Invertase SUC2 Is the Key Hydrolase for Inulin Degradation in Saccharomyces cerevisiae

Abstract

Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae.

TEXT

Inulin is a conserved polysaccharide in tubers, roots, or leaves of many plants. It consists of linear chains of β-2,1-linked d-fructofuranose molecules terminated by a glucose residue. Inulin has recently drawn increased attention due to its potential for fuel ethanol production. Selection of yeast strains capable of producing inulinases (EC 3.2.1.80) has become a preferred choice since such yeast strains can be used to produce ethanol by low-cost consolidated bioprocessing (1, 2).

The budding yeast Saccharomyces cerevisiae is a suitable and widely used industrial microorganism for large-scale ethanol fermentations. However, it has traditionally been considered to be inulin-negative yeast (3, 4). This is mainly because no genes encoding inulinase has been previously found in S. cerevisiae. In addition, invertase in S. cerevisiae preferentially catalyzes hydrolysis of disaccharide sucrose and trisaccharide raffinose with limited activity toward inulin (5, 6). With the aid of genomic tools, a significantly wide range of genotype and phenotype variations has been realized for diversified yeast populations (7–10). Recently, strains of S. cerevisiae have been reported to be able to utilize inulin and convert inulin-type sugars to ethanol (11). We also identified strains able to utilize inulin from varied S. cerevisiae populations (12). However, genetic mechanisms of inulin metabolism in such strains are unknown. The present study aims to identify specific enzymes responsible for inulin utilization in S. cerevisiae.

A homothallic S. cerevisiae strain, JZ1C (CGMCC AS 2.3878), from China General Microbiological Culture Collection Center, Academia Sinica, Beijing, China, was used in this study. Strain JZ1C has been previously reported to have a high efficiency of inulin utilization (12). In order for yeast to utilize inulin, cells must be able to produce secretable inulinase to break down inulin into fructose and glucose. Since no inulinase gene has been identified for S. cerevisiae, we performed protein purification to identify enzymes with inulin hydrolytic activity in strain JZ1C. The extracellular crude enzyme solutions were produced by cultivating strain JZ1C cells in YPI medium (1% yeast extract, 2% peptone, and 2% inulin [wt/vol], pH 5.5) at 30°C for 48 h with shaking at 200 rpm. The supernatant of 1,000 ml crude enzyme solutions was concentrated by ultrafiltration (10-kDa cutoff) and applied to a Q-Sepharose Fast Flow anion exchange column (0.7 by 2.5 cm). The column was equilibrated in 0.02 M Tris-HCl buffer (pH 7.4), and the bound proteins were eluted with a step gradient of NaCl from 0 to 1 M in the equilibration. The positive eluant was concentrated by ultrafiltration and applied to a Sephadex 200 PG gel filtration column (2.5 by 120 cm). The enzyme activity toward inulin or sucrose was assayed by determining the concentration of reducing sugars released. The reaction mixture, containing 50 μl of diluted enzyme solution and 450 μl of 2% chicory inulin (Bio Basic Inc., Canada) or sucrose (dissolved in 0.1 M acetate buffer, pH 5.0), was incubated at 50°C for 15 min. An equal amount of enzyme solution inactivated by boiling at 100°C for 10 min was used as a control. The reaction was terminated at 100°C for 10 min, and the concentration of the reducing sugar in the mixture was measured by spectrophotometry at 540 nm. The reaction was linear up to 14.5% of inulin degraded. One inulinase unit (U) was defined as the amount of enzyme which yields 1 μmol fructose per min under the assay conditions used in this study. The protein concentration was determined using the Bradford assay as described previously (13).

After gel filtration, a single protein band with inulin hydrolytic activity was detected. The protein was purified to homogeneity, with a 6.8-fold increase in enzyme activity (see Table S1 in the supplemental material). Peptides of the purified protein were detected by electrospray ionization mass spectrometry (ESI-MS/MS) after in-gel trypsin digestion. The identified peptides covered 50% of the total amino acid sequences of invertase SUC2 in strain S288c (GenBank accession number NP_012104) and showed 99% (265/267) amino acid identity with the corresponding peptides of SUC2. The molecular mass of these identified peptides based on ESI-MS/MS analysis was 31.3 kDa, which is comparable to the expected molecular mass of 30.9 kDa. A SUC2 gene from strain JZ1C (GenBank accession number JQ836661) was cloned and sequence verified using the respective primers (see Table S2). The DNA sequence of SUC2 from strain JZ1C displayed 11 nucleotide substitutions compared with that of S288c, including 4 nonsynonymous sites. The deduced amino acid sequence from the cloned SUC2 gene in JZ1C was identical to that detected by ESI-MS/MS analysis for the purified protein. The relative molecular mass of the purified protein from JZ1C was more than 116 kDa, estimated by SDS-PAGE, while the deduced molecular mass of SUC2 was 60.7 kDa (Fig. 1). Deglycosylation by endoglycosidase Endo H_f (NEB P0703) indicated that the discrepancy in mass should be attributed to glycosylation of the secreted enzyme, and the diffuse band on SDS-PAGE pointed to glycosylation heterogeneity (Fig. 1). These results confirmed that the purified enzyme from JZ1C was SUC2.

