Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene

ABSTRACT The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

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Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109

Applied and Environmental Microbiology ◽

10.1128/aem.70.2.999-1007.2004 ◽

2004 ◽

Vol 70 (2) ◽

pp. 999-1007 ◽

Cited By ~ 52

Author(s):

Christopher T. Nomura ◽

Kazunori Taguchi ◽

Seiichi Taguchi ◽

Yoshiharu Doi

Keyword(s):

Escherichia Coli ◽

Chain Length ◽

Acyl Carrier Protein ◽

Short Chain ◽

Pha Synthase ◽

Short Chain Length ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Pha Copolymers

ABSTRACT Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C3 to C5, medium-chain-length (MCL) PHAs consist of monomer units of C6 to C14, and SCL-MCL PHAs consist of monomers ranging in size from C4 to C14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C4 to C10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.

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Engineering of Stable Recombinant Bacteria for Production of Chiral Medium-Chain-Length Poly-3-Hydroxyalkanoates

Applied and Environmental Microbiology ◽

10.1128/aem.65.8.3265-3271.1999 ◽

1999 ◽

Vol 65 (8) ◽

pp. 3265-3271 ◽

Cited By ~ 33

Author(s):

Maria A. Prieto ◽

Michele B. Kellerhals ◽

Gian B. Bozzato ◽

Dragan Radnovic ◽

Bernard Witholt ◽

...

Keyword(s):

Molecular Weight ◽

Chain Length ◽

Expression System ◽

Average Molecular Weight ◽

Polydispersity Index ◽

Recombinant Bacteria ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Pha Production

ABSTRACT In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans. We used the mini-Tn5 system as a tool to constructEscherichia coli 193MC1 and P. oleovoransPOMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level. E. coli 193MC1 produced considerably shorter polyesters thanP. oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that E. coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.

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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 polyhydroxyalkanoate (PHA) synthase 1 of Pseudomonas sp. H9 synthesized a 3-hydroxybutyrate-dominant hybrid of short- and medium-chain-length PHA

Enzyme and Microbial Technology ◽

10.1016/j.enzmictec.2020.109719 ◽

2021 ◽

Vol 143 ◽

pp. 109719

Author(s):

Chung-Hsien Liu ◽

Houng-Yung Chen ◽

Yuh-Ling Lee Chen ◽

Der-Shyan Sheu

Keyword(s):

Chain Length ◽

Pha Synthase ◽

Pseudomonas Sp ◽

Medium Chain ◽

Medium Chain Length

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FabG, an NADPH-Dependent 3-Ketoacyl Reductase ofPseudomonas aeruginosa, Provides Precursors for Medium-Chain-Length Poly-3-Hydroxyalkanoate Biosynthesis inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.182.10.2978-2981.2000 ◽

2000 ◽

Vol 182 (10) ◽

pp. 2978-2981 ◽

Cited By ~ 63

Author(s):

Qun Ren ◽

Nicolas Sierro ◽

Bernard Witholt ◽

Birgit Kessler

Keyword(s):

Escherichia Coli ◽

Chain Length ◽

Reductase Activity ◽

Inescherichia Coli ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Encoding Gene ◽

Coa Reductase ◽

Formation Step

ABSTRACT Escherichia coli hosts expressing fabG ofPseudomonas aeruginosa showed 3-ketoacyl coenzyme A (CoA) reductase activity toward R-3-hydroxyoctanoyl-CoA. Furthermore, E. coli recombinants carrying the poly-3-hydroxyalkanoate (PHA) polymerase-encoding gene phaCin addition to fabG accumulated medium-chain-length PHAs (mcl-PHAs) from alkanoates. When E. coli fadB orfadA mutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-CoA formation step during β-oxidation, respectively, were transformed with fabG, higher levels of PHA were synthesized in E. coli fadA, whereas similar levels of PHA were found in E. coli fadB, compared with those of the corresponding mutants carrying phaC alone. These results strongly suggest that FabG of P. aeruginosais able to reduce mcl-3-ketoacyl-CoAs generated by the β-oxidation to 3-hydroxyacyl-CoAs to provide precursors for the PHA polymerase.

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Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) from Gluconate by Recombinant Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.540-548.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 540-548 ◽

Cited By ~ 56

Author(s):

Stefan Klinke ◽

Qun Ren ◽

Bernard Witholt ◽

Birgit Kessler

Keyword(s):

Escherichia Coli ◽

Chain Length ◽

De Novo ◽

Dry Weight ◽

Acid Synthesis ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Encoding Gene ◽

Encoding Genes

ABSTRACT It was shown recently that recombinant Escherichia coli, defective in the β-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303–309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinant E. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I (′thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., thebad promoter and alk promoter, for the ′thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the ′thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinantE. coli consisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinant E. coli JMU193, harboring PHA polymerase and ′thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and β-oxidation.

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Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico

PeerJ ◽

10.7717/peerj.4780 ◽

2018 ◽

Vol 6 ◽

pp. e4780 ◽

Cited By ~ 5

Author(s):

Carolina A. Martínez-Gutiérrez ◽

Hever Latisnere-Barragán ◽

José Q. García-Maldonado ◽

Alejandro López-Cortés

Keyword(s):

Chain Length ◽

Microbial Mats ◽

Pha Synthase ◽

Rrna Gene ◽

Bacterial Strains ◽

Plastic Properties ◽

Medium Chain ◽

Medium Chain Length ◽

Pha Production ◽

Hypersaline Microbial Mats

Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs) in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs), which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to aHalomonassp. In addition,Paracoccussp.,Planomicrobiumsp. andStaphylococcussp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those ofPseudomonas putida, suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence ofphaCandphaC1sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

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Fructose-Based Production of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Copolymer by Arctic Pseudomonas sp. B14-6

Polymers ◽

10.3390/polym13091398 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1398

Author(s):

Tae-Rim Choi ◽

Ye-Lim Park ◽

Hun-Suk Song ◽

Sun Mi Lee ◽

Sol Lee Park ◽

...

Keyword(s):

Molecular Weight ◽

Chain Length ◽

Average Molecular Weight ◽

Short Chain ◽

Pha Synthase ◽

Pseudomonas Sp ◽

Short Chain Length ◽

Medium Chain ◽

Medium Chain Length ◽

Pha Production

Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.

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PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.2117-2124.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 2117-2124 ◽

Cited By ~ 52

Author(s):

Silke Fiedler ◽

Alexander Steinbüchel ◽

Bernd H. A. Rehm

Keyword(s):

Fatty Acid ◽

Carbon Source ◽

Chain Length ◽

De Novo ◽

Carbon Sources ◽

Pha Synthase ◽

Pseudomonas Fragi ◽

Medium Chain ◽

De Novo Biosynthesis ◽

Medium Chain Length

ABSTRACT Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (C6 to C14) (PHAMCL), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044–24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressedphaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) fromP. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol−1 and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 andphaG established a new pathway for PHAMCLbiosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the β-oxidation inhibitor acrylic acid mediated PHAMCL accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHAMCL synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).

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