Shan Yuan,Chao Xu,Miaomiao Jin,Xinglin Jiang,Wei Liu,Mo Xian,Ping Jin
Aldehydes are ubiquitous metabolites in living cells. As reactive electrophiles, they have the capacity to form adducts with cellular protein thiols and amines, leading to potential toxicity. Dynamic regulation has proven to be an effective strategy for addressing the accumulation of toxic metabolites. However, there are limited reports on applying dynamic control specifically to mitigate aldehyde accumulation. In this study, the cinnamaldehyde accumulation in the biosynthesis of cinnamylamine was used as a model to evaluate a two-way dynamic regulation strategy. First, we utilized whole-genome transcript arrays to identify the cinnamaldehyde-responsive promoters: the upregulated promoter P 4 and the downregulated promoter P d . They were then employed as biosensors to dynamically regulate the synthesis and consumption of cinnamaldehyde, mitigating its toxic effects on the host. This strategy successfully reduced cinnamaldehyde accumulation by 50% and increased the production of cinnamylamine by 2.9 times. This study demonstrated a cinnamaldehyde-induced autoregulatory system that facilitated the conversion of cinnamic acid into cinnamylamine without the need for costly external inducers, presenting a promising and economically viable approach. The strategy also serves as a reference for alleviating the inhibitory effects of other toxic aldehydes on microorganisms. Additionally, the biosensors (P d and P 4 ) can respond to a range of aldehyde compounds, offering a rapid and sensitive method for detecting toxic aldehydes in both environmental samples and microorganisms, thus provide a valuable tool for screening strains enhanced aldehyde yield.
Background Aromatic primary amines (APAs) are key intermediates in the chemical industry with numerous applications. Efficient and mild biocatalytic synthesis is an excellent complement to traditional chemical synthesis. Our lab previously reported a whole-cell catalytic system for the synthesis of APAs catalyzed by carboxylic acid reductase from Neurospora crassa (ncCAR) and ω-transaminase from Ochrobactrum anthropi (OATA). However, the accumulation of toxic intermediates (aromatic aldehydes) during biocatalytic synthesis affected yields of APAs due to metabolic imbalance. Results In this work, the biocatalytic synthesis of APAs (taking cinnamylamine as an example) was metabolically regulated by the overexpression or knockout of five native global transcription factors (TFs), the overexpression of eight native resistance genes, and optimization of promoters. Transcriptome analysis showed that knockout of the TF arcA increased the fluxes of NADPH and ATP in E. coli , while the rate of pyruvate metabolism was accelerated. In addition, the genes related to stress and detoxification were upregulated with the overexpression of resistance gene marA , which reduced the NADPH level in E. coli . Then, the expression level of soluble OATA increased by promoter optimization. Overall, arcA and marA could regulate the catalytic rate of NADPH- dependent ncCAR, while arcA and optimized promoter could regulate the catalytic rate of OATA. Lastly, the cinnamylamine yield of the best metabolically engineered strain S020 was increased to 90% (9 mM, 1.2 g/L), and the accumulation of cinnamaldehyde was below 0.9 mM. This work reported the highest production of cinnamylamine by biocatalytic synthesis. Conclusion This regulatory process provides a common strategy for regulating the biocatalytic synthesis of other APAs. Being entirely biocatalytic, our one-pot procedure provides considerable advantages in terms of environmental and safety impacts over reported chemical methods.
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