Dataset Information

Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli.

ABSTRACT: Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in bacterial and mammalian cells. The family of CDNs includes c-di-GMP, c-di-AMP and two distinct versions of hybrid cGAMPs. Studies related to these CDNs require large doses that are relatively expensive to generate by current methods. Here we report what to our knowledge is the first feasible microbial-based method to prepare these CDNs including c-di-GMP, 3'3'-cGAMP and 2'3'-cGAMP. The method mainly includes two parts: producing high yield of CDNs by engineering the overexpression of the proper dinucleotide cyclases (DNCs) and other related proteins in Escherichia coli, and purifying the bacteria-produced CDNs by a unified and simple process involving a STING affinity column, macroporous adsorption resin and C18 reverse-phase liquid chromatography. After purification, we obtained the diammonium salts of c-di-GMP, 3'3'-cGAMP and 2'3'-cGAMP with weight purity of >99, >96, >99% and in yields of >68, >26, and >82 milligrams per liter of culture, respectively. This technological platform enables the production of CDNs from cheaper material, provides a sustainable source of CDNs for scientific investigation, and can easily be further developed to prepare CDNs on a large scale for industry.

SUBMITTER: Lv Y

PROVIDER: S-EPMC6753226 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Highly Efficient Preparation of Cyclic Dinucleotides <i>via</i> Engineering of Dinucleotide Cyclases in <i>Escherichia coli</i>.

Lv Yun Y Sun Qichao Q Wang Xiaodan X Lu Yi Y Li Yaoyao Y Yuan Huiqing H Zhu Jing J Zhu Deyu D

Frontiers in microbiology 20190913

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in bacterial and mammalian cells. The family of CDNs includes c-di-GMP, c-di-AMP and two distinct versions of hybrid cGAMPs. Studies related to these CDNs require large doses that are relatively expensive to generate by current methods. Here we report what to our knowledge is the first feasible microbial-based method to prepare these CDNs including c-di-GMP, 3'3'-cGAMP and 2'3'-cGAMP. The method mainly includes two parts: ...[more]

PMID: 31572324

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Project description:BackgroundHeme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme.ResultsHere, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold.ConclusionHigh intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.

Project description:BackgroundThe 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.ResultsIn 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 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::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, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) 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.ConclusionsThe 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

Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli.

Publications

Highly Efficient Preparation of Cyclic Dinucleotides <i>via</i> Engineering of Dinucleotide Cyclases in <i>Escherichia coli</i>.

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