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Critical residues of class II PHA synthase for expanding the substrate specificity and enhancing the biosynthesis of polyhydroxyalkanoate

Paper ID Volume ID Publish Year Pages File Format Full-Text
17031 42633 2014 7 PDF Available
Title
Critical residues of class II PHA synthase for expanding the substrate specificity and enhancing the biosynthesis of polyhydroxyalkanoate
Abstract

•Development of a class II PHA synthase protein model for structure predition.•Identification of beneficial sites Gln481 and Ala547 for enhancing PHA production.•The residue Leu484 changes the substrate specificity of PhaC1Pp from 8C shift to 4C.

This study describes protein model of type II Pseudomonas putida GPo1 synthase (PhaC1Pp) and using single or multiple points mutagenesis to identify the beneficial amino acid residues that change the PHA accumulation and the substrate chain-length specificity of type II PHA synthase. The P. putida GPp104 PHA− was used as a host for evaluating the substrate specificity and PHA yield of the mutated PhaC1Pp. The evolved PhaC1Pp were coexpressed with β-ketothiolase (phbARe) and the acetoacetyl-CoA reductase (phbBRe) to supply sufficient short-chain length (R)-3-hydroxyacyl-CoA as a substrate. A single point mutation at L484V remarkably enhanced the monomer ratio of (R)-3-hydroxybutyrate in a PHA accumulation experiment. Saturation mutagenesis experiment at 484 concluded that Val is the most favorable amino acid in PhaC1Pp for incorporating (R)-3-hydroxybutyrate unit synthesis. In addition, a single mutation at Q481M, S482G and A547V obviously increased PHA yields. Q481M and S482G enhanced the (R)-3-hydroxyhexanoate monomer composition in the PHA accumulation by P. putida GPp104 PHA−. This is the first data that spotlighted the important effect of Leu484 on substrate specificity of PHA synthase and Ala547 on the PHA accumulation.

Keywords
Polyhydroxyalkanoates; Class II PHA synthase; Saturation mutagenesis; Site-directed mutagenesis; Substrate specificity
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Critical residues of class II PHA synthase for expanding the substrate specificity and enhancing the biosynthesis of polyhydroxyalkanoate
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 56, 5 March 2014, Pages 60–66
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us