Manchester Institute of Biotechnology, Faculty of Science and Engineering, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
Microb Cell Fact. 2024 Nov 19;23(1):312. doi: 10.1186/s12934-024-02582-z.
Flavonoids are a structurally diverse group of secondary metabolites, predominantly produced by plants, which include a range of compounds with pharmacological importance. Pinocembrin is a key branch point intermediate in the biosynthesis of a wide range of flavonoid subclasses. However, replicating the biosynthesis of these structurally diverse molecules in heterologous microbial cell factories has encountered challenges, in particular the modest pinocembrin titres achieved to date. In this study, we combined genome engineering and enzyme candidate screening to significantly enhance the production of pinocembrin and its derivatives, including chrysin, pinostrobin, pinobanksin, and galangin, in Escherichia coli.
By implementing a combination of established strain engineering strategies aimed at enhancing the supply of the building blocks phenylalanine and malonyl-CoA, we constructed an E. coli chassis capable of accumulating 353 ± 19 mg/L pinocembrin from glycerol, without the need for precursor supplementation or the fatty acid biosynthesis inhibitor cerulenin. This chassis was subsequently employed for the production of chrysin, pinostrobin, pinobanksin, and galangin. Through an enzyme candidate screening process involving eight type-1 and five type-2 flavone synthases (FNS), we identified Petroselinum crispum FNSI as the top candidate, producing 82 ± 5 mg/L chrysin. Similarly, from a panel of five flavonoid 7-O-methyltransferases (7-OMT), we found pinocembrin 7-OMT from Eucalyptus nitida to yield 153 ± 10 mg/L pinostrobin. To produce pinobanksin, we screened seven enzyme candidates exhibiting flavanone 3-hydroxylase (F3H) or F3H/flavonol synthase (FLS) activity, with the bifunctional F3H/FLS enzyme from Glycine max being the top performer, achieving a pinobanksin titre of 12.6 ± 1.8 mg/L. Lastly, by utilising a combinatorial library of plasmids encoding G. max F3H and Citrus unshiu FLS, we obtained a maximum galangin titre of 18.2 ± 5.3 mg/L.
Through the integration of microbial chassis engineering and screening of enzyme candidates, we considerably increased the production levels of microbially synthesised pinocembrin, chrysin, pinostrobin, pinobanksin, and galangin. With the introduction of additional chassis modifications geared towards improving cofactor supply and regeneration, as well as alleviating potential toxic effects of intermediates and end products, we anticipate further enhancements in the yields of these pinocembrin derivatives, potentially enabling greater diversification in microbial hosts.
类黄酮是一类结构多样的次生代谢产物，主要由植物产生，其中包括一系列具有药理学重要性的化合物。松属素是广泛存在的黄酮类亚类生物合成中的关键分支点中间体。然而，在异源微生物细胞工厂中复制这些结构多样的分子的生物合成遇到了挑战，特别是迄今为止实现的适度松属素产量。在这项研究中，我们结合了基因组工程和酶候选物筛选，以显著提高大肠杆菌中松属素及其衍生物（包括白杨素、乔松素、根皮素和高良姜素）的产量。
通过实施一系列旨在增强苯丙氨酸和丙二酰辅酶 A 供应的既定菌株工程策略，我们构建了一种大肠杆菌底盘，能够从甘油中积累 353±19mg/L 的松属素，而无需前体补充或脂肪酸生物合成抑制剂 cerulenin。该底盘随后用于白杨素、乔松素、根皮素和高良姜素的生产。通过涉及 8 种 1 型和 5 种 2 型黄酮合酶（FNS）的酶候选物筛选过程，我们鉴定出 Petroselinum crispum FNSI 为最佳候选物，可产生 82±5mg/L 的白杨素。同样，在 5 种黄酮 7-O-甲基转移酶（7-OMT）的筛选中，我们发现来自桉树的 pinocembrin 7-OMT 可产生 153±10mg/L 的乔松素。为了生产根皮素，我们筛选了 7 种具有黄酮醇 3-羟化酶（F3H）或 F3H/黄酮醇合酶（FLS）活性的酶候选物，其中大豆的双功能 F3H/FLS 酶表现最佳，根皮素产量为 12.6±1.8mg/L。最后，通过利用编码大豆 F3H 和柑橘 FLS 的质粒组合文库，我们获得了 18.2±5.3mg/L 的高良姜素最大产量。
通过微生物底盘工程的整合和酶候选物的筛选，我们大大提高了微生物合成的松属素、白杨素、乔松素、根皮素和高良姜素的产量。通过引入额外的底盘修饰，以改善辅助因子的供应和再生，以及减轻中间体和最终产物的潜在毒性影响，我们预计这些松属素衍生物的产量将进一步提高，这可能使微生物宿主的多样性更大。
