Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases

Nambu, Yuka; Ishii-Hyakutake, Manami; Harada, Ken; Matsumoto, Shoji; Tsuge, Takeharu

doi:10.1002/1873-3468.13651

Cited by 12 publications

(8 citation statements)

References 26 publications

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“…Figures 3C–F show time courses of PHB granules accumulated in E. coli TOP10 grown in LB medium at 6, 12, 18, and 24 h visualized by Sudan Black B staining. Finally, LB medium was chosen for further optimization ( Nambu et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

Napathorn

Visetkoop

Pinyakong

et al. 2021

Front. Bioeng. Biotechnol.

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Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04 and pColdTF-phaCABA–04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

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Section: Resultsmentioning

confidence: 99%

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

Napathorn

Visetkoop

Pinyakong

et al. 2021

Front. Bioeng. Biotechnol.

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“…The estimated sizes of the PhaC 2 polymerases differ considerably from PhaC 1 with predicted molecular weights ranging from 93.6 to 106.4 kDa (Table 3 ). All PhaC polymerases contain a PhaC box sequence (G/S)XCX(G/A)G with similarity to the lipase box motif found in lipases and PhaZ depolymerases [ 92 ]. Most important is the conserved cysteine residue in the center of the box, which constitutes the active center of the enzyme, while the X’s denounce non-conserved amino acids (AA).…”

Section: Resultsmentioning

confidence: 99%

“…Most important is the conserved cysteine residue in the center of the box, which constitutes the active center of the enzyme, while the X’s denounce non-conserved amino acids (AA). The PhaC boxes in Halomonas are described to contain either an SYCLG or GYCLG motif, while a GFCVGG motif is found in C. necator PhaC 1 [ 86 , 87 , 91 , 92 ]. All Cobetia PhaC 1 and PhaC 2 sequences analyzed in this study contained a GYCLG or SYCIG motif, respectively (Table 3 ).…”

Section: Resultsmentioning

confidence: 99%

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant

et al. 2021

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Background Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. Results Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. Conclusions Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

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“…Analysis of CI-PhaC amino acids sequences of different halophiles PHA-producing Halomonas strains showed similar lipase box-like sequence (Ser-X-Cys-X-Gly) (figX). From the database analysis the most common described pattern is Glycine-X-Cysteine-X-Glycine (G-X-C-X-G) which can be modified to (GS)-X-C-X-(GA)-(GA) ( Nambu et al, 2019 ). The PhaC Hd has a lipase box-like sequence, S-X-C-X-G located between the residue 371 and the residue 375.…”

Section: Discussionmentioning

confidence: 99%

Extremophilic Bacterium Halomonas desertis G11 as a Cell Factory for Poly-3-Hydroxybutyrate-co-3-Hydroxyvalerate Copolymer’s Production

Hammami

Souissi

Souii

et al. 2022

Front. Bioeng. Biotechnol.

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Microbial polyhydroxyalkanoates (PHA) are biodegradable and biocompatible bio-based polyesters, which are used in various applications including packaging, medical and coating materials. In this study, an extremophilic hydrocarbonoclastic bacterium, previously isolated from saline sediment in the Tunisian desert, has been investigated for PHA production. The accumulation of intracellular PHA granules in Halomonas desertis G11 was detected by Nile blue A staining of the colonies. To achieve maximum PHA yield by the strain G11, the culture conditions were optimized through response surface methodology (RSM) employing a Box-Behnken Design (BBD) with three independent variables, namely, substrate concentration (1–5%), inoculum size (1–5%) and incubation time (5–15 days). Under optimized conditions, G11 strain produced 1.5 g/L (68% of DCW) of PHA using glycerol as a substrate. Application of NMR (1H and 13C) and FTIR spectroscopies showed that H. desertis accumulated PHA is a poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). The genome analysis revealed the presence of typical structural genes involved in PHBV metabolism including phaA, phaB, phaC, phaP, phaZ, and phaR, coding for acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase, class I polyhydroxyalkanoates synthases, phasin, polyhydroxyalkanoates depolymerase and polyhydroxyalkanoates synthesis repressor, respectively. Glycerol can be metabolized to 1) acetyl-CoA through the glycolysis pathway and subsequently converted to the 3HB monomer, and 2) to propionyl-CoA via the threonine biosynthetic pathway and subsequently converted to the 3HV monomer. In silico analysis of PhaC1 from H. desertis G11 indicated that this enzyme belongs to Class I PHA synthase family with a “lipase box”-like sequence (SYCVG). All these characteristics make the extremophilic bacterium H. desertis G11 a promising cell factory for the conversion of bio-renewable glycerol to high-value PHBV.

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Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases

Cited by 12 publications

References 26 publications

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant

Extremophilic Bacterium Halomonas desertis G11 as a Cell Factory for Poly-3-Hydroxybutyrate-co-3-Hydroxyvalerate Copolymer’s Production

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