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13 pages, 1689 KiB

Open AccessArticle

Enhanced Production of (R)-3-Hydroxybutyrate Oligomers by Coexpression of Molecular Chaperones in Recombinant Escherichia coli Harboring a Polyhydroxyalkanoate Synthase Derived from Bacillus cereus YB-4

by Saki Goto, Yuki Miyahara, Seiichi Taguchi, Takeharu Tsuge and Ayaka Hiroe

Microorganisms 2022, 10(2), 458; https://doi.org/10.3390/microorganisms10020458 - 16 Feb 2022

Cited by 1 | Viewed by 2525

Abstract

The biodegradable polyester poly-(R)-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a (R)-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by [...] Read more.

The biodegradable polyester poly-(R)-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a (R)-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant Escherichia coli expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from Bacillus cereus YB-4 (PhaRC_YB4) was examined. First, genes encoding enzymes related to 3HBO biosynthesis (phaRC_YB4, phaA and phaB derived from Ralstonia eutropha H16) and two types of molecular chaperones (groEL, groES, and tig) were introduced into the E. coli strains BW25113 and BW25113ΔadhE. As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2–3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaR_YB4 and PhaC_YB4) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaR_YB4 and PhaC_YB4. These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO. Full article

(This article belongs to the Special Issue Development of Eco-friendly Bioplastics Using Microbes)

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14 pages, 2036 KiB

Open AccessArticle

Screening Method for Polyhydroxyalkanoate Synthase Mutants Based on Polyester Degree of Polymerization Using High-Performance Liquid Chromatography

by Manami Ishii-Hyakutake, Tetsuo Sakurai and Takeharu Tsuge

Microorganisms 2021, 9(9), 1949; https://doi.org/10.3390/microorganisms9091949 - 14 Sep 2021

Viewed by 2136

Abstract

A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC). In this method, PHA production was achieved using recombinant Escherichia coli supplemented with benzyl alcohol as a chain terminal compound. The [...] Read more.

A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC). In this method, PHA production was achieved using recombinant Escherichia coli supplemented with benzyl alcohol as a chain terminal compound. The cultured cells containing benzyl alcohol-capped PHA were decomposed by alkaline treatment, and the peaks of the decomposed monomer and benzyl alcohol were detected using HPLC. The DP of PHA could be determined from the peak ratio of the decomposed monomer to terminal benzyl alcohol. The measured DP was validated by other instrumental analyses using purified PHA samples. Using this system, mutants of PHA synthase from Bacillus cereus YB-4 (PhaRC_YB4) were screened, and some enzymes capable of producing PHA with higher DP than the wild-type enzyme were obtained. The PHA yields of two of these enzymes were equivalent to the yield of the wild-type enzyme. Therefore, this screening method is suitable for the selection of beneficial mutants that can produce high molecular weight PHAs. Full article

(This article belongs to the Special Issue Development of Eco-friendly Bioplastics Using Microbes)

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Review

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29 pages, 8853 KiB

Open AccessReview

Genome-Wide Metabolic Reconstruction of the Synthesis of Polyhydroxyalkanoates from Sugars and Fatty Acids by Burkholderia Sensu Lato Species

by Natalia Alvarez-Santullano, Pamela Villegas, Mario Sepúlveda Mardones, Roberto E. Durán, Raúl Donoso, Angela González, Claudia Sanhueza, Rodrigo Navia, Francisca Acevedo, Danilo Pérez-Pantoja and Michael Seeger

Microorganisms 2021, 9(6), 1290; https://doi.org/10.3390/microorganisms9061290 - 12 Jun 2021

Cited by 18 | Viewed by 4516

Abstract

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha [...] Read more.

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner–Doudoroff (ED), pentose-phosphate (PP), and lower Embden–Meyerhoff–Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via β-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions. Full article

(This article belongs to the Special Issue Development of Eco-friendly Bioplastics Using Microbes)

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