In Vivo Characterization and Application of the PHA Synthase from Azotobacter vinelandii for the Biosynthesis of Polyhydroxyalkanoate Containing 4-Hydroxybutyrate

Polyhydroxyalkanoate (PHA) is a biodegradable thermoplastic naturally synthesized by many microorganisms, and the PHA synthase (PhaC) is known to be the key enzyme involved in determining the material properties and monomer composition of the produced PHA. The ability to exploit widely distributed, commonly found soil microorganisms such as Azotobacter vinelandii to synthesize PHA containing the lipase-degradable 4-hydroxybutyrate (4HB) monomer will allow for convenient production of biocompatible and flexible PHA. Comparisons between the A. vinelandii wild type and mutant strains, with and without a surface layer (S-layer), respectively, in terms of gene or amino acid sequences, synthase activity, granule morphology, and PHA productivity, revealed that the S-layer is the sole factor affecting PHA biosynthesis by A. vinelandii. Based on PHA biosynthesis using different carbon sources, the PhaC of A. vinelandii showed specificity for short-chain-length PHA monomers, making it a member of the Class I PHA synthases. In addition, it was proven that the PhaC of A. vinelandii has the inherent ability to polymerize 4-hydroxybutyrate (4HB) and the mediated accumulation of PHA with 4HB fractions ranging from 10 mol% to as high as 22 mol%. The synthesis of biocompatible PHA containing tailorable amounts of 4HB with an expanded range of elasticity and lipase-degradability will enable a wider range of applications in the biomedical field.

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Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

Applied and Environmental Microbiology ◽

10.1128/aem.00564-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3813-3821 ◽

Cited By ~ 24

Author(s):

Jo-Ann Chuah ◽

Satoshi Tomizawa ◽

Miwa Yamada ◽

Takeharu Tsuge ◽

Yoshiharu Doi ◽

...

Keyword(s):

Chain Length ◽

Fold Increase ◽

Short Chain ◽

Pha Synthase ◽

Wild Type ◽

Short Chain Length ◽

Medium Chain ◽

Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

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The Azotobacter vinelandii AlgE mannuronan C-5-epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation

Environmental Microbiology ◽

10.1111/j.1462-2920.2008.01597.x ◽

2008 ◽

Vol 10 (7) ◽

pp. 1760-1770 ◽

Cited By ~ 25

Author(s):

Magnus Steigedal ◽

Håvard Sletta ◽

Soledad Moreno ◽

Mali Mærk ◽

Bjørn E. Christensen ◽

...

Keyword(s):

Azotobacter Vinelandii ◽

Cyst Formation ◽

Monomer Composition

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Wide Distribution among Halophilic Archaea of a Novel Polyhydroxyalkanoate Synthase Subtype with Homology to Bacterial Type III Synthases

Applied and Environmental Microbiology ◽

10.1128/aem.01117-10 ◽

2010 ◽

Vol 76 (23) ◽

pp. 7811-7819 ◽

Cited By ~ 62

Author(s):

Jing Han ◽

Jing Hou ◽

Hailong Liu ◽

Shuangfeng Cai ◽

Bo Feng ◽

...

Keyword(s):

Amino Acid ◽

Halophilic Archaea ◽

Wide Distribution ◽

Amino Acid Sequences ◽

Pha Synthase ◽

Haloarcula Marismortui ◽

Type Iii ◽

C Terminus ◽

Genes Encoding

ABSTRACT Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaCHm) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaCHm were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.

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Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6789-6799.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6789-6799 ◽

Cited By ~ 19

Author(s):

Nuttawee Niamsiri ◽

Soazig C. Delamarre ◽

Young-Rok Kim ◽

Carl A. Batt

Keyword(s):

Amino Acid ◽

Ralstonia Eutropha ◽

Molar Fraction ◽

Class Ii ◽

Pha Synthase ◽

Sequence Analyses ◽

Burkholderia Glumae ◽

Pcr Products ◽

Key Enzyme

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 (phaC1 Po ) 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 phaC1 Po genes that produced more PHA than the native phaC1 Po . 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 (M w) 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.

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Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2411-2419.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2411-2419 ◽

Cited By ~ 77

Author(s):

Tomoyasu Kichise ◽

Seiichi Taguchi ◽

Yoshiharu Doi

Keyword(s):

Random Mutagenesis ◽

Pha Synthase ◽

Evolution System ◽

In Vitro Evolution ◽

Limited Region ◽

Monomer Composition ◽

Flexible Material ◽

Aeromonas Caviae ◽

Key Enzyme

ABSTRACT By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaCAc, was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaCAc gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaCAc gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).

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Polyhydroxyalkanoate (PHA) Production in Pseudomonas sp. phDV1 Strain Grown on Phenol as Carbon Sources

Microorganisms ◽

10.3390/microorganisms9081636 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1636

Author(s):

Iliana Kanavaki ◽

Athina Drakonaki ◽

Ermis Dionisios Geladas ◽

Apostolos Spyros ◽

Hao Xie ◽

...

Keyword(s):

Aromatic Compounds ◽

Carbon Sources ◽

Biodegradable Plastics ◽

Pha Synthase ◽

Pseudomonas Sp ◽

Degrading Bacterium ◽

Cell Extracts ◽

Transmission Electron ◽

Key Enzyme ◽

Pha Production

Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200–600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. The isolated biopolymer was confirmed to be polyhydroxybutyrate (PHB) by FTIR, NMR and MALDI-TOF/TOF analyses. The ability of strain Pseudomonas sp. phDV1 to remove phenol and to produce PHB makes the strain a promising biocatalyst in bioremediation and biosynthesis of biodegradable plastics.

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