Designing a microbial factory suited for plant chloroplast-derived enzymes to efficiently and green synthesize natural products: Capsanthin and capsorubin as examples

Abstract

Specific cellular microenvironment, multi-enzyme complex and expensive essential cofactor make the biological manufacturing of plant chloroplast natural products (PCNPs) extremely challenging. The above difficulties have hampered the biosynthesis of capsanthin and capsorubin in the past 30 years. Here, we take capsanthin and capsorubin as examples to design an innovative microbial factory to promote the heterologous synthesis of PCPNs. Our main strategy is mimicking the microenvironment of chloroplasts in microbial factory. First, accumulation of violaxanthin, which is the key precursor, was increased by 587.9%, through introducing oxidative microenvironment and thioredoxin. The initial capsanthin-producing strain with 0.28 mg g⁻¹ DCW were obtained by introducing capsanthin/capsorubin synthase (CCS). Subsequently, chloroplast-derived chaperones Cpn60α, Cpn60β and Cpn20 created a folding-promoting microenvironment for CCS. At the same time, by imitating the quasi-natural CCS, an artificial homotrimer was constructed and obtained 5.15 mg g⁻¹ DCW capsanthin, and 1.62 mg g⁻¹ DCW capsorubin. Finally, sufficient FADH₂ was provided for CCS by feeding 20 mM formate. This process was realized by the continuous catalysis of formate dehydrogenase and flavin reductase. The engineered strain accumulated 6.77 mg g⁻¹ DCW of capsanthin and 2.18 mg g⁻¹ DCW of capsorubin. Compared with the initial strain, the yield of capsanthin was increased by 24.18 times, and 13.54 times of the highest yield reported so far. Artificially designed microbial cell factory and low-cost cofactor supply methods are in line with the current sustainable and green wave of biochemicals. This work not only provides a platform strain for low-cost and sustainable biosynthesis, but also provides a paradigm for heterologous expression of chloroplast-derived enzymes.