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Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.

ABSTRACT: Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. However, functional integration of synthetic pathways into the respective host cell metabolism and optimization of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropanoid p-coumaryl alcohol in Escherichia coli.Basis for the construction of a p-coumaryl alcohol producing strain was the development of Operon-PLICing as method for the rapid combinatorial assembly of synthetic operons. This method is based on the chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution to generate complementary, single-stranded overhangs and subsequent hybridization of multiple DNA-fragments. Furthermore, during the assembly of these DNA-fragments, Operon-PLICing offers the opportunity for balancing gene expression of all pathway genes on the level of translation for maximizing product titers by varying the spacing between the Shine-Dalgarno sequence and START codon. With Operon-PLICing, 81 different clones, each one carrying a different p-coumaryl alcohol operon, were individually constructed and screened for p-coumaryl alcohol formation within a few days. The absolute product titer of the best five variants ranged from 48 to 52 mg/L p-coumaryl alcohol without any further optimization of growth and production conditions.Operon-PLICing is sequence-independent and thus does not require any specific recognition or target sequences for enzymatic activities since all hybridization sites can be arbitrarily selected. In fact, after PCR-amplification, no endonucleases or ligases, frequently used in other methods, are needed. The modularity, simplicity and robustness of Operon-PLICing would be perfectly suited for an automation of cloning in the microtiter plate format.

SUBMITTER: van Summeren-Wesenhagen PV

PROVIDER: S-EPMC4464236 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.

van Summeren-Wesenhagen Philana V PV Voges Raphael R Dennig Alexander A Sokolowsky Sascha S Noack Stephan S Schwaneberg Ulrich U Marienhagen Jan J

Microbial cell factories 20150611

<h4>Background</h4>Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. However, functional integration of synthetic pathways into the respective host cell metabolism and optimization of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropanoid p-coumaryl alcohol in Esche ...[more]

PMID: 26062542

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Project description:BackgroundMulti-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under PT7, Ptrc, and PlacUV5 promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-D-glucose synthesis, all cloned in a multi-monocistronic fashion under PT7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses.ResultsThe engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-D-glucose synthesis were fed with 2 mM (+)-catechin and D-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under Ptrc promoter and UDP-D-glucose biosynthesis genes under PT7 promoter led to the production of ~?439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) D-glucose. This system did not require exogenous supplementation of UDP-D-glucose.ConclusionA synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-D-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.

Dataset Information

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.

Publications

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.

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