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 13 C nuclear magnetic resonance spectrum.

Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

“…One microgram of EcoRI-digested genomic DNA was ligated with 50 ng of EcoRI and alkaline phosphatase (New England BioLabs)-treated pBluescript II KS by T4 DNA ligase (Fermentas) at 8°C. The ligation product was transformed into E. coli XL1-Blue cells by electroporation as described previously (33). Transformants were spread onto an LB agar plate containing 1.5% glucose and 100 g/ml ampicillin and incubated at 37°C for 24 to 48 h. Colonies with PHA accumulation were screened based on an opaque colony appearance.…”

“…The lyophilized cells were subjected to methylation. The PHA content was determined by gas chromatography as described in a previous report (33).…”

“…Nonrational design methods, i.e., in vitro and in vivo evolution approaches, are commonly applied to many PHA synthases, including those of C. necator H16 (41), Aeromonas caviae (1,18), Pseudomonas sp. strain 61-3 (35,36), and Pseudomonas putida GPo1 (33). The above-mentioned studies succeeded in obtaining point mutations which improve the performance of PHA synthase by changing the substrate specificity or enhancing the enzyme activity.…”

Biodegradable Polymers: Recent Developments and New Perspectives

“…Furthermore, the useful evolvants obtained through systematic enzyme evolution have been supplied to other organisms, including plants [69,70]. The impacts of these reports inspired additional research groups to apply directed evolution to individual PHA synthases of interests [71][72][73]. …”

Isolamento de bactéria produtoras de polihidroxialcanoatos de cadeia curta e média a partir de óleos vegetais

“…Por outro lado, PHA sintases do tipo II que apresentam maior especificidade por HA MCL , são capazes de incorporar pequenas frações de 3HB ao PHA. Neste último caso, diversos trabalhos têm demonstrado a possibilidade de aumentar expressivamente a fração de 3HB desde que vias efetivas de suprimento desses monômeros estejam disponíveis (ABE et al, 1994;KATO et al, 1996a;KATO et al, 1996b;LEE et al, 1995;SCHROLL et al, 1996;CHEN et al, 2006;SHEU et al, 2004) …”