Abstract
Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by β-1,4 and β-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors’ ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.
Highlights
Microorganisms have been widely used to obtain pharmaceuticals, biopolymers, vaccines, enzymes, and various other chemicals [1]
O. polymorpha was engineered for hyaluronic acid (HA) production
A genetic switch using a serine-type integrase-13 to control the expression of both hasAp and hasB was demonstrated
Summary
Microorganisms have been widely used to obtain pharmaceuticals, biopolymers, vaccines, enzymes, and various other chemicals [1]. Through metabolic engineering and synthetic biology approaches, it is possible to generate recombinant strains to produce desired compounds and bypass the chemical synthesis [2]. One of the value-added products that can be obtained through microorganism-based processes is hyaluronic acid (HA). This biopolymer features high viscosity and elasticity and is abundant in the extracellular matrix of vertebrates’ connective tissues. Due to its high biocompatibility, HA has various applications in the medical, cosmetic, and pharmaceutical areas (see details in [3]). According to Grand View Research Inc., the global HA market may reach USD 16.6 billion by
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