Altering the substrate specificity of polyhydroxyalkanoate synthase 1 derived from Pseudomonas putida GPo1 by localized semirandom mutagenesis

Abstract

The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Pp), class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA- as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB(-)4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB(-)4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB(-)4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

FIG. 1.

The outline of predicted positions and amino acid variants introduced to the phaC1_Pp gene. F1, F2, F3, F4, F5, and F6 are the conserved regions among PHA synthases. There are 23 amino acid sites distributed throughout six conserved regions involved in the protein evolution. In the designations, the first letter (left) indicates the residue of the wild-type protein, the number indicates the position of the amino acid residue, and the final letters (right) indicate the possible residues capable of being introduced by degenerate primers.

FIG. 2.

The strategy of constructing and screening the mutant library. (A) The DNA fragments, their corresponding position within the phaC1_Pp gene, and their predicted sizes. A, B, C, D, E, F, and G are the DNA fragments produced by PCR with the corresponding primers EcoRISDF/R6, F1/R5, F2/R4, F3/R3, F4/R2, F5/R1, and F6/23BamHR, respectively. phaC1_Pp, the PHA synthase 1 gene of P. putida GPo1. (B) The agarose gel electrophoresis profile of purified DNA fragments generated from phaC1_Pp by PCR. Lane 1, A fragment (721 bp); lane 2, B fragment (224 bp); lane 3, C fragment (281 bp); lane 4, D fragment (89 bp); lane 5, E fragment (269 bp); lane 6, F fragment (146 bp); lane 7, G fragment (158 bp); lane M, 100 bp DNA ladder (BioLab). (C) Reamplification of reassembly PCR products with primer pair EcoRISDF/23BamHR. Lane M, λ/HindIII DNA marker. The reaction volume is 20 μl. The arrow indicates the predicted whole phaC1_Pp DNA size (∼1.7 kb).

FIG. 3.

The 125-MHz ¹³C-NMR spectrum of the acetone-soluble fraction of polyester yielded by R. eutropha PHB⁻4/pPS-E1 in chloroform. 3HHx, 3-hydroxyhexonoate; 3HO, 3-hydroxyoctanote.