Biosynthesis of poly(3-hydroxybutyrateco-3-hydroxy-4-methylvalerate) by Strain Azotobacter chroococcum 7B

Abstract

Production of novel polyhydroxyalkanoates (PHAs), biodegradable polymers for biomedical applications, and biomaterials based on them is a promising trend in modern bioengineering. We studied the ability of an effective strain-producer Azotobacter chroococcum 7B to synthesize not only poly(3-hydroxybutyrate) homopolymer (PHB) and its main copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), but also a novel copolymer, poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB4MV). For the biosynthesis of PHB copolymers, we used carboxylic acids as additional carbon sources and monomer precursors in the chain of synthesized copolymers. The main parameters of these polymers' biosynthesis were determined: strain-producer biomass yield, polymer yield, molecular weight and monomer composition of the synthesized polymers, as well as the morphology of A. chroococcum 7B bacterial cells. The physico-chemical properties of the polymers were studied using nuclear magnetic resonance spectroscopy (NMR), differential scanning calorimetry (DSC), contact angle test, and other methods. In vitro biocompatibility of the obtained polymers was investigated using stromal cells isolated from the bone marrow of rats with the XTT cell viability test. The synthesis of the novel copolymer PHB4MV and its chemical composition were demonstrated by NMR spectroscopy: the addition of 4-methylvaleric acid to the culture medium resulted in incorporation of 3-hydroxy-4-methylvalerate (3H4MV) monomers into the PHB polymer chain (0.6 mol%). Despite the low molar content of 3H4MV in the obtained copolymer, its physico-chemical properties were significantly different from those of the PHB homopolymer: it has lower crystallinity and a higher contact angle, i.e. the physico-chemical properties of the PHB4MV copolymer containing only 0.6 mol% of 3H4MV corresponded to a PHBV copolymer with a molar content ranging from 2.5% to 7.8%. In vitro biocompatibility of the obtained PHB4MV copolymer, measured in the XTT test, was not statistically different from the cell growth of PHB and PHBV polymers, which make its use possible in biomedical research and development.

Keywords: Azotobacter chroococcum 7B; biocompatibility; biosynthesis; bone marrow stromal cells; crystallinity; poly(3-hydroxybutyrate); poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate).

Fig. 1

Scheme of biosynthesis of PHB and its copolymers by A. chroococcum 7B. 1 – pyruvate dehydrogenase complex; 2 – β-ketothiolase; 3 – NADPH-dependent acetoacetyl-CoA reductase; 4 – short chain carboxylic acids PHA-polymerase; 5 – acyl-CoA synthase; 6 – acyl-CoA dehydrogenase; 7 – enoyl-CoA hydratase; 8 – NADH-dependent acetoacetyl-CoA reductase. Abbreviations: 4M – 4-methyl-; 2M – 2-methyl-; 3HB – 3-hydroxybutyrate-; 3HV – 3-hydroxyvalerate; 3H4MV – 3-hydroxy-4-methylvalerate; Poly-3HB-3H4MV – poly(3-hydroxybutyrate-co-3-hydroxy- 4-methylvalerate); Poly-3HB-3HV – poly(3-hydroxybutyrate-co-3-hydroxyvalerate); poly-3HB – poly(3-hydroxybutyrate).

Fig. 2

¹H 500 MHz NMR spectrum of PHB4MV copolymer. A – PHB polymer chain: a – CH₃ (s), b – CH (b), c – CH₂ (b), poly(3-hydroxy-4-methylvalerate) polymer chain: d – CH₂ (s), e – CH₃ (s), f – CH (b), g – CH₂ (b), 1 – side groups, 2 – polymer backbone; *an enlarged section of the graph is shown in the inset (B)

Fig. 3

The effect of adding carboxylic acids to the culture medium on the morphology of strain-producer A. chroococcum cells (light microscopy, ×900). A – S + 5 mM VA (added after 12 hours), after 72 hours of culturing; B – S + 20 mM VA (added t 0 h) after 72 h of culturing; C – S + 20 mM HxA (added after 12 hours) after 72 hours of culturing; D – S + 20 mM 4MVA (added after 12 hours), after 72 hours of culturing

Fig. 4

DSC thermograms of PHB4MV obtained by biosynthesis by A. chroococcum 7B: 1 – curve of the first heating cycle; 2 – curve of the first cooling cycle; 3 – curve of the second heating cycle; 4 – curve of the second cooling cycle; areas of the melting and crystallization peaks are shaded, respectively

Fig. 5

Changes in the number of viable bone marrow stromal cells of rats cultured on PHB, PHBV1 and PHB4MV polymer films according to the XTT test. * P < 0.05 when compared to PHB group, n = 6