Cloning,Expression And Functional Characterization Of Enzyme Genes From Scheffersomyces Stipitis Involved In The Detoxification Of Aldehyde Inhibitors Derived From Lignocellulosic Biomass Conversion

Lignocellulosic biomass, due to its renewable, low-cost, and most abundant and widely distribution on earth, is considered as the most promising feedstock for industrial bioethanol production. However, prior to the ethanol fermentation process, lignocellulosic biomass must be hydrolysed to simple sugars via a pre-treatment process. This harsh pretreatment method generates numerous compounds, i.e. Inhibitors, which inhibit cell growth and the subsequent ethanol fermentation. Aldehyde inhibitors are the most critical ones among numerous inhibitors. Compared with native fermentation microorganism of Saccharomyces cerevisiae, Scheffersomyces (Pichia) stipitis it is not only able to ferment both hexose (such as glucose, mannose, and galactose) and pentose (such as xylose and arabinose) derived from biomass, but also able to secrete various cellulases and hemicellulases for enzymatic hydrolysis of lignocellulosic biomass to sugars. Therefore, S. stipitis is considered as the most promising fermentation microorganism for industrial bioethanol production from lignocellulosic biomass. Recently, the functional enzyme genes involved in the detoxification of aldehyde have been extensively studied in S. cerevisiae, but remain unknown in S. stipitis. Moreover, the two species of S. stipitis and S. cerevisiae have relatively far distance in phylogenetic relationship. Therefore, it is necessary to carry out a study on functional characterization of enzyme genes involved in the detoxification of aldehyde in S. stipitis.In this study, the S. stipitis type strain CBS 6054 was used as the experimental material. Genes from three families of SsADH, SsAAD, and SsALD, and a single gene SsGRE2 were selected as research targets based on previous reports on S. cerevisiae. The objectives of this study were to 1) investigate the transcription responses of the 20 genes to key aldehyde inhibitors of furfural and HMF; 2) overexpress these genes in S. cerevisiae and test their enzyme activities and properties for reduction of furfural and HMF in details; 3) test their enzyme activities in brief for reduction of other nine aldehydes - some of them are inhibitors generated during pretreatment of lignocellulosic biomass; and 4) analyze the amino acid sequences of all the target proteins. The new findings obtained in this study were presented as follows:(1) Comparative analyses indicated that cell growth of S. stipitis was inhibited by both furfural and HMF at early growth stage, but resumed after experiencing a lag-phase stage. Using quantitative real-time PCR method, transcription responses of the 20 target genes (mentioned above) to furfural and HMF at lag-phase stage were determined. Our research results showed that, among 7 genes in SsADH gene family, SsADH4 and SsADH6 were significantly induced under furfural or HMF stress condition, and the most change of transcription level increased by 150 fold. Among 5 genes in SsAAD gene family, SsAAD3 was the only one gene significantly up-regulated under both furfural and HMF stress conditions, and the highest up-regulated expression increased by 8 fold. SsGRE2 was significantly induced under both furfural and HMF stress conditions. The highest transcription level of SsGRE2 appeared under furfural stress condition, displaying 26.5-fold increase compared to control. Among 7 genes in SsALD gene family, transcription level of SsALD6 displayed 5.5-fold increase under furfural stress condition, and transcription level of SsALD3 displayed 1.5-fold increase under HMF stress condition, compared to control. Transcription levels of the rest candidate genes were not up-regulated significantly, and some of them even decreased slightly.(2) The coding region sequences of the 20 candidate genes were amplified from S. stipitis by PCR, and then inserted into the pYES2/NT B plasmid. Finally, the recombinant shuttle vectors containing each candidate gene were obtained. The newly constructed plasmids were transformed into S. cerevisiae strain INVScl and overexpressed successfully in galactose induction medium. Our study results on enzymatic characteristics of the overexpressed proteins showed that in SsADH family, SsAdh4p, SsAdh5p, SsAdh6p, and SsAdh7p showed both NADH and NADPH-dependent activities for furfural reduction, and SsAdhlp showed only NADH-dependent activity for furfural reduction. For HMF reduction, only SsAdh4p, SsAdh5p, SsAdh6p, and SsAdh7p showed NADPH-dependent activities, and no significant NADH dependent activity was observed in all SsADH proteins. The optimum catalytic activities were observed at pH 6.0 and 30? for all of the SsADH proteins displaying