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Abstract
Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps) synthesizes PHA copolymer containing 3-hydroxybutyrate (3HB) and a small amount (0.5 mol%) of 3-hydroxy-4-methylvalerate (3H4MV) from fructose as a carbon source. In this study, enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural and self-producible precursor. We found that the incorporation of 3H4MV was enhanced by the supplementation of excess amount (10 g/L) of leucine in the culture medium. This finding indicates that 3H4MV can be derived from leucine. To increase metabolic flux to leucine biosynthesis in the host strain by eliminating the feedback inhibition, the cells were subjected to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and leucine analog resistant mutants were generated. The mutants showed statistically higher 3H4MV fraction than the parent strain without supplementing leucine. Additionally, by supplying excess amount of leucine, the mutants synthesized 3HB-based PHA copolymer containing 3.1 mol% 3H4MV and 1.2 mol% 3-hydroxyvalerate (3HV) as minor constituents, which significantly affected the thermal properties of the copolymer. This study demonstrates that it is possible to enhance the monomer supply of 3H4MV into PHA by manipulating leucine metabolism.
Highlights
Polyhydroxyalkanoate (PHA) is a kind of aliphatic polyester synthesized by a wide variety of microorganisms as intracellular storage and carbon source (Sudesh et al 2000)
Effect of Amino Acid Supplementation on 3H4MV Fraction Because the carbon back bone of 3H4MV is the same as that of branched amino acid leucine (Figure 1), we expected that leucine and its structurally related amino acids could function as 3H4MV precursors
To evaluate the feasibility of 3H4MV provision from amino acids, R. eutropha PHB-4 expressing phaC1Ps was cultivated in mineral salt (MS) plus fructose medium supplemented with 10 g/L of various amino acids
Summary
Polyhydroxyalkanoate (PHA) is a kind of aliphatic polyester synthesized by a wide variety of microorganisms as intracellular storage and carbon source (Sudesh et al 2000). It can be biosynthesized from renewable carbon sources such as sugars and plant oils, and can be completely biodegraded in the environment. P (3HB) is a brittle and rigid material with low flexibility depends on the substrate specificity of the polymerizing enzyme, PHA synthase (PhaC). Since the other PhaCs from pseudomonads are unable to polymerize 3HB unit, PhaC1Ps has been useful for the synthesis of 3HBbased PHA copolymer incorporating various types of 3HA
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