Heterologous Production of Hyaluronic Acid in an ε-Poly- l -Lysine Producer, Streptomyces albulus

Abstract

bHyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an -poly-Llysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.H yaluronic acid (HA) is a linear, uniformly repetitive glycosaminoglycan composed of up to 20,000 disaccharide units of D-glucuronic acid (GluUA) and N-acetylglucosamine (GlcNAc) (1) and ubiquitously exists as hydrogel in vertebrate tissues. HA is particularly abundant in cartilage, synovial fluid, dermis, and the vitreous humor of the eye. In these tissues, the polysaccharide has significant structural, physiological, and biological functions due to its viscoelastic property and ability to retain a large volume of water (2). These distinctive properties have been exploited in a wide range of medical applications, including in the areas of ophthalmology, orthopedics, and wound healing (3), where the performance and therapeutic efficacy of HA are highly dependent on its molecular weight. Product quality is also considered a key factor for these biomedical applications in order to avoid serious allergic and inflammatory reactions caused by contaminants, especially upon injection (4, 5). Thus, to meet the growing demand for HA in the field of biomedicine (6), it is critical to produce high-molecular-weight HA from a safe source that does not carry those concerns.Traditionally, HA had been obtained commercially from rooster corms, but in recent years it has mainly been produced through the fermentation process of an attenuated pathogenic strain of group C streptococci that naturally synthesize the identical macromolecule as a part of the extracellular capsule in order to conceal it from the immune system of...

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

“…Similarly, purified SeHAS, reconstituted into Escherichia-coli-derived or synthetic proteoliposomes, synthesized and translocated high MW HA into the vesicle lumen, requiring only exogenous UDP-GlcUA and UDP-GlcNAc (Hubbard, McNamara, Azumaya, Patel, & Zimmer, 2012). These observations, along with the demonstration of HA production in a variety of bacteria heterologously expressing HAS (Chien & Lee, 2007b;Mao, Shin, & Chen, 2009;Widner et al, 2005;Yoshimura, Shibata, Hamano, & Yamanaka, 2015), indicate that a single HAS protein produces HA autonomously via a process requiring as many as seven different functions to initiate and elongate the HA chain, and extrude it across the membrane (Weigel, 2002).…”

“…This is not entirely surprising given that a cls (encoding CL synthase) deficient S. pyogenes mutant retained the full ability to produce the HA capsule, relative to the parent strain which constitutively produced HA due to a mutation of the transcriptional regulator CovR (Rosch et al, 2007). Similarly, heterologous HA production was achieved in a variety of bacterial hosts expressing Class I HAS from group C streptococci without alterations to the host membrane (Chien & Lee, 2007b;Yoshimura et al, 2015;Yu & Stephanopoulos, 2008). Similarly, heterologous HA production was achieved in a variety of bacterial hosts expressing Class I HAS from group C streptococci without alterations to the host membrane (Chien & Lee, 2007b;Yoshimura et al, 2015;Yu & Stephanopoulos, 2008).…”

“…However, recent work showed that HAS derived from S. equisimilis (SeHAS; encoded by seHas) could mediate the release of the small fluorescent dye Cascade Blue from inside liposomes, and that mutations, which decreased HAS activity and HA MW in an earlier study, in one of its transmembrane domains resulted in reduced Cascade Blue efflux (Kumari, Baggenstoss, Parker, & Weigel, 2006;Medina, Lin, & Weigel, 2012). These observations, along with the demonstration of HA production in a variety of bacteria heterologously expressing HAS (Chien & Lee, 2007b;Mao, Shin, & Chen, 2009;Widner et al, 2005;Yoshimura, Shibata, Hamano, & Yamanaka, 2015), indicate that a single HAS protein produces HA autonomously via a process requiring as many as seven different functions to initiate and elongate the HA chain, and extrude it across the membrane (Weigel, 2002). These observations, along with the demonstration of HA production in a variety of bacteria heterologously expressing HAS (Chien & Lee, 2007b;Mao, Shin, & Chen, 2009;Widner et al, 2005;Yoshimura, Shibata, Hamano, & Yamanaka, 2015), indicate that a single HAS protein produces HA autonomously via a process requiring as many as seven different functions to initiate and elongate the HA chain, and extrude it across the membrane (Weigel, 2002).…”

Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance heterologous production of hyaluronic acid in engineered Bacillus subtilis

“…Accordingly, the enhanced HA production upon supplementation of n-hexadecane may not be associated with the catalytic activities of HAS, but the increased ATP generation resulting from increased oxygen transfer (Yoshimura, Shibata, Hamano, & Yamanaka, 2015). On the other hand, the lower MW of HA produced in cultures containing n-hexadecane suggests premature release of the HA chain.…”

Recent advances in the biotechnological production of microbial poly(ɛ-l-lysine) and understanding of its biosynthetic mechanism

“…Actually, a powerful ATP supply was detected from all the popular ɛ-PL-producing strains investigated. As an extension of those studies, Yoshimura et al took full advantage of this excellent characteristic of ɛ-PL-producing strains and focused on their heterologous hyaluronic acid production, which resulted in the efficient production of hyaluronic acid with high molecular weight (Yoshimura et al 2015). Probably, ɛ-PL-producing strains (of course, the pls should be deleted) could be superior hosts for heterologous production of a wide variety of bioproducts in the future, as their biosynthesis pathways require amounts of ATP in vivo.…”