Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis

One of the primary goals of microbial metabolic engineering is to achieve high titer, yield and productivity (TYP) of engineered strains. This TYP index requires optimized carbon flux toward desired molecule with minimal by-product formation. De novo redesign of central carbon and redox metabolism holds great promise to alleviate pathway bottleneck and improve carbon and energy utilization efficiency. The engineered strain, with the overexpression or deletion of multiple genes, typically can't meet the TYP index, due to overflow of central carbon and redox metabolism that compromise the final yield, despite a high titer or productivity might be achieved. To solve this challenge, we reprogramed the central carbon and redox metabolism of Bacillus subtilis and achieved high TYP production of N-acetylglucosamine. Specifically, a "push-pull-promote" approach efficiently reduced the overflown acetyl-CoA flux and eliminated byproduct formation. Four synthetic NAD(P)-independent metabolic routes were introduced to rewire the redox metabolism to minimize energy loss. Implementation of these genetic strategies led us to obtain a B. subtilis strain with superior TYP index. GlcNAc titer in shake flask was increased from 6.6 g L-1 to 24.5 g L-1, the yield was improved from 0.115 to 0.468 g GlcNAc g(-1) glucose, and the productivity was increased from 0.274 to 0.437 g L-1 h(-1). These titer and yield are the highest levels ever reported and, the yield reached 98% of the theoretical pathway yield (0.478 g g(-1) glucose). The synthetic redesign of carbon metabolism and redox metabolism represent a novel and general metabolic engineering strategy to improve the performance of microbial cell factories.

微生物代谢工程的主要目标之一是实现高滴度，产量和生产力（典型）工程菌株。该典型指数需要最小的副产品形成的优化碳通量朝所需的分子。中央碳和氧化还原代谢的从头重新设计具有巨大的希望，可以减轻途径瓶颈并提高碳和能源利用效率。由于中央碳和氧化还原代谢的溢出，工程菌株的过表达或删除了多个基因，通常无法满足键入指数，尽管可能会达到高滴度或生产力，但仍会损害最终产量。为了解决这一挑战，我们重新编程了枯草芽孢杆菌的中央碳和氧化还原代谢，并获得了N-乙酰葡萄糖的高典型产生。具体而言，“推pull-promote”方法有效地降低了溢出的乙酰-COA通量并消除了副产品形成。引入了四个合成NAD（P）独立的代谢途径，以重新连接氧化还原代谢，以最大程度地减少能量损失。这些遗传策略的实施使我们获得了具有出色典型指数的枯草芽孢杆菌菌株。奶昔中的GlcNAC滴度从6.6 g L-1增加到24.5 g L-1，产量从0.115升至0.468 g GlcNAC G（-1）葡萄糖，生产率从0.274提高到0.437 G L-1 H（-1）。这些滴度和产量是有史以来报告的最高水平，产量达到了理论途径产量的98％（0.478 g g（-1）葡萄糖）。碳代谢和氧化还原代谢的合成重新设计代表了一种新颖而通用的代谢工程策略，可改善微生物细胞工厂的性能。