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Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases.

ABSTRACT: Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (?10?mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology-based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l-arabinose-inducible expression of the T7 phage-derived Gp2 protein from a genome-integrated site. The glycosyltransferase genes are encoded on conventional pET-based expression plasmids that allow T7 RNA polymerase-driven inducible expression by isopropyl-?- d-galactoside. Laboratory batch and scaled-up (20?L) fed-batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12-fold as a result of production under growth-decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ?5 and ?1% of total intracellular protein. Fed-batch production gave sucrose synthase in a yield of 2,300?U/L of culture (830?mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3-fold) and quality (2.3-fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth-arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

SUBMITTER: Lemmerer M

PROVIDER: S-EPMC6767175 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases.

Lemmerer Martin M Mairhofer Juergen J Lepak Alexander A Longus Karin K Hahn Rainer R Nidetzky Bernd B

Biotechnology and bioengineering 20190205 6

Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology-based strategy of decoupling the enzyme expression from the Es ...[more]

PMID: 30659592

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Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Project description:The ability to control the synthesis of products in the absence of cell division remains an attractive alternative for the optimization of microbial processes. If this could be achieved, it would dramatically expand the productivity of many microbial processes by increasing product synthesis in the absence of an increase in cell mass. In the present work we have looked at the factors involved in the design of a host where the fluxes would be channeled towards product formation rather than biomass synthesis. To identify the genes responsible for diverting the metabolic flux specifically towards product formation we have used for this study is quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabolically active cells. These cells channel the metabolic flux towards recombinant protein production and therefore the specific product yield per unit biomass is significantly higher. Indole which is previously known to be a signalling molecule is here used as an inducer of Quiescence. E.coli L-Asparaginase is used as a model protein in this work. Fed batch cultures were carried out in Sartorius BIOSTAT B plus 5liter bioreactor. The data acquisition was done with the MFCS Shell software (version) provided with the bioreactor. The Sartorius BIOSTAT B plus had proportional integral derivative-based control loops for temperature, pH, antifoam, and dissolved-oxygen (DO) regulation. The culture was grown in a batch mode till 10-12 OD (µ=0.5) and then the feed was attached. In the next hour the culture was induced for product formation with 1M IPTG. In case of Q culture quiescence was induced with 0.5M Indole just 5min after induction with IPTG. Post induction every hour samples were collected and frozen with liquid nitrogen and stored at -80ºC for further analytical use. Total RNA extraction, RNA quality control (Agilent BioAnalyser), total RNA labeling, microarray hybridization and scanning were performed according to the Affymetrix GeneChip Expression analysis .

Dataset Information

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases.

Publications

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases.

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