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Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.

ABSTRACT: Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systematic approach was used to increase glycoprotein production titres of an exemplar protein, AcrA, and the resulting glycosylation efficiency was quantified using a combination of Western blots and pseudo Selective Reaction Monitoring (pSRM). Western blot and pSRM results demonstrate that codon optimising the oligosaccharyltransferase, PglB, for E. coli expression, increases efficiency by 77% and 101%, respectively. Furthermore, increasing expression of glycosyltransferase, WecA, in E. coli improves efficiency by 43% and 27%, respectively. However, increasing the amount of donor lipid used in the glycosylation process did not impact on the glycosylation efficiency in this system, with this specific protein.

SUBMITTER: Pandhal J

PROVIDER: S-EPMC3401370 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.

Pandhal Jagroop J Desai Pratik P Walpole Caroline C Doroudi Leyla L Malyshev Dmitry D Wright Phillip C PC

Biochemical and biophysical research communications 20120210 3

Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systemat ...[more]

PMID: 22342719

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Project description:BACKGROUND: Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. RESULTS: The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. CONCLUSIONS: The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

Project description:BACKGROUND:The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. RESULTS:In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>?0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (?ptsG::glk, ?galR::zglf, ?poxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, ?pgi, ?pgm, and RBSL5-zwf). Strain R04 (SG104 and ?pgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. CONCLUSIONS:The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

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Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.

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Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.

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