Methane or methanol-oxidation dependent synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by obligate type II methanotrophs

• Pure cultures of Type II methanotrophs enabled PHBV synthesis.
• Cells incubated with both C1 substrates and odd-carbon VFAs produced PHBV.
• In the absence of C1 substrates, neither P3HB nor PHBV was synthesized.
• Oxidation of C1 substrates generates the energy required for 3HV incorporation.
• Incorporation of 13C-labeled valerate was confirmed using NMR.

Obligate methanotrophs capable of producing polyhydroxyalkanoates (PHAs), a sustainable alternative to non-biodegradable petroleum-based plastics, under nutrient-limited conditions are currently limited to synthesis of poly(3-hydroxybutyrate) (P3HB). Diversifying monomers would greatly expand the range of applications. Here we report the first pure culture evidence of methanotrophic synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). When grown with methane as sole substrate, the Type II obligate methanotroph Methylocystis parvus OBBP produces P3HB, but not PHBV, under nutrient-limiting conditions. We observed synthesis of PHBV when strain OBBP was incubated with C1 substrates (methane, methanol, or formate) and propionate or valerate. PHBV production was confirmed by gas chromatography, gel permeation chromatography, differential scanning calorimetry, and nuclear magnetic resonance with [1-13C]valerate. In the absence of C1-substrate oxidation, neither P3HB nor PHBV was synthesized. The mol% 3-hydroxyvalerate depended upon the amount of propionate or valerate consumed, and ranged from 0 to 40 mol%. Valerate addition resulted in a higher mol% 3-hydroxyvalerate and higher wt% PHA than propionate addition. To assess the generality of this capability among Type II obligate methanotrophs, we added methane and valerate to Methylosinus trichosporium OB3b, with similar results. We hypothesize that oxidation of C1 substrates generates the energy required for activation of co-substrates and incorporation of 3-hydroxyvalerate.

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