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

Hiroshi Habe,Hideaki Koike,Yuya Sato,Yasufumi Iimura ,Tomoyuki Hori,Nobutada Kimura,Manabu Kanno,Kohtaro Kirimura

doi:10.1186/s13568-019-0853-y

Hiroshi Habe, Hideaki Koike + Show 6 more

Open Access

https://doi.org/10.1186/s13568-019-0853-y

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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.

Highlights

In recent years, much attention has been paid to the catalytic transformation of lignocellulose into key building block compounds under relatively mild conditions
Comparative transcriptome analysis Identification of genes involved in levulinic acid (LA) catabolism was carried out using a comparative RNA-seq technique with the transcriptomes of P. citronellolis LA18T grown on LA and pyruvic acid (Additional file 1: Figure S1)
The comparison of LA- and PA-grown LA18T cells allowed identification of the two most strongly upregulated genes (PCLA_07r0385 and PCLA_07f0384), which were highly homologous to cation acetate symporter and aminolevulinic acid dehydratases (ALADs), respectively (Table 1)

Summary

Introduction

Much attention has been paid to the catalytic transformation of lignocellulose into key building block compounds under relatively mild conditions. Rand et al clarified a seven-gene operon that is essential for assimilating LA into the β-oxidation pathway in Pseudomonas putida KT2440 (Rand et al 2017) This excellent study reported that lvaABCDE genes were upregulated in the presence of LA and were involved in the conversion of LA to an intermediate of the β-oxidation pathway, 3-hydroxyvaleryl-coenzyme A (3HV-CoA; Fig. 1). LvaC (putative oxidoreductase) may be involved in the conversion of 4PV-CoA to 3HV-CoA via pentanoyl-CoA These results suggest a unique catabolic pathway for LA, especially in that the isomerization of 4HV-CoA to 3HV-CoA proceeds through a phosphorylated intermediate, 4PV-CoA (Zhang et al 2009; Harris et al 2011; Rand et al 2017)

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