aldehyde reduction activities with either NADH or NADPH as the co-factor. When NADH was used as the co-factor, SsAdh7p showed the highest affinity to furfural (Km,1.94±0.12 mM). When NADPH was used as the co-factor, SsAdh4p showed the highest affinities to furfural and HMF (Km,0.24±0.12 mM and 0.26±0.03 mM). In SsAAD family, all of the five candidate proteins showed NADH-dependent activities for furfural reduction. As for HMF reduction, only NADPH-dependent activity was observed from SsAAD2p. The optimum catalytic activities for all SsAAD proteins were observed in weak acid condition (pH 6.0) or neutral condition (pH 7.0) with either NADH or NADPH as the co-factor. The temperature for optimum catalytic activity was at 30? for most SsAAD proteins except for SsAadlp at 40? and SsAad2p at 20?. When NADH was used as the co-factor, SsAAD4p showed the highest affinity to furfural (Km,3.24±0.03 mM), however, SsAAD5p showed the highest catalytic efficiency to furfural (Kcat/Km,78.51±1.82 mM-1 min-1). SsGre2p showed both NADH- and NADPH-dependent activities for furfural reduction, but displayed only NADPH-dependent activity for HMF reduction. The optimum catalytic activity was observed at pH 6.0 for furfural reduction with NADH as the co-factor, and at pH 7.0 for both furfural and HMF reduction with NADPH as the co-factor. The temperature of optimum catalytic activity of SsGre2p was at 20? for furfural reduction with either NADH or NADPH as the co-factor, but was at 40? for HMF reduction with NADPH as the co-factor. SsGre2p showed the highest affinity to furfural when NADH was used as the co-factor (Km,0.36 mM). In SsALD family, enzyme activities of all the proteins tested in this study were absolutely weak for both furfural and HMF reduction with either NADH or NADPH as the co-factor.(3) In this study, enzyme activities for reduction of other nine aldehydes were tested, and some of them are inhibitors commonly detected in lignocellulosic hydrolysates. In SsADH family, most proteins showed catalytic activities to most of the nine aldehydes, and SsAdhlp showed the highest specific activity (883.07±8.23 U/mg) with NADH as the cofactor for reduction of acetaldehyde, much higher than any other SsADH proteins with either NADH or NADPH as cofactor. In SsAAD family, SsAad2p and SsAad3p showed enzyme activities for reduction of 7 aldehydes when NADH was used as the co-factor, and SsAad4p showed enzyme activities for reduction of 6 aldehydes when NADPH was used as the co-factor. Among SsAAD family, SsAad2p showed the highest enzyme activity (208.45±5.36 U/mg) when acetaldehyde and NADH were used as the substrate and the co-factor, respectively. SsGre2p showed enzyme activities toward all the nine aldehydes, and displayed the highest of specific activity (25.7 U/mg) for phenylacetaldehyde reduction with NADPH as the co-factor. In SsALD family, SsAldlp, SsAld4p, and SsAld7p showed enzyme activities to a broad range of substrates, and the highest specific activity (112.83±4.02 U/mg) was observed for SsAldlp in acetaldehyde reduction with NADH as the co-factor.(4) Amino acid sequences analysis of target proteins showed that, in SsADH family, all of the seven SsADH proteins were identified as zinc-dependent medium-chain dehydrogenase/reductase (MDR) protein. In SsAAD family, four SsAAD proteins were identified as MDR protein except for SsAadlp as short-chain dehydrogenase/reductase (SDR) protein. SsGre2p was identified as SDR protein. Meanwhile, some conserved sequences, such as co-factor-binding and catalytic domains, were found in all of the 20 target proteins.Prior to this study, functions of the 20 target genes from S. stipitis were only annotated putatively based on homologous analysis of DNA sequence. In this study, functional genes that showed enzyme activities for detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion were successively identified from the target genes. Transcription responses of the 20 genes to furfural and HMF were also briefly investigated, and their enzyme properties for reduction of these two aldehyde inhibitors were tested in details. The key enzyme genes involved in the detoxification of furfural and HMF in S. stipitis were found out from the 20 target genes according to comprehensively comparative analyses, and their detoxification mechanisms to furfural and HMF were discovered. Some aldehyde reductase genes which display potential for other alcohol-fuel production was also found in this study. Research achievements from this study can help us to better understand the mechanism of in situ detoxification of aldehyde inhibitors in S. stipitis, meanwhile, contribute to the development and utilization of new enzymes and aldehyde tolerant microorganism. This study will undoubtedly promote the industrial production of bioethanol from lignocellulose.