Dataset Information

MRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli.

ABSTRACT: The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3' untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5'-TAATG-3' between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes.

SUBMITTER: Bui LM

PROVIDER: S-EPMC6651523 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in <i>Escherichia coli</i>.

Bui Le Minh LM Geraldi Almando A Nguyen Thi Thuy TT Lee Jun Hyoung JH Lee Ju Young JY Cho Byung-Kwan BK Kim Sun Chang SC

International journal of molecular sciences 20190628 13

The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3' untranslated region of ...[more]

PMID: 31261687

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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.

Dataset Information

MRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli.

Publications

mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in <i>Escherichia coli</i>.

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