1. Introduction
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 2027 [
4].
HA is a glycosaminoglycan formed by disaccharide repeats of UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) linked by β-1,4 and β-1,3 glycosidic bonds [
5]. Its synthesis is catalyzed by the enzyme hyaluronan synthase (HAS), encoded by the gene
hasA, and responsible for assembling the two precursors in the cytosol, elongating the polymer chain and releasing it into the extracellular matrix. Gram-positive bacteria and yeasts are the most used microorganisms for producing heterologous HA, especially those with Generally Recognized as Safe (GRAS) status. Many such strains have been developed in recent years, using different approaches to improve HA titers [
6].
Bacillus subtilis [
7,
8],
Corynebacterium glutamicum [
9], and
Lactococcus lactis [
10] are the main bacteria used for the production of this polymer. Bacteria possess the complete pathway for the synthesis of both precursors, whereas yeasts are only naturally capable of synthesizing UDP-GlcNAc. Thus, in addition to
hasA, HA production in yeast cells requires the expression of the
hasB gene, which encodes the UDP-glucose 6-dehydrogenase needed for UDP-GlcUA synthesis (
Figure 1). To the best of our knowledge, only two yeasts have been engineered for HA production so far:
Kluyveromyces lactis [
11] and
Komagataella phaffii (previously known as
Pichia pastoris) [
12]. Here, we describe the heterologous production of HA by
Ogataea (
Hansenula)
polymorpha.
In terms of available genetic tools, a set of endogenous promoters was previously described for
O. polymorpha with different regulatory mechanisms that allow for developing strategies to regulate gene expression [
16]. As a methylotrophic yeast, many of these characterized promoters are related to methanol metabolism, controlling the expression of genes encoding enzymes required to metabolize methanol. Additionally, promoters regulating genes related to nitrate metabolism and inducible by this nitrogen source (
YNR1 [
17] and
YNI1 [
18]) as well as related to temperature increase (
TPS1 and
HSA1 [
19]) were described in
O. polymorpha. Although a set of promoters is available for this non-conventional yeast, the methanol-inducible one is preferred to control heterologous gene expression. Cultivation strategies using these promoters usually are based on two phases, although other approaches based on derepression of these promoters were also reported [
20]. The first step is the growth phase, which focuses on biomass production using glucose or glycerol, followed by a methanol induction phase to produce the heterologous protein. Therefore, the utilization of methanol-inducible promoters allows developing strategies that decouple growth and product synthesis, because in the absence of methanol no significant levels of heterologous protein are achieved. Such decoupled strategies are advantageous for synthesis of compounds which metabolic pathways compete with biomass production, such as HA.
In
O. polymorpha, the promoter which controls the methanol oxidase gene (pMOX, also referred as pAOX, since the methanol oxidase is the primary alcohol oxidase described in this organism [
17]) is preferred for heterologous gene expression [
21], and its regulatory mechanisms are widely studied [
22]. Although it is mainly regulated by methanol and repressed by glucose, it is active under limiting-glucose conditions [
23], leading to promoter leakage, which might affect strategies that require gene regulation. Additionally, pMOX is derepressed in the presence of other carbon sources such as glycerol, xylose, ribose, and sorbitol [
24]. Other promoters upregulated by methanol and repressed by glucose include pDHAS (dihydroxyacetone synthase) and pFMD (formaldehyde dehydrogenase) [
25]. The latter is an alternative to pMOX since it is considered a strong promoter in the presence of methanol. However, a high level of enzyme production (13.5 g/L) has been achieved using glucose as the carbon source [
26]. For HA production, the utilization of methanol-inducible promoters is a feasible strategy to avoid competition between its synthesis and biomass production, which is the main limitation for the heterologous production of HA (
Figure 1) [
27,
28].
Although methanol-inducible promoters are widely used in heterologous protein expression by
O. polymorpha, their use may be discouraged due to its leakage under some carbon sources and methanol toxicity and flammability, especially at an industrial scale. Thus, promoters regulated by other inducers or constitutive promoters are available alternatives. The strong constitutive pGAP promoter is commonly used for gene regulation in
O. polymorpha [
29]. Other endogenous constitutive promoters already described for this yeast include pTEF1 [
13] and pADH1 [
30]. However, this type of promoter does not allow one to tune gene regulation, and the heterologous protein is produced similarly regardless of cultivation conditions. Therefore, an alternative genetic tool must be considered for tunning gene regulation. For example, the utilization of genetic switches such as serine integrases enables the building of genetic circuits through targeted DNA rearrangement. If two recognizing sites (
attB and
attP) are inserted flanking the desired DNA sequence, the integrase is capable of identifying these sites and flip the sequence at 180° (see details in [
31]). Thus, this strategy works as a genetic tool for gene regulation once any genetic element (promoter, coding sequence, and/or terminator) can be constructed flanked by these
att sites. The rotation of the desired DNA sequence can occur in a specific condition. Recently, a system using the sites
attB and
attP and different serine integrases was applied to build a genetic switch in other eukaryotic cells [
32].
In this work, the genetic modifications necessary for the heterologous production of HA using
O. polymorpha as a chassis organism are described (
Figure 1). For this, the
hasA and
hasB genes were integrated into the
O. polymorpha genome under different promoters’ regulation. Various combinations were tested to evaluate their influence on HA titers. Two
hasA genes were used, referred to as
hasAs (from
Streptococcus zooepidemicus) and
hasAp (from
Pasteurella multocida). The
hasAp gene was evaluated because, as with the
hasB (from
Xenopus laevis) used, we previously demonstrated that the enzymes encoded by these genes were active in
K. lactis, another non-conventional yeast [
11]. The
hasAs was selected since it is widely used for heterologous production of HA [
7,
33,
34].
Moreover, to better control HA production, we have also employed a genetic switch using a serine integrase to control both
hasAp and
hasB gene expression. The integrase-13 (Int13) was selected due to its versatility, successfully applied to design genetic switches in human, bovine, and plant cells [
32]. Besides, no point mutations or changes in cell viability caused by the Int13 were detected in cells analyzed in the previous study. Thus, we evaluated a genetic switch using the Int13 as a proof-of-concept in
O. polymorpha to control the expression of both
hasB and
hasAp genes aiming at HA production by this yeast. A capsule-like layer could be seen around cells of the strain containing the genetic switch controlling
hasB and
hasAp expression named here as EMB103, using a scanning electron microscopy analysis. In the other strains expressing only
hasB and
hasA, the cell surface was similar to the wild-type strain. However, after 48 h of cultivation in an optimized medium, HA was quantified from the culture broth of all three constructed strains tested. This is the first report of an
O. polymorpha strain developed for HA production to the best of our knowledge.