Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3

Abstract

<i>Pseudomonas</i> sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate-<i>co</i>-3-hydroxyalkanoate) [P(3HB-<i>co</i>-3HA)], consisting of 3HA units of 4-12 carbon atoms. <i>Pseudomonas</i> sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the <i>phb</i> and <i>pha</i> loci, respectively. The P(94 mol% 3HB-<i>co</i>-6 mol% 3HA) copolymer synthesized by the recombinant strain of <i>Pseudomonas</i> sp. 61-3 (<i>phbC</i>::<i>tet</i>) harboring additional copies of <i>phaC1</i> gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB-<i>co</i>-3HA) copolymer using steamed soybean wastewater, a by-product in brewing <i>miso</i>, which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB-<i>co</i>-3HA) by the <i>Pseudomonas</i> sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB-<i>co</i>-3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of <i>Pseudomonas</i> sp. 61-3 (<i>phbC</i>::<i>tet</i>) harboring additional copies of the <i>phaC1</i> gene in the presence of 2% glucose and 10-20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0-1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB-<i>co</i>-3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater.