Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Highlights

• Halomonas bluephagenesis was engineered to express PhaCJ4AK4 for the production of scl-co-mcl PHA.

• It produces 3-hydroxybutyrate copolymers containing functional 3-hydroxy-5-hexenoate (3HHxE) monomer.

• The functional 3HHxE can be finely tuned and achieved a remarkably high of 35 mol% in its copolymers.

• The 5-Hexenoic acid conversion efficiency reached up to 91% in a 48-h of open and unsterile fermentation.

• The resulted PHA containing 12.5 mol% 3HHxE exhibits more than 1000% elongation at break.

Abstract

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.

Introduction

Polyhydroxyalkanoates (PHA) are a class of diverse polyesters with biodegradability and biocompatibility (Anderson and Dawes, 1990; Blank et al., 2020; Lenz and Marchessault, 2005; Li et al., 2016b; López et al., 2015). Various microorganisms can produce PHA as intracellular carbon and energy storage compounds from multiple sources (Dodds and Gross, 2007; Gross et al., 1989; Koller et al., 2005; Lu et al., 2009; Nikel et al., 2008). As one of the most promising bioplastics, PHA have been developed into many types of products aiming for various usages (Chen and Patel, 2012; Gao et al., 2011; Gross and Kalra, 2002; Wierckx et al., 2015; Zinn et al., 2001). Commercially available PHA include poly(3-hydroxybutyrate) (PHB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) (Koller et al., 2017; Sudesh et al., 2000; Tsuge, 2002). All of them contain saturated side chains largely restricting the possibility of functional modification for more new properties and applications (Hazer and Steinbuchel, 2007; Radivojevic et al., 2016; Tortajada et al., 2013).

Some microbial cultures can be fed with various functionalized substrates to produce PHA containing unsaturated bonds, carbonyl, epoxy, halogens, hydroxyl and/or phenyl groups (Arkin et al., 2000; Hartmann et al., 2004; Kai and Loh, 2014). Among them, the unsaturated bonds in PHA can be chemically modified using small molecules, grafting with responsive groups via thiol-ene click reaction, atom transfer radical polymerization (ATRP), or reversible addition fragmentation chain transfer polymerization (RAFT) (Arslan et al., 2010; Hazer, 2015; Ma et al., 2016). The resulting functional PHA generate new properties including thermal-sensitivity, amphiphilicity or protein absorption (Babinot et al., 2012; Ma et al., 2016; Yao et al., 2016).

Unsaturated PHA such as poly(3-hydroxy-10-undecenoate) is a medium-chain-length (mcl) PHA using 9-decenol or 10-undecenoic acid as substrates (Follonier et al., 2015; Lageveen et al., 1988; Sun et al., 2009). The fatty acid substrates were degraded via the β-oxidation pathway to remove two carbon atoms in each cycle, leading to shortened 3HA monomers in the resultant PHA (Escapa et al., 2012; Sudesh et al., 2000). It is, however, difficult to control ratios of mcl monomers include 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD), leading to inconsistent physical properties of the PHA. (Green et al., 2002; Taguchi et al., 1999). The mcl-PHA are usually soft, flexible due to their amorphous properties, resulting in the low melting temperature and weak processing properties (Gumel et al., 2012; Koller et al., 2008; Sudesh et al., 2000). PHA with short-chain-length (scl) monomers of 3–5 carbon atoms are basically stiff, brittle and crystalline. The combination of scl- and mcl-monomers in PHA forms significantly improved their physical properties (Phithakrotchanakoon et al., 2013; Witholt and Kessler, 1999).

One promising candidate of scl-co-mcl PHA copolymer is P(3HB-co-3HHx) or PHBHHx, which is synthesized by a PHA synthase with specific affinity for monomers of 4–6 carbon atoms (Arikawa and Matsumoto, 2016; Fukui and Doi, 1997; Park et al., 2001; Tsuge et al., 2004). PHBHHx possesses biocompatibility, biodegradability and reasonable mechanical strength depending on the molar ratio of 3HHx monomers in the PHA copolymer (Chen and Wu, 2005; Sato et al., 2004), which can be turned into nanoparticles or tissue engineering scaffolds for medical fields (Chen and Wu, 2005; Wu et al., 2013). Hence, if PHBHHx can integrate the advantages of unsaturated PHA, it could become a more valuable biomaterial.

Some strains especially Cupriavidus necator or Aeromonas hydrophila have been employed to produce PHBHHx, but they frequently suffer microbial contamination during the growth (Arikawa and Matsumoto, 2016; Li et al., 2014). The next generation industrial biotechnology (NGIB) based on an extremophile Halomonas bluephagenesis (Chen and Jiang, 2018; Tan et al., 2011), allows PHA production to be conducted under open, unsterile and continuous fermentation conditions (Fu et al., 2014; Tan et al., 2014; Yin et al., 2015). The wild type H. bluephagenesis is able to accumulate PHB up to 80% of cell dry mass (CDM) (Tan et al., 2011). Moreover, H. bluephagenesis has also been successfully engineered to produce P3HB4HB on a large scale of 5000 L (Ye et al., 2018). Additionally, to widen the potential of H. bluephagenesis as a production chassis, CRISPR/cas9 has been developed for further metabolic engineering. (Calero and Nikel, 2019; Qin et al., 2018).

This study aimed to engineer H. bluephagenesis for the production of PHBHHx and its functional forms via synthetic biology and metabolic engineering methods. The produced materials were characterized in detail.