Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli

Abstract Background Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllusenabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli. Results We first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4′-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3′,4′-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F. Conclusion We established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds

Manipulation of the carbon storage regulator system for metabolite remodeling and biofuel production in Escherichia coli.

BackgroundMicrobial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.ResultsWe engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO(2).ConclusionsWe have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts

Manipulation of the carbon storage regulator system for metabolite remodeling and biofuel production in <it>Escherichia coli</it>

Abstract Background Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO2 make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways. Results We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO2. Conclusions We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.</p

Manipulation of the Carbon Storage Regulator System for Metabolite Remodeling and Biofuel Production in Escherichia coli

AbstractBackgroundMicrobial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO2 make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.ResultsWe engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO2.ConclusionsWe have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts

Modular Engineering of l-Tyrosine Production in Escherichia coli

Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways