Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway

Abstract

BackgroundVanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin.ResultsTime-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and l-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-l-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33 % (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25 % increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability.ConclusionsResults from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or methionine biosynthesis, we pursued two orthogonal strategies that are each aimed at deregulating multiple reactions. Our results increase the working knowledge of SAM biosynthesis engineering and provide a framework for improving titers of metabolic products dependent upon methylation reactions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0459-x) contains supplementary material, which is available to authorized users.

CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production

“…Finally, we found that coordinated, global perturbation of SAM biosynthesis was obtainable through a single CRISPRi intervention, leading to increased conversion through the anthocyanin O -methylation tailoring reaction. The metJ dysregulation strategy described in this and another recent report [25] should be useful for improving methylation of any natural product, but the general steps taken here should also be applicable for studying unique anthocyanin tailoring reactions that require co-option of distinct cosubstrates. Future efforts to integrate anthocyanin pathway genes into the genome of E. coli should also be undertaken to generate stable production strains lacking plasmids.…”

“…Despite modest titers outlined here for the novel anthocyanin product P3G, very impressive and extensive work published during the preparation of this manuscript examined several solutions to SAM limitation in E. coli K-12 MG1655(DE3) lineages for production of vanillate, an O -methylated natural product that had been produced in E. coli previously but suffered from low titers [25]. Combined deregulation of SAM biosynthesis through metJ deletion, feedback desensitization of enzymes catalyzing committed or important biosynthetic steps, and deregulation of SAM regeneration improved de novo vanillate titers by twofold to approximately 400 mg/L [25]. These findings imply that SAM-dependent natural product biosyntheses in E. coli at industrially relevant titers will likely be achievable.…”

“…Interestingly, and in contrast to our work with anthocyanins, deletion of metJ alone was not sufficient to improve vanillate titers. It is possible that this difference stems from disparities in metabolite pools or regulation between B (used in this work) and K (used in [25]) E. coli lineages; alternatively, this could be attributed simply to distinct enzyme properties between the selected AOMT and catechol OMT used for vanillate production. Other strategies outlined in literature that have been reported to augment SAM production include supplementation of media with methionine and improvement of precursor ATP pools by reducing ATP usage in other reactions [54].…”

“…Given the existence of S -adenosylmethionine (SAM or AdoMet, the cosubstrate used by AOMTs to methylate anthocyanins primarily at the 3′- and 5′-hydroxyls) in the natural metabolism of E. coli , coupled with the proven capability of E. coli to express active AOMTs, we posited that endogenous SAM pools might be sufficient to achieve in vivo anthocyanin O -methylation. Further supporting this hypothesis, E. coli has been utilized as a host for heterologous production of other natural compounds that require SAM as a cosubstrate such as vanillate [25], N -acyl-homoserine lactones [26], polyketides [27], and even for biotransformations of flavonoids to their O -methylated counterparts [28–30]. Until recently, however, there has been little effort aimed at improving SAM pools for co-option by exogenous, SAM-dependent methyltransferase reactions.…”

Isolation and Analysis of Salt Response of Lactobacillus plantarum FS5-5 from Dajiang

“…Protein concentration was initially determined according to the Bradford method [11], with bovine serum albumin (BSA) as the protein standard. The SDS-PAGE consisted of 5 % stacking gel and 12 % separation gel.…”