Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida

Hiroshi Habe, Hideaki Koike, Yuya Sato, Yosuke Iimura, Tomoyuki Hori, Manabu Kanno, Nobutada Kimura, Kotaro Kirimura

Research output: Contribution to journalArticle

Abstract

Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

Original languageEnglish
Article number127
JournalAMB Express
Volume9
Issue number1
DOIs
Publication statusPublished - 2019 Dec 1

Fingerprint

Coenzyme A Ligases
Pseudomonas putida
Pseudomonas
Genes
Coenzyme A
Fatty Acids
levulinic acid
Porphobilinogen Synthase
Escherichia coli
Symporters
Amino Acid Sequence Homology
Gene Expression Profiling
Complex Mixtures
Pyruvic Acid
Biomass
Ligation
Cations
Acetates
Adenosine Triphosphate
High Pressure Liquid Chromatography

Keywords

  • Acyl-CoA synthetase
  • Levulinic acid
  • Levulinyl-CoA synthetase
  • Lignocellulose
  • Pseudomonas citronellolis

ASJC Scopus subject areas

  • Biophysics
  • Applied Microbiology and Biotechnology

Cite this

Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida. / Habe, Hiroshi; Koike, Hideaki; Sato, Yuya; Iimura, Yosuke; Hori, Tomoyuki; Kanno, Manabu; Kimura, Nobutada; Kirimura, Kotaro.

In: AMB Express, Vol. 9, No. 1, 127, 01.12.2019.

Research output: Contribution to journalArticle

Habe, Hiroshi ; Koike, Hideaki ; Sato, Yuya ; Iimura, Yosuke ; Hori, Tomoyuki ; Kanno, Manabu ; Kimura, Nobutada ; Kirimura, Kotaro. / Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida. In: AMB Express. 2019 ; Vol. 9, No. 1.
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abstract = "Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35{\%} identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.",
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AU - Iimura, Yosuke

AU - Hori, Tomoyuki

AU - Kanno, Manabu

AU - Kimura, Nobutada

AU - Kirimura, Kotaro

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AB - Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

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KW - Lignocellulose

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