Synergistic improvement of cinnamylamine production by metabolic regulation

Abstract

Abstract 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.

Keywords

• Cinnamylamine
• Metabolic engineering
• Transcriptome analysis
• Escherichia coli
• One-pot
• Whole-cell catalysis