Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

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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Development of a New Strategy for Production of Medium-Chain-Length Polyhydroxyalkanoates by Recombinant Escherichia coli via Inexpensive Non-Fatty Acid Feedstocks

Applied and Environmental Microbiology ◽

10.1128/aem.07020-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 519-527 ◽

Cited By ~ 66

Author(s):

Qin Wang ◽

Ryan C. Tappel ◽

Chengjun Zhu ◽

Christopher T. Nomura

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Chain Length ◽

Acyl Carrier Protein ◽

Carbon Sources ◽

Content Type ◽

Medium Chain ◽

Medium Chain Length ◽

Coa Transferase ◽

Fatty Acid Carbon

ABSTRACTPseudomonas putidaKT2440 is capable of producing medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when grown on unrelated carbon sources during nutrient limitation. Transcription levels of genes putatively involved in PHA biosynthesis were assessed by quantitative real-time PCR (qRT-PCR) inP. putidagrown on glycerol as a sole carbon source. The results showed that two genes,phaGand the PP0763 gene, were highly upregulated among genes potentially involved in the biosynthesis of MCL-PHAs from unrelated carbon sources. Previous studies have describedphaGas a 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase, and based on homology, the PP0763 gene was predicted to encode a medium-chain-fatty-acid CoA ligase. High expression levels of these genes during PHA production inP. putidaled to the hypothesis that these two genes are involved in PHA biosynthesis from non-fatty acid carbon sources, such as glucose and glycerol. ThephaGppand PP0763 genes fromP. putidawere cloned and coexpressed with the engineeredPseudomonassp. 61-3 PHA synthase genephaCl(STQK)psin recombinantEscherichia coli. Up to 400 mg liter−1MCL-PHAs was successfully produced from glucose. This study has produced the largest amount of MCL-PHAs reported from non-fatty acid carbon sources in recombinantE. colito date and opens up the possibility of using inexpensive feedstocks to produce MCL-PHA polymers.

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Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid β-oxidation cycle in Escherichia coli

Metabolic Engineering ◽

10.1016/j.ymben.2014.05.004 ◽

2014 ◽

Vol 24 ◽

pp. 78-86 ◽

Cited By ~ 41

Author(s):

Qianqian Zhuang ◽

Qian Wang ◽

Quanfeng Liang ◽

Qingsheng Qi

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Chain Length ◽

Medium Chain ◽

Medium Chain Length

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Synthesis of medium chain length fatty acid ethyl esters in engineered Escherichia coli using endogenously produced medium chain fatty acids

Enzyme and Microbial Technology ◽

10.1016/j.enzmictec.2013.03.012 ◽

2013 ◽

Vol 53 (2) ◽

pp. 128-133 ◽

Cited By ~ 12

Author(s):

Liping Fan ◽

Junfeng Liu ◽

Kaili Nie ◽

Luo Liu ◽

Fang Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Fatty Acid ◽

Chain Length ◽

Fatty Acid Ethyl Esters ◽

Ethyl Esters ◽

Medium Chain ◽

Medium Chain Fatty Acids ◽

Medium Chain Length ◽

Engineered Escherichia Coli

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Optimizing a Fed-Batch High-Density Fermentation Process for Medium Chain-Length Poly(3-Hydroxyalkanoates) in Escherichia coli

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.618259 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ryan A. Scheel ◽

Truong Ho ◽

Yuki Kageyama ◽

Jessica Masisak ◽

Seamus McKenney ◽

...

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Chain Length ◽

Fermentation Process ◽

Octanoic Acid ◽

Decanoic Acid ◽

High Density ◽

Fed Batch ◽

Medium Chain ◽

Medium Chain Length

Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by β-oxidation deficient recombinant Escherichia coli LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L–1). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L–1, 0.42 g L–1 h–1), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L–1). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.

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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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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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YfcX Enables Medium-Chain-Length Poly(3-Hydroxyalkanoate) Formation from Fatty Acids in Recombinant Escherichia coli fadB Strains

Journal of Bacteriology ◽

10.1128/jb.184.20.5696-5705.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5696-5705 ◽

Cited By ~ 44

Author(s):

Kristi D. Snell ◽

Feng Feng ◽

Luhua Zhong ◽

David Martin ◽

Lara L. Madison

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Chain Length ◽

Open Reading Frame ◽

Gene Encoding ◽

Reading Frame ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Insertional Inactivation

ABSTRACT Expression of Escherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHAMCL) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional β-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHAMCLs when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB− YfcX− PhaC+ strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.

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