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.
Highlights
Hyaluronic acid is a viscoelastic and hygroscopic glycosaminoglycan polymer comprising glucuronic acid and N-acetyl glucosamine (Jongsareejit et al, 2007; Yu and Stephanopoulos, 2008) and is found in skin and intercellular space of tissues of human (Kogan et al, 2007; Marcellin et al, 2014)
To test whether hyaluronic acid could be produced from glucose in this manner, we cultured E. coli strain HA01 in LB medium supplemented with 3 g/L glucose (3 g/L galactose was added to keep consistency for all cultures, HA01 strain cannot use galactose under both sugars condition)
We demonstrate the production of hyaluronic acid from the simultaneous consumption of glucose and galactose in cultures of engineered E. coli capable of enhancing the biosynthesis of UDP-glucuronic acid
Summary
Hyaluronic acid is a viscoelastic and hygroscopic glycosaminoglycan polymer comprising glucuronic acid and N-acetyl glucosamine (Jongsareejit et al, 2007; Yu and Stephanopoulos, 2008) and is found in skin and intercellular space of tissues of human (Kogan et al, 2007; Marcellin et al, 2014). Industrial-scale production of hyaluronic acid has been performed using a pathogen Streptococcus zooepidemicus (Kim et al, 1996; Schiraldi et al, 2010), which produces a pathogenic factor together hyaluronic acid that contributes to forming the extracellular capsule (Kim et al, 2019). Because of this risk, there has been increasing interest in engineering of non-pathogenic microorganisms, including genus of Escherichia, Bacillus, Lactococcus, and Agrobacterium, for hyaluronic acid production (Widner et al, 2005; Chien and Lee, 2007; Mao and Chen, 2007; Yu and Stephanopoulos, 2008)
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