Dataset Information

Metabolic Engineering of Escherichia coli for the Production of Hyaluronic Acid From Glucose and Galactose.

ABSTRACT: Hyaluronic acid is a glycosaminoglycan biopolymer widely present throughout connective and epithelial tissue, and has been of great interest for medical and cosmetic applications. In the microbial production of hyaluronic acid, it has not been established to utilize galactose enabling to be converted to UDP-glucuronic acid, which is a precursor for hyaluronic acid biosynthesis. In this study, we engineered Escherichia coli to produce hyaluronic acid from glucose and galactose. The galactose-utilizing Leloir pathway was activated by knocking out the galR and galS genes encoding the transcriptional repressors. Also, the hasA gene from Streptococcus zooepidemicus was introduced for the expression of hyaluronic acid synthase. The consumption rates of glucose and galactose were modulated by knockout of the pfkA and zwf genes, which encode 6-phosphofructokinase I and glucose-6-phosphate dehydrogenase, respectively. Furthermore, the precursor biosynthesis pathway for hyaluronic acid production was manipulated by separately overexpressing the gene clusters galU-ugd and glmS-glmM-glmU, which enable the production of UDP-glucuronic acid and UDP-N-acetyl-glucosamine, respectively. Batch culture of the final engineered strain produced 29.98 mg/L of hyaluronic acid from glucose and galactose. As a proof of concept, this study demonstrated the production of hyaluronic acid from glucose and galactose in the engineered E. coli.

SUBMITTER: Woo JE

PROVIDER: S-EPMC6881274 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Metabolic Engineering of <i>Escherichia coli</i> for the Production of Hyaluronic Acid From Glucose and Galactose.

Woo Ji Eun JE Seong Hyeon Jeong HJ Lee Sang Yup SY Jang Yu-Sin YS

Frontiers in bioengineering and biotechnology 20191121

Hyaluronic acid is a glycosaminoglycan biopolymer widely present throughout connective and epithelial tissue, and has been of great interest for medical and cosmetic applications. In the microbial production of hyaluronic acid, it has not been established to utilize galactose enabling to be converted to UDP-glucuronic acid, which is a precursor for hyaluronic acid biosynthesis. In this study, we engineered <i>Escherichia coli</i> to produce hyaluronic acid from glucose and galactose. The galacto ...[more]

PMID: 31824939

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Project description:Hydroxy fatty acids (HFAs) are valuable chemicals for a broad variety of applications. However, commercial production of HFAs has not been established so far due to the lack of low cost routes for their synthesis. Although the microbial transformation pathway of HFAs was extensively studied decades ago, these attempts mainly focused on converting fatty acids or vegetable oils to their hydroxyl counterparts. The use of a wider range of feedstocks to produce HFAs would reduce the dependence on oil crops and be expected to cut down the manufacturing cost.In this study, the industrially important microorganism Escherichia coli was engineered to produce HFAs directly from glucose. Through the coexpression of the acetyl-CoA carboxylase (ACCase) and the leadless acyl-CoA thioesterase ('TesA), and knockout of the endogenous acyl-CoA synthetase (FadD), an engineered E. coli strain was constructed to efficiently synthesize free fatty acids (FFAs). Under shake-flask conditions, 244.8 mg/L of FFAs were obtained by a 12 h induced culture. Then the fatty acid hydroxylase (CYP102A1) from Bacillus megaterium was introduced into this strain and high-level production of HFAs was achieved. The finally engineered strain BL21?fadD/pE-A1'tesA&pA-acc accumulated up to 58.7 mg/L of HFAs in the culture broth. About 24 % of the FFAs generated by the thioesterase were converted to HFAs. Fatty acid composition analysis showed that the HFAs mainly consisted of 9-hydroxydecanoic acid (9-OH-C10), 11-hydroxydodecanoic acid (11-OH-C12), 10-hydroxyhexadecanoic acid (10-OH-C16) and 12-hydroxyoctadecanoic acid (12-OH-C18). Fed-batch fermentation of this strain further increased the final titer of HFAs to 548 mg/L.A robust HFA-producing strain was successfully constructed using glucose as the feedstock, which demonstrated a novel strategy for bioproduction of HFAs. The results of this work suggest that metabolically engineered E. coli has the potential to be a microbial cell factory for large-scale production of HFAs.

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.

Dataset Information

Metabolic Engineering of Escherichia coli for the Production of Hyaluronic Acid From Glucose and Galactose.

Publications

Metabolic Engineering of <i>Escherichia coli</i> for the Production of Hyaluronic Acid From Glucose and Galactose.

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