253-022-12210-8
Tina Baur ₁ , Alexander Wentzel ₂ , Peter Dürre ₁

Affiliation

摘要

羧酸丙酸酯是一种有价值的平台化学品，在各个领域都有应用。这种酸的生物生产已经引起了极大的兴趣，因为它可以被认为是石化合成的可持续替代品。在这项工作中，Clostridium saccharoperbutylacetonicum被代谢改造为通过丙烯酸酯途径产生丙酸盐。总的来说，已建立的合成途径包含八个基因，这些基因编码催化丙酮酸转化为丙酸的酶。这些包括来自新丙酸厌氧菌的丙酸辅酶 A 转移酶、乳酰辅酶 A 脱水酶和丙烯酰辅酶 A 还原酶，以及来自肠膜明串珠菌亚种的D-乳酸脱氢酶。肠系膜。由于难以在标准启动子的控制下将所有基因组装到一个质粒上，将来自艰难梭菌的 P _tcdB - tcdR启动子系统整合到携带丙烯酸酯途径基因的双质粒系统中。使用荧光激活和吸收转移标签 (FAST) 作为荧光报告基因，分析了几种启动子在C. saccharoperbutylacetonicum中的活性，以确定驱动tcdR表达的合适候选者。选择乳糖诱导型 P _bgaL启动子后，工程化的C. saccharoperbutylacetonicum菌株在诱导基因表达后产生 0.7 mM 丙酸。低生产力被怀疑是导致细胞中丙烯酰辅酶 A 积累的代谢失衡的结果。为了平衡所提出的不平衡，丙酸合成操纵子被重新排列，从而使丙酸浓度增加了近四倍。这项研究是第一个报告使用梭菌宿主菌株生产重组丙酸盐的研究，该菌株为生物基丙酸盐开辟了一条新途径，并在后续工作中进一步改进。

关键点

• 使用FAST 测定C. saccharoperbutylacetonicum 中的启动子活性。

• 在C. saccharoperbutylacetonicum 中实施丙酸盐生产。

• 丙酸产量增加375% 至3 mM 的浓度。

"点击查看英文标题和摘要"

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum

Abstract

The carboxylic acid propionate is a valuable platform chemical with applications in various fields. The biological production of this acid has become of great interest as it can be considered a sustainable alternative to petrochemical synthesis. In this work, Clostridium saccharoperbutylacetonicum was metabolically engineered to produce propionate via the acrylate pathway. In total, the established synthetic pathway comprised eight genes encoding the enzymes catalyzing the conversion of pyruvate to propionate. These included the propionate CoA-transferase, the lactoyl-CoA dehydratase, and the acryloyl-CoA reductase from Anaerotignum neopropionicum as well as a D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. mesenteroides. Due to difficulties in assembling all genes on one plasmid under the control of standard promoters, the P_tcdB-tcdR promoter system from Clostridium difficile was integrated into a two-plasmid system carrying the acrylate pathway genes. Several promoters were analyzed for their activity in C. saccharoperbutylacetonicum using the fluorescence-activating and absorption-shifting tag (FAST) as a fluorescent reporter to identify suitable candidates to drive tcdR expression. After selecting the lactose-inducible P_bgaL promoter, engineered C. saccharoperbutylacetonicum strains produced 0.7 mM propionate upon induction of gene expression. The low productivity was suspected to be a consequence of a metabolic imbalance leading to acryloyl-CoA accumulation in the cells. To even out the proposed imbalance, the propionate-synthesis operons were rearranged, thereby increasing the propionate concentration by almost four-fold. This study is the first one to report recombinant propionate production using a clostridial host strain that has opened a new path towards bio-based propionate to be improved further in subsequent work.