The Dalian Institute of Chemistry, Chinese Academy of Sciences, developed cofactor engineering strategies to promote biosynthesis of natural products

The cover image was produced by the Science Visualization Center of China Science Newspaper

Recently, the team of researcher Zhou Yongjin of the Synthetic Microbiology Research Group of the Dalian Institute of Chemistry of the Chinese Academy of Sciences cooperated with Professor Zhang Lei of the Department of Pharmacy of the Naval Military Medical University to make new progress in the synthetic biology of phenolic acid natural products. The collaborative team constructed and optimized the phenolic acid biosynthesis pathway in Saccharomyces cerevisiae, strengthened the supply and turnover of key cofactors, and realized the efficient synthesis of phenolic acid compounds.

On April 28, 2022, the study was published in Nature Chemical Biology under the title “Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast.” The first author of the paper is Chen Ruibing, a postdoctoral fellow at the Dalian Institute of Chemicals.

In recent years, the rapid development of synthetic biology has made the sustainable supply of natural products a reality, and the construction of a complete biosynthetic pathway in microbial cells can realize the efficient biosynthesis of a series of complex natural products such as artemisinic acid and alkaloids. Saccharomyces cerevisiae is widely used in food brewing, and with its strong plasticity, robustness and reliable safety, it has gradually become one of the main platforms for building cell factories. At present, although enzyme engineering and pathway engineering have been widely used to improve the performance of yeast cell factories, their efficiency for synthesizing natural products needs to be further improved.

In the above context, the cooperative team is committed to improving the efficiency of the synthesis of phenolic acid natural products. Studies have found that the catalytic enzyme activity involved in intracellular cofactors is not only determined by enzyme expression, but also related to the level of cofactors, especially when expressing exogenous enzymes, they often face cofactor mismatch or insufficient supply restrictions. As a result, Zhou Yongjin’s team systematically reviewed the important value of cofactors in the synthesis of natural products, and provided four feasible cofactor engineering protocols: reconstructing the biosynthesis of cofactors; increasing the metabolic level of intracellular/intra-organelle cofactors; balancing the homeostasis of cofactors; and improving the active form of cofactors (iScience, 2020).

Caffeine biosynthesis, one of the phenolic acid compounds in yeast, requires cofactors FAD (H2) and NADPH, while ferulic acid biosynthesis requires SAM as a methyl donor. In this work, the team significantly improved the efficiency of caffeic acid biosynthesis by modifying the central metabolism to improve NADPH supply, constructing cytoplasmic FAD(H2) synthesis pathways, and directing mitochondrial FAD(H2) to cytoplasm to improve cytoplasmic FAT(H2) supply, so that its yield reached 5.5g/L, which was much higher than the 0.8g/L reported in the literature. On this basis, the team used high expression methyltransferase to construct a ferulic acid biosynthesis pathway, further strengthened the methyl cycle to remove the inhibition effect of methyltransferase, and improved the SAM level and SAM turnover of the methyl donor cofactor, so that the ferulic acid yield reached 3.8 g/L, which was much higher than the 0.04 g/L reported in the literature. This work reveals the regulation of different cofactors in yeast, especially the cofactor allocation between different organelles in the cell, which provides theoretical guidance for the regulation of cofactors. Moreover, this work will provide sufficient precursors for the efficient synthesis of complex active natural products (lignans and polyphenolic acids, etc.), which is expected to provide guarantees for the development of new natural product resources and the sustainable use of endangered Chinese medicine resources.

The work has been funded by the National Key Research Program, the National Natural Science Foundation of China, the National Natural Science Foundation of China Outstanding Youth Fund, the Xingliao Talents Program, the Dalian Institute of Chemicals And Research Innovation Fund and other projects. (Source: Science Network)

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