Journal of Microbiology

Journal Article

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources: Woo Young Kim, Seung-Jin Kim, Hye-Rin Seo, Yoonyong Yang, Jong Seok Lee, Moonsuk Hur, Byoung-Hee Lee, Jong-Geol Kim, Min-Kyu Oh; J. Microbiol. 2024;62(7):569-579. Published online May 3, 2024; DOI: https://doi.org/10.1007/s12275-024-00136-x

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Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

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Research Support, Non-U.S. Gov'ts

NOTE] Biosynthetic Pathway for Poly(3-Hydroxypropionate) in Recombinant Escherichia coli: Qi Wang , Changshui Liu , Mo Xian , Yongguang Zhang , Guang Zhao; J. Microbiol. 2012;50(4):693-697. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2234-y

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Abstract PDF

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.

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NOTE] Biosynthesis of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Copolyesters with a High Molar Fraction of 3-Hydroxyvalerate by an Insect-Symbiotic Burkholderia sp. IS-01: Do Young Kim , Doo-Sang Park , Soon Bum Kwon , Moon Gyu Chung , Kyung Sook Bae , Ho-Yong Park , Young Ha Rhee; J. Microbiol. 2009;47(5):651-656. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0109-7

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Abstract PDF: Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB- co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).

Review

Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates: Do Young Kim , Hyung Woo Kim , Moon Gyu Chung , Young Ha Rhee; J. Microbiol. 2007;45(2):87-97.; DOI: https://doi.org/2528 [pii]

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Abstract PDF: Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chain length of C6-C14, are polyesters that are synthesized and accumulated in a wide variety of Gram-negative bacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications because they have useful mechanical properties and are biodegradable and biocompatible. The versatile metabolic capacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain various functional substituents; these MCL-PHAs are of great interest because these functional groups can improve the physical properties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituents can be modified by chemical reactions to obtain more useful groups that can extend the potential applications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedical fields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded by microorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relatively uncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases have been investigated at the molecular level. All known MCL-PHA depolymerases share a highly significant similarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recent advances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group.

Research Support, Non-U.S. Gov'ts

Molecular Characterization of Extracellular Medium-chain-length Poly(3-hydroxyalkanoate) Depolymerase Genes from Pseudomonas alcaligenes Strains: Do Young Kim , Hyun Chul Kim , Sun Young Kim , Young Ha Rhee; J. Microbiol. 2005;43(3):285-294.; DOI: https://doi.org/2211 [pii]

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Abstract PDF: A bacterial strain M4-7 capable of degrading various polyesters, such as poly(e-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ_PalM4-7) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ_PalM4-7 was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35^oC. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ_PalLB19) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M_r of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ_PalM4-7) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced M_r of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ_PalLB19 and the PhaZ_PalM4-7 commonly share the lipase box, GISSG, in their catalytic domains, and utilize ^111Asn and ^110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.

Enzymatic and Non-enzymatic Degradation of Poly (3-Hydroxybutyrate-co-3-Hydroxyvalerate) Copolyesters Produced by Alcaligenes sp. MT-16: Gang Guk Choi , Hyung Woo Kim , Young Ha Rhee; J. Microbiol. 2004;42(4):346-352.; DOI: https://doi.org/2100 [pii]

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Abstract PDF: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters with a variety of 3HV contents (ranging from 17 to 60 mol%) were produced by Alcaligenes sp. MT-16 grown on a medium containing glucose and levulinic acid in various ratios, and the effects of hydrophilicity and crystallinity on the degradability of the copolyesters were evaluated. Measurements of thermo-mechanical properties and Fourier-transform infrared spectroscopy in the attenuated total reflectance revealed that the hydrophilicity and crystallinity of poly(3HB-co-3HV) copolyesters decreased as 3HV content in the copolyester increased. When the prepared copolyester film samples were non-enzymatically hydrolysed in 0.01 N NaOH solution, the weights of all samples were found to have undergone no changes over a period of 20 weeks. In contrast, the copolyester film samples were degraded by the action of extracellular polyhydroxybutyrate depolymerase from Emericellopsis minima W2. The overall rate of weight loss was higher in the films containing higher amounts of 3HV, suggesting that the enzymatic degradation of the copolyester is more dependent on the crystallinity of the copolyester than on its hydrophilicity. Our results suggest that the degradability characteristics of poly(3HB-co-3HV) copolyesters, as well as their thermo-mechanical properties, are greatly influenced by the 3HV content in the copolyesters.

Effect of Levulinic Acid on the Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha KHB-8862: Sun Ho Chung , Gang Guk Choi , Hyung Woo Kim , Young Ha Rhee; J. Microbiol. 2001;39(1):79-82.

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Abstract PDF: The influence of levulinic acid (LA) on the production of copolyester consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) by Ralstonia eutropha was investigated. Addition of LA into the culture medium greatly increased the molar fraction of 3HV in the copolyester, indicating that LA can be utilized as a precursor of 3HV. In shake flask culture, the 3HV content in the copolyester increased from 7 to 75 mol% by adding 0.5 to 4.0 g/L LA to the medium containing fructose syrup as a main carbon source. A maximal copolyester concentration of 3.6 g/L (69% of dry cell weight) was achieved with a 3HV content of 40 mol% in a jar fermentor culture containing 4.0 g/L of LA. When LA (total concentration, 4 g/L) was added repeatedly into a fermentor culture to maintain its concentration at a low level, the copolyester content and the 3HV yield from LA reached up to 85% of dry cell weight and 5.0 g/g, respectively, which were significantly higher than those when the same concentration of the LA was supplied all at once. The present results indicated that LA is more effective than propionate or valerate as a cosubstrate for the production of copolyesters with varying molar fractions of 3HV by R. eutropha.

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