Dataset Information

Simultaneous production of N-acetylheparosan and recombinant chondroitin using gene-engineered Escherichia coli K5.

ABSTRACT: N-Acetylheparosan and chondroitin are increasingly needed as alternative sources of animal-derived sulfated glycosaminoglycans (GAGs) and as inert substances in medical devices and pharmaceuticals. The N-acetylheparosan productivity of E. coli K5 has achieved levels of industrial applications, whereas E.coli K4 produces a relatively lower amount of fructosylated chondroitin. In this study, the K5 strain was gene-engineered to co-express K4-derived, chondroitin-synthetic genes, namely kfoA and kfoC. The productivities of total GAG and chondroitin in batch culture were 1.2 g/L and 1.0 g/L respectively, which were comparable to the productivity of N-acetylheparosan in the wild K5 strain (0.6-1.2 g/L). The total GAG of the recombinant K5 was partially purified by DEAE-cellulose chromatography and was subjected to degradation tests with specific GAG-degrading enzymes combined with HPLC and 1H NMR analyses. The results indicated that the recombinant K5 simultaneously produced both 100-kDa chondroitin and 45-kDa N-acetylheparosan at a weight ratio of approximately 4:1. The content of chondroitin in total GAG partially purified was 73.2%. The molecular weight of recombinant chondroitin (100 kDa) was 5-10 times higher than that of commercially available chondroitin sulfate. These results indicated that the recombinant K5 strain acquired the chondroitin-producing ability without altering the total GAG productivity of the host.

SUBMITTER: Suzuki K

PROVIDER: S-EPMC10121815 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Simultaneous production of N-acetylheparosan and recombinant chondroitin using gene-engineered Escherichia coli K5.

Suzuki Kiyoshi K Kaseyama-Takemoto Hiromi H

Heliyon 20230324 4

N-Acetylheparosan and chondroitin are increasingly needed as alternative sources of animal-derived sulfated glycosaminoglycans (GAGs) and as inert substances in medical devices and pharmaceuticals. The N-acetylheparosan productivity of E. coli K5 has achieved levels of industrial applications, whereas E.coli K4 produces a relatively lower amount of fructosylated chondroitin. In this study, the K5 strain was gene-engineered to co-express K4-derived, chondroitin-synthetic gene ...[more]

PMID: 37095938

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Project description:BackgroundThe biosynthesis of human milk oligosaccharides (HMOs) using several microbial systems has garnered considerable interest for their value in pharmaceutics and food industries. 2'-Fucosyllactose (2'-FL), the most abundant oligosaccharide in HMOs, is usually produced using chemical synthesis with a complex and toxic process. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce 2'-FL, but the low stoichiometric yields (2'-FL/glucose or glycerol) are still far from meeting the requirements of industrial production. The sufficient carbon flux for 2'-FL biosynthesis is a major challenge. As such, it is of great significance for the construction of recombinant strains with a high stoichiometric yield.ResultsIn the present study, we designed a 2'-FL biosynthesis pathway from fructose with a theoretical stoichiometric yield of 0.5 mol 2'-FL/mol fructose. The biosynthesis of 2'-FL involves five key enzymes: phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-D-mannose 4,6-dehydratase (Gmd), and GDP-L-fucose synthase (WcaG), and α-1,2-fucosyltransferase (FucT). Based on starting strain SG104, we constructed a series of metabolically engineered E. coli strains by deleting the key genes pfkA, pfkB and pgi, and replacing the original promoter of lacY. The co-expression systems for ManB, ManC, Gmd, WcaG, and FucT were optimized, and nine FucT enzymes were screened to improve the stoichiometric yields of 2'-FL. Furthermore, the gene gapA was regulated to further enhance 2'-FL production, and the highest stoichiometric yield (0.498 mol 2'-FL/mol fructose) was achieved by using recombinant strain RFL38 (SG104ΔpfkAΔpfkBΔpgi119-lacYΔwcaF::119-gmd-wcaG-manC-manB, 119-AGGAGGAGG-gapA, harboring plasmid P30). In the scaled-up reaction, 41.6 g/L (85.2 mM) 2'-FL was produced by a fed-batch bioconversion, corresponding to a stoichiometric yield of 0.482 mol 2'-FL/mol fructose and 0.986 mol 2'-FL/mol lactose.ConclusionsThe biosynthesis of 2'-FL using recombinant E. coli from fructose was optimized by metabolic engineering strategies. This is the first time to realize the biological production of 2'-FL production from fructose with high stoichiometric yields. This study also provides an important reference to obtain a suitable distribution of carbon flux between 2'-FL synthesis and glycolysis.

Project description:BackgroundVanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details.ResultsEffect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 micromoles/g x min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass.ConclusionFerulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions.

Dataset Information

Simultaneous production of N-acetylheparosan and recombinant chondroitin using gene-engineered Escherichia coli K5.

Publications

Simultaneous production of <i>N</i>-acetylheparosan and recombinant chondroitin using gene-engineered <i>Escherichia coli</i> K5.

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