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Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system.

ABSTRACT: BACKGROUND:Cinnamyl alcohol is not only a kind of flavoring agent and fragrance, but also a versatile chemical applied in the production of various compounds. At present, the preparation of cinnamyl alcohol depends on plant extraction and chemical synthesis, which have several drawbacks, including limited scalability, productivity and environmental impact. It is therefore necessary to develop an efficient, green and sustainable biosynthesis method. RESULTS:Herein, we constructed a recombinant Escherichia coli BLCS coexpressing carboxylic acid reductase from Nocardia iowensis and phosphopantetheine transferase from Bacillus subtilis. The strain could convert cinnamic acid into cinnamyl alcohol without overexpressing alcohol dehydrogenase or aldo-keto reductase. Severe product inhibition was found to be the key limiting factor for cinnamyl alcohol biosynthesis. Thus, a biphasic system was proposed to overcome the inhibition of cinnamyl alcohol via in situ product removal. With the use of a dibutyl phthalate/water biphasic system, not only was product inhibition removed, but also the simultaneous separation and concentration of cinnamyl alcohol was achieved. Up to 17.4 mM cinnamic acid in the aqueous phase was totally reduced to cinnamyl alcohol with a yield of 88.2%, and the synthesized cinnamyl alcohol was concentrated to 37.4 mM in the organic phase. This process also demonstrated robust performance when it was integrated with the production of cinnamic acid from L-phenylalanine. CONCLUSION:We developed an efficient one-pot two-step biosynthesis system for cinnamyl alcohol production, which opens up possibilities for the practical biosynthesis of natural cinnamyl alcohol at an industrial scale.

SUBMITTER: Zhang C

PROVIDER: S-EPMC7424670 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system.

Zhang Chen C Xu Qian Q Hou Hongliang H Wu Jiawei J Zheng Zhaojuan Z Ouyang Jia J

Microbial cell factories 20200812 1

<h4>Background</h4>Cinnamyl alcohol is not only a kind of flavoring agent and fragrance, but also a versatile chemical applied in the production of various compounds. At present, the preparation of cinnamyl alcohol depends on plant extraction and chemical synthesis, which have several drawbacks, including limited scalability, productivity and environmental impact. It is therefore necessary to develop an efficient, green and sustainable biosynthesis method.<h4>Results</h4>Herein, we constructed a ...[more]

PMID: 32787860

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Project description:BACKGROUND: With the increasing stress from oil price and environmental pollution, aroused attention has been paid to the microbial production of chemicals from renewable sources. The C12/14 and C16/18 alcohols are important feedstocks for the production of surfactants and detergents, which are widely used in the most respected consumer detergents, cleaning products and personal care products worldwide. Though bioproduction of fatty alcohols has been carried out in engineered E. coli, several key problems have not been solved in earlier studies, such as the quite low production of C16/18 alcohol, the lack of optimization of the fatty alcohol biosynthesis pathway, and the uncharacterized performance of the engineered strains in scaled-up system. RESULTS: We improved the fatty alcohol production by systematically optimizing the fatty alcohol biosynthesis pathway, mainly targeting three key steps from fatty acyl-acyl carrier proteins (ACPs) to fatty alcohols, which are sequentially catalyzed by thioesterase, acyl-coenzyme A (CoA) synthase and fatty acyl-CoA reductase. By coexpression of thioesterase gene BTE, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene acr1, 210.1?mg/L?C12/14 alcohol was obtained. A further optimization of expression level of BTE, fadD and acr1 increased the C12/14 alcohol production to 449.2?mg/L, accounting for 75.0% of the total fatty alcohol production (598.6?mg/L). In addition, by coexpression of thioesterase gene 'tesA, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene FAR, 101.5?mg/L?C16/18 alcohol was obtained, with C16/18 alcohol accounting for 89.2% of the total fatty alcohol production. CONCLUSIONS: To our knowledge, this is the first report on selective production of C12/14 and C16/18 alcohols by microbial fermentation. This work achieved high-specificity production of both C12/14 and C16/18 alcohols. The encouraging 598.6?mg/L of fatty alcohols represents the highest titer reported so far. In addition, the 101.5?mg/L 89.2%?C16/18 alcohol suggests an important breakthrough in C16/18 alcohol production. A more detailed optimization of the expression level of fatty alcohol biosynthesis pathway may contribute to a further improvement of fatty alcohol production.

Project description:BackgroundPhenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.ResultsA pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure.ConclusionPhenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

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Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system.

Publications

Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system.

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