The open reading frame 02797 from Candida tropicalis encodes a novel NADH-dependent aldehyde reductase

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Highlights

  • CTRG_02797 from Candida tropicalis encodes a broad-substrate aldehyde reductase.

  • Ctrg_02797p is strictly NADH dependent for enzymatic conversion of aldehydes to their corresponding alcohols.

  • Ctrg_02797p is localized to the cytoplasm in C. tropicalis cells.

  • Ctrg_02797p is a medium-chain dehydrogenase/reductase (MDR) and can be further grouped into the CADH subfamily.

Abstract

Owing to its high-temperature tolerance, robustness, and wide use of carbon sources, Candida tropicalis is considered a good candidate microorganism for bioconversion of lignocellulose to ethanol. It also has the intrinsic ability to in situ detoxify aldehydes derived from lignocellulosic hydrolysis. However, the aldehyde reductases that catalyze this bioconversion in C. tropicalis remain unknown. Herein, we found that the uncharacterized open reading frame (ORF), CTRG_02797, from C. tropicalis encodes a novel and broad substrate-specificity aldehyde reductase that reduces at least seven aldehydes. This enzyme strictly depended on NADH rather than NADPH as the co-factor for catalyzing the reduction reaction. Its highest affinity (Km), maximum velocity (Vmax), catalytic rate constant (Kcat), and catalytic efficiency (Kcat/Km) were observed when reducing acetaldehyde (AA) and its enzyme activity was influenced by different concentrations of salts, metal ions, and chemical protective additives. Protein localization assay demonstrated that Ctrg_02797p was localized in the cytoplasm in C. tropicalis cells, which ensures an effective enzymatic reaction. Finally, Ctrg_02797p was grouped into the cinnamyl alcohol dehydrogenase (CADH) subfamily of the medium-chain dehydrogenase/reductase family. This research provides guidelines for exploring more uncharacterized genes with reduction activity for detoxifying aldehydes.

Introduction

The unrestrained use of fossil energy causes resource depletion, making it imperative to find more alternative energy sources. Lignocellulose, which can be used to produce a series of energy products by microbial fermentation, is a desirable substitute for fossil resources [1]. As an abundant biomass resource, it also has the advantages of low cost, renewable, widespread distribution and great exploitation potential [2]. However, the intricate and rigid structure of lignocellulose hinders its fermentation by traditional microorganisms. Thus, lignocellulose needs to be pretreated to decompose into simple sugars before its fermentation [3]. Diluted-acid pretreatment is considered a simple, fast and low-cost method for hydrolyzing lignocellulose, but it also inevitably generates abundant by-products, which can inhibit microbial growth and ethanol fermentation [4]. Among the numerous chemical compounds, aldehydes are regarded as the most hazardous and dominant inhibitors in lignocellulosic hydrolysates [5].

Although aldehydes inhibit the cell growth of Saccharomyces cerevisiae, they can be detoxified to their corresponding alcohol by multiple aldehyde reductases [6]. On the basis of their protein chain length, structural features, and conserved functional motifs, the reductase superfamily can be classified into: short-chain dehydrogenase/reductase (SDR), medium-chain dehydrogenase/reductase (MDR), long-chain dehydrogenase/reductase (LDR), and aldo-keto reductase (AKR) families [7,8]. A previous study has shown that aldehyde reductases play indispensable roles in in situ detoxification of aldehydes into their corresponding alcohols in S. cerevisiae [6]. Thus, the discovery of aldehyde reductases is of great significance for exploring the detoxification mechanism of aldehydes in S. cerevisiae.

Owing to its high-temperature tolerance, robustness, and wide use of carbon sources, Candida tropicalis, which is considered a good candidate microorganism for bioconversion of lignocellulose to ethanol, is able to in situ detoxify aldehydes derived from lignocellulosic hydrolysis [9,10]. However, the C. tropicalis aldehyde reductases that catalyze this bioconversion remain unknown. The genome of C. tropicalis has been completely sequenced [11], but many uncharacterized open reading frames (ORFs), putative proteins, and proteins with unknown functions need to be further characterized. Basic Local Alignment Search Tool (BLAST) analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi) of Ctrg_02797p demonstrated that it is an MDR protein that can be further grouped into the cinnamyl alcohol dehydrogenase (CADH) subfamily. Thus, we speculated that CTRG_02797 most probably encodes a CADH protein related to the reduction of aldehydes, and/or ketones and quinones in C. tropicalis. However, because of the unknown function of this gene, more information and data need to be revealed.

Section snippets

Strains, plasmids, media, and reagents

The C. tropicalis yeast strain, SHC-03, from a liquor fermentation factory in Shehong (Sichuan, China) and the S. cerevisiae yeast strain, INVSc1 (his3Δ1/his3Δ1 leu2/leu2 trpl-289 ura3-52/ura3-52), from Invitrogen (Carlsbad, CA, USA) were used to amplify and overexpress the target gene, CTRG_02797 (GenBank accession no. NW_003020055), respectively. The Escherichia coli strain, DH5α, from Sangon Biotech (Shanghai, China) was used as the host strain for overexpressing and screening of plasmids.

Gene cloning and protein expression

The DNA sequence of CTRG_02797 amplified from C. tropicalis SHC-03 and the plasmid pYES2/NT C digested by BamHI and XhoI were used to construct the recombinant plasmid, pYES2/NT C-CTRG_02797 (Fig. 1). Using diagnostic PCR and restriction endonuclease analysis, the recombinant expression plasmid, pYES2/NT C-CTRG_02797, was confirmed to be successfully constructed (Fig. S1). The newly constructed plasmid was transformed into the INVSc1 strain for overexpression of Ctrg_02797p. The target protein

Discussion

Numerous studies have reported that dozens of reductases can reduce aldehydes in S. cerevisiae [18]. However, aldehyde reductases in C. tropicalis remain unknown. In this study, we cloned the uncharacterized ORF, CTRG_02797, and obtained its encoded protein by overexpressing it under the GAL1 promoter in induction medium. Enzymatic tests showed that CTRG_02797 encodes a functional protein that can reduce multiple aldehydes. Conversion of aldehydes to alcohols catalyzed by aldehyde reductases in

Funding

This work was supported by the National Natural Science Foundation of China (No. 31570086) and the 2011 Collaborative Innovation Center for Farmland Protection and Agricultural Product Safety in Sichuan Province (No. 2011).

CRediT authorship contribution statement

Xiangdong Hu: Data curation, Writing - original draft. Xuebing Han: Data curation, Writing - original draft. Lan Wu: Methodology, Software. Hanyu Wang: Methodology, Software. Yidan Ouyang: Software, Validation. Qian Li: Software, Validation. Xiaolin Kuang: Software, Validation. Quanju Xiang: Visualization, Investigation. Xiumei Yu: Visualization, Investigation. Xi Li: Visualization, Investigation. Yunfu Gu: Formal analysis. Ke Zhao: Formal analysis. Qiang Chen: Formal analysis. Menggen Ma:

Declaration of competing interest

The authors declare no conflicts of interest associated with this manuscript.

Acknowledgement

We thank Michal Bell, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing a draft of this manuscript.

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    Xiangdong Hu and Xuebing Han contributed equally to this work.

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