Background Protein-based bioconversion has been demonstrated as a sustainable approach to produce higher alcohols and ammonia fertilizers. However, owing to the switchover from transcription mediated by the bacterial RNA polymerase σ70 to that mediated by alternative σ factors, the biofuel production driven by σ70-dependent promoters declines rapidly once cells enter the stationary phase or encounter stresses. To enhance biofuel production, in this study the growth phase-independent and nitrogen-responsive transcriptional machinery mediated by the σ54 is exploited to drive robust protein-to-fuel conversion. Results We demonstrated that disrupting the Escherichia coli ammonia assimilation pathways driven by glutamate dehydrogenase and glutamine synthetase could sustain the activity of σ54-mediated transcription under ammonia-accumulating conditions. In addition, two σ54-dependent promoters, argTp and glnAp2, were identified as suitable candidates for driving pathway expression. Using these promoters, biofuel production from proteins was shown to persist to the stationary phase, with the net production in the stationary phase being 1.7-fold higher than that derived from the optimal reported σ70-dependent promoter P LlacO1. Biofuel production reaching levels 1.3- to 3.4-fold higher than those of the σ70-dependent promoters was also achieved by argTp and glnAp2 under stressed conditions. Moreover, the σ54-dependent promoters realized more rapid and stable production than that of σ70-dependent promoters during fed-batch fermentation, producing up to 4.78 g L - 1 of total biofuels. Conclusions These results suggested that the nitrogen-responsive transcriptional machinery offers the potential to decouple production from growth, highlighting this system as a novel candidate to realize growth phase-independent and stress-resistant biofuel production.

中文翻译：

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由氮响应转录机制驱动的基于蛋白质的生物精炼。

背景 基于蛋白质的生物转化已被证明是一种生产高级醇和氨肥的可持续方法。然而，由于从细菌 RNA 聚合酶 σ70 介导的转录转换为由替代 σ 因子介导的转录，一旦细胞进入静止期或遇到压力，由 σ70 依赖性启动子驱动的生物燃料生产迅速下降。为了提高生物燃料的生产，在本研究中，由 σ54 介导的与生长阶段无关且对氮响应的转录机制被用来驱动强大的蛋白质到燃料的转化。结果我们证明，在氨积累条件下，破坏由谷氨酸脱氢酶和谷氨酰胺合成酶驱动的大肠杆菌氨同化途径可以维持 σ54 介导的转录活性。此外，两个 σ54 依赖性启动子 argTp 和 glnAp2 被确定为驱动通路表达的合适候选者。使用这些启动子，蛋白质的生物燃料生产被证明持续到固定相，固定相的净产量比最佳报告的 σ70 依赖性启动子 P LlacO1 的净产量高 1.7 倍。argTp 和 glnAp2 在压力条件下也实现了比 σ70 依赖性启动子高 1.3 到 3.4 倍的生物燃料产量。而且，在补料分批发酵过程中，σ54依赖性启动子比σ70依赖性启动子实现了更快和更稳定的生产，产生了高达4.78 g L - 1的总生物燃料。结论 这些结果表明，氮响应转录机制提供了将生产与生长脱钩的潜力，突出了该系统作为实现生长阶段独立和抗应激生物燃料生产的新候选者。