Metabolic Engineering

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Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli

O-Methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ∼30 enzymes against five substrates in 3 days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus far uncharacterized O-methyltransferases for future use as biocatalysts

Development of a sustainable bioprocess for the production of novel Xylooligosaccharides (XOS) and their potential application

The growing demand of novel food products for well-being and age related issues coupled with increasing health care expenditure has attracted global attention on prebiotics. Xylooligosaccarides (XOS) are the only nutraceuticals that can be produced from lignocellulosic biomass. Indeed, XOS can be produced from agricultural crop residues, which is encouraging to the food ingredient industries, as these raw materials are inexpensive, abundant and renewable in nature. XOS beneficial effects include, besides the selective growth stimulation of beneficial gut microflora, enhanced mineral absorption, cholesterol lowering, glucose homeostasis, pathogen exclusion, immune modulation, antioxidant and anticarcinogenic activities, among others. The precursor for XOS is xylan. Xylan is the polysaccharide accounting for 25 to 50% of the dry mass of lignocellulosic-based agriculture residues. XOS can then be produced through chemical or enzymatic processes. The microbial or enzymatic conversion of xylan into value-added useful products, as XOS, holds a great promise for the use of a variety of agro-food and industrial residues. The goal of this PhD project is to develop a sustainable bioprocess by exploring the use of agro-industrial residues for the production of novel XOS and to evaluate their effect on the probiotics viability under simulated gastric conditions. The proposed tasks involve several design and engineering approaches to optimize the production process.info:eu-repo/semantics/publishedVersio

Design and construction of a new biosynthetic pathway for the production of curcuminoids in Escherichia coli

Curcuminoids are natural pigments from plants that have been reported as potential cancer-fighting drugs. The aim of this work is to engineer an artificial biosynthetic pathway for curcuminoids production by Escherichia coli. Starting from the substrate tyrosine, the curcumin pathway involves several enzymatic steps: conversion of tyrosine to p-coumaric acid; conversion of p-coumaric acid to caffeic acid; production of caffeoyl-CoA from caffeic acid; production of feruloyl-CoA from caffeoyl-CoA; and finally the production of curcumin from feruloyl-CoA and possibly other curcuminoids, due to enzyme promiscuity. The enzymes involved in the two first enzymatic steps are tyrosine ammonia lyase from Rhodotorula glutinis, P450 CYP199A2 from Rhodopseudomonas palustris, and the redox partners pdr from Pseudomonas putida and pux from R. palustris. These two steps were successfully accomplished. Two CoA ligases from different sources are being explored for the conversion of the different carboxylic acids into their corresponding CoA esters. Different combinations of these enzymes and caffeoyl-CoA 3-methyl transferase may lead to the production of different curcuminoids. For the last step of the pathway two approaches are being studied: the use of diketide-CoA synthase and curcuminoid synthase from Curcuma longa, and curcumin synthase from Oryza sativa that itself catalyzes both steps. Successful construction of the curcuminoids biosynthetic pathway would mark a significant step forward in the in situ production of these poorly soluble, anti-carcinogenic compounds

Biosynthetic production of curcuminoids

Curcuminoids are natural phenylpropanoids from plants that have been reported as potential cancer-fighting drugs. Nevertheless, these compounds present a poor bioavailability. Cellular uptake is low and curcuminoids are quickly metabolized once inside the cell, requiring repetitive oral doses to achieve an effective concentration for therapeutic activity [1]. Herein, we report an engineered artificial pathway for the production of curcuminoids in Escherichia coli. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used and 188 µM (70 mg/L) of curcumin was obtained from ferulic acid [2]. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used [3]. Caffeoyl-CoA 3-O-methyl-transferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 µM of curcumin were produced, respectively. This is the first report on the use of DCS and CURS1 in vivo to produce curcuminoids. In addition, curcumin, the most studied curcuminoid for therapeutic purposes and considered in many studies as the most potent and active, was produced by feeding tyrosine using a pathway involving caffeic acid. We anticipate that by using a tyrosine overproducing strain, curcumin can be produced in E. coli without the need of adding expensive precursors to the medium, thus decreasing the production cost. Therefore, this alternative pathway represents a step forward in the heterologous production of curcumin using E. coli. Aiming at greater production titers and yields, the construction of this pathway in another model organism such as Saccharomyces cerevisiae is being considered

Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2018.09.088.The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4-6 was obtained, presenting high stability after a static in vitro digestion (98.5±0.2%). The maximum production yield expressed as total XOS per amount of xylan (306±4mg/g) was achieved after 8h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5°C, using 2.5g/L of initial concentration of xylan increased up to 5.0g/L at 3h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield.CA, SCS, ACP, EC and LRR acknowledge their grants (PD/BD/ 105870/2014, SFRH/BPD/88584/2012, SFRH/BPD/ 101181/2014, SFRH/BPD/70589/2010 and SFRH/BSAB/142873/2018) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit; COMPETE 2020 (POCI-010145-FEDER-006684); QOPNA research Unit (FCT UID/QUI/00062/ 2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project MultiBiorefinery (POCI-01-0145-FEDER-016403); the Project FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01-0145FEDER-029773). The authors also acknowledge BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli

In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways

Production of d-Glyceric acid from d-Galacturonate in Escherichia coli

A microbial production platform has been developed in Escherichia coli to synthesize d-glyceric acid from d-galacturonate. The expression of uronate dehydrogenase (udh) from Pseudomonas syringae and galactarolactone isomerase (gli) from Agrobacterium fabrum, along with the inactivation of garK, encoding for glycerate kinase, enables d-glyceric acid accumulation by utilizing the endogenous expression of galactarate dehydratase (garD), 5-keto-4-deoxy-D-glucarate aldolase (garL), and 2-hydroxy-3-oxopropionate reductase (garR). Optimization of carbon flux through the elimination of competing metabolic pathways led to the development of a ΔgarKΔhyiΔglxKΔuxaC mutant strain that produced 4.8 g/l of d-glyceric acid from d-galacturonate, with an 83% molar yield. Cultivation in a minimal medium produced similar yields and demonstrated that galactose or glycerol serve as possible carbon co-feeds for industrial production. This novel platform represents an alternative for the production of d-glyceric acid, an industrially relevant chemical, that addresses current challenges in using acetic acid bacteria for its synthesis: increasing yield, enantio-purity and biological stability.U.S. Army Research Office (Contract W911NF-09-0001

Substrate‐activated expression of a biosynthetic pathway in Escherichia coli

Microbes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of d-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer. Activation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production d-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13 ± 0.03 g L−1 d-glyceric acid within 6 h of galacturonate substrate addition. This work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis