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Engineering a probiotic strain of Escherichia coli to induce the regression of colorectal cancer through production of 5-aminolevulinic acid.

ABSTRACT: Bacterial vectors can be engineered to generate microscopic living therapeutics to produce and deliver anticancer agents. Escherichia coli Nissle 1917 (Nissle 1917) is a promising candidate with probiotic properties. Here, we used Nissle 1917 to develop a metabolic strategy to produce 5-aminolevulinic acid (5-ALA) from glucose as 5-ALA plays an important role in the photodynamic therapy of cancers. The coexpression of hemA^M and hemL using a low copy-number plasmid led to remarkable accumulation of 5-ALA. The downstream pathway of 5-ALA biosynthesis was inhibited by levulinic acid (LA). Small-scale cultures of engineered Nissle 1917 produced 300 mg l^-1 of 5-ALA. Recombinant Nissle 1917 was applied to deliver 5-ALA to colorectal cancer cells, in which it induced the accumulation of antineoplastic protoporphyrin X (PpIX) and specific cytotoxicity towards colorectal cancer cells irradiated with a 630 nm laser. Moreover, this novel combination therapy proved effective in a mouse xenograft model and was not cytotoxic to normal tissues. These findings suggest that Nissle 1917 will serve as a potential carrier to effectively deliver 5-ALA for cancer therapy.

SUBMITTER: Chen J

PROVIDER: S-EPMC8449674 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Engineering a probiotic strain of Escherichia coli to induce the regression of colorectal cancer through production of 5-aminolevulinic acid.

Chen Junhao J Li Xiaohong X Liu Yumei Y Su Tianyuan T Lin Changsen C Shao Lijun L Li Lanhua L Li Wanwei W Niu Guoyu G Yu Jing J Liu Ling L Li Miaomiao M Yu Xiaoli X Wang Qian Q

Microbial biotechnology 20210716 5

Bacterial vectors can be engineered to generate microscopic living therapeutics to produce and deliver anticancer agents. Escherichia coli Nissle 1917 (Nissle 1917) is a promising candidate with probiotic properties. Here, we used Nissle 1917 to develop a metabolic strategy to produce 5-aminolevulinic acid (5-ALA) from glucose as 5-ALA plays an important role in the photodynamic therapy of cancers. The coexpression of hemA<sup>M</sup> and hemL using a low copy-number plasmid led to remarkable ac ...[more]

PMID: 34272828

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Project description:BackgroundFlavonoids 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.ResultsBy 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.ConclusionThrough 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.

Dataset Information

Engineering a probiotic strain of Escherichia coli to induce the regression of colorectal cancer through production of 5-aminolevulinic acid.

Publications

Engineering a probiotic strain of Escherichia coli to induce the regression of colorectal cancer through production of 5-aminolevulinic acid.

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