RESUMO
Vanillyl alcohol is a phenolic alcohol and is used as a flavoring agent in foods and beverages. In this paper, we propose a novel artificial pathway for microbial production of vanillyl alcohol from simple carbon sources. The pathway extends from 4-hydroxybenzoic acid (4-HBA), and needs only three heterologous enzymes, p-hydroxybenzoate hydroxylase (PobA), carboxylic acid reductase (CAR) and caffeate O-methyltransferase (COMT). First, we examined the promiscuous activity of COMT toward 3,4-dihydroxybenzyl alcohol and found a kcat value of 0.097 s-1. Meanwhile, 499.36 mg/L vanillyl alcohol was produced by COMT in vivo catalysis when fed with 1000 mg/L 3,4-dihydroxybenzyl alcohol. In the following experiment, de novo biosynthesis of vanillyl alcohol was carried out and 240.69 mg/L vanillyl alcohol was produced via modular optimization of pathway genes. This work was to date the first achievement for microbial production of vanillyl alcohol. Additionally, the present study demonstrates the application of enzyme promiscuity of COMT in the design of an artificial pathway for the production of high-value methylated aromatic compounds.
Assuntos
Álcoois Benzílicos/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Sintéticos/genética , Engenharia Metabólica/métodos , Álcoois Benzílicos/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Metiltransferases/genética , Modelos Genéticos , Parabenos/metabolismoRESUMO
Derived from the bacterial adaptive immune system, CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering. In this review, we outline the fundamental CRISPR tools that have been employed for strain optimization. These tools include CRISPR editing, CRISPR interference, CRISPR activation and protein imaging. To further characterize the CRISPR technology, we present current applications of these tools in microbial systems, including model- and non-model industrial microorganisms. Specially, we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation. To address these challenges, we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering. Lastly, multiple promising research directions were proposed, mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.