Engineering of chimeric class II polyhydroxyalkanoate synthases

Abstract

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

FIG. 1.

(A) Construction of plasmids used in this study. (B) PCR detection and amplification of putative phaC gene fragments from soil DNA extracts using a set of generic primers. Lane 1, no DNA; lanes 2 to 6, PCR products from soil DNA extracts 1 (S1), S2, S3, S4, and S5, respectively; lane 7, phaC amplified from B. megaterium cell lysate as a control. (C) Fluorescence measurements of Nile blue A dye screening assay for functional chimeric PHA synthases; shown are active chimeric S3-44 and S3-69, wild-type phaC1_Po (W1), deleted phaC1_PoI225L-linker (D1), and one point mutation I225LphaC1_Po (W2).

FIG. 2.

Amino acid analyses of the wild-type PhaC1_Po, all five active chimeric synthases, and the wild-type PhaC_Re. Amino acid differences are shaded in yellow. The solid red line indicates conserved amino acid residues found in medium-chain-length PHA synthases. *, amino acids that are highly conserved among all PHA synthases.

FIG. 3.

Phylogenetic tree analysis of different phaC fragments recovered from soils, which contribute to active (P1 to -5) and inactive (N1 to -14) chimeric PHA synthase genes. The analysis was done based on a comparison of amino acid sequence data using the DNAStar MegAlign program.

FIG. 4.

Alignment of the P. oleovorans PHA synthase 1 (PhaC1_Po) with the B. glumae lipase (1THA_A). The PhaC1_Po sequence was threaded onto the bacterial lipase structures using SWISS-MODEL, and the alignment was performed using MegAlign. Identical residues in the sequences are shown as boldface letters, and conservative replacements are shown as light-gray letters. The open arrowheads indicate the catalytic cysteine of PhaC1_Po, the catalytic serine of the lipase, the conserved H479 of PhaC1_Po, the conserved H285 of the lipase, and the conserved D451 of PhaC1_Po. The lipase catalytic region is boxed. The two unique restriction sites (MfeI and MluI) are shown. The arrows indicate two highly conserved regions found in all PHA synthases that were used to design primer For-MfeI-general phaC and primer Rev-MluI-general phaC. The asterisks indicate residues where amino acid substitutions occurred in all five active chimeras.

FIG. 5.

Threading model of PHA synthase 1 from P. oleovorans. (A) Localization of all point mutations inherent in the construction of hybrid PhaC1_Po. The genetically engineered internal 540-bp region is represented by blue color (I225 to R403). Inherent mutations are indicated by arrows. Catalytic residues are shown as stick side chains and indicated by red letters. The upper model represents one set of mutations. The lower model represents a second set of mutations with the PhaC1_Po model rotated by 180°. (B) Threading model of localization of amino acid residue substitutions of all five PhaC1_Po chimeras. Substituted amino acids are located in the colored regions. Blue indicates the amino acid substitutions found in all five active chimeras. Green indicates the amino acid substitutions found in some of active chimeras. Pink indicates the amino acid substitutions found only in the chimeric S3-44.