Fig 1.

SDS-PAGE (8%) depicted the purified enzyme and the deglycosylation product after digestion by endoglycosidase Endo H_f (P0703; New England BioLabs, Ltd. [NEB]). The deglycosylation was performed according to the instructions provided by NEB.

The kinetic hydrolysis of inulin by purified SUC2 was measured using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as described previously (12). Analyses were performed on a Dionex ICS-3000 chromatography system (Dionex Corporation). The reaction mixture containing 1.2 μg of inulin and 5 U of the purified protein was incubated at 37°C for 22 h. Then, fractions were collected at various time points and heated to a boil to inactivate the reaction. The solution without enzyme addition was used as a control. As shown in Fig. 2, the inulin consisted of fructooligosaccharides with a variety of degree of polymerization (DP) values. After 22 h of hydrolysis, the concentrations of each fructooligosaccharide were reduced to below the detection limits. It was clear that any kind of fructooligosaccharide with a DP value lower than 20 was digested by the purified SUC2 (Fig. 2). The hydrolysis products of inulin were fructose and glucose at a ratio of 10.3:1, which indicate exoinulinase activity of the purified SUC2. Invertase has been reported to have low activity with inulin, but the DP bound of the digestion was previously unknown (6). Generally, S. cerevisiae strains are only able to utilize fructooligosaccharides with a DP value up to around 6 (6, 11). Our study demonstrated that SUC2 from strain JZ1C hydrolyzed fructooligosaccharides with a DP value of 20. Strain JZ1C showed 4.3-fold higher extracellular enzyme activity when inulin was used as a carbon source than with sucrose as the carbon source. This suggested that invertase SUC2 can be induced by inulin in strain JZ1C.

Fig 2.

Kinetic hydrolysis of commercial chicory inulin by purified SUC2, determined by HPAEC-PAD. G, F, and S represent glucose, fructose, and sucrose, respectively. The numbers denote the assigned DP values based on the assumption described by Corradini et al. (23).

Nine SUC genes (SUC1 to SUC5 and SUC7 to SUC10) have been found in S. cerevisiae (14). A yeast strain often has more than one SUC gene, although not necessarily with all the invertase genes. It is unclear whether SUC2 is the key enzyme for inulin utilization in strain JZ1C. To address this issue, gene deletion and complementation assays were performed with JZ1C and a standard laboratory strain, BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (a gift from EUROSCARF, Frankfurt, Germany) (Table 1). To delete the SUC2 gene, a PCR-mediated gene disruption method based on homologous recombination was employed (16). Double homologous arms were initially amplified by using the oligonucleotide primer pairs SDUP_F/SDUP_R and SDD_F/SDD_R (see Table S2 in the supplemental material). The homologous arms were inserted into the pUG6 vector through restricted endoenzyme digestion and ligation to generate a plasmid, pUG6-del (see Fig. S1). The deletion cassette conferring Geneticin resistance was obtained by performing a PCR on plasmid pUG6-del using the primers SDUP_F and SDD_R (see Table S2). Yeast transformation was performed according to the lithium acetate method (17). The replacement of the SUC2 open reading frame (ORF) with the amplified modules was verified by PCR analysis of total DNA isolated from the G418-resistant transformants using the primer pairs Dup_F/Dup_R, Ddn_F/Ddn_R, and Dup_F/Ddn_R (see Table S2).

Table 1.

Strains used in this study

Strain	Genotype	Source or reference
JZ1C	Wild type, homothallic diploid	15
BY4741	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0	EUROSCARF
JZ1C-R	JZ1C SUC2::kanMX	This study
BY4741-R	BY4741 SUC2::kanMX	This study
JZ1C-D	JZ1C SUC2-Δ	This study
BY4741-D	BY4741 SUC2-Δ	This study
JZ1C-C	JZ1C SUC2-Δ containing pYC230-SUC2	This study
BY4741-C	BY4741 SUC2-Δ containing pYC230-SUC2	This study

After the disruption of SUC2::kanMX, the heterozygotes of the diploid JZ1C transformants were confirmed by PCR. Cell growth of these JZ1C transformants on inulin or sucrose was not affected by the replacement of one of the two SUC2 alleles. In contrast, growth of the haploid BY4741 transformants was severely damaged (see Fig. S2 in the supplemental material). This demonstrated a clear negative effect of the SUC2 disruption on BY4741. To confirm the effect of SUC2 deletion on JZ1C, we further generated homozygous SUC2::kanMX transformants. Sporulation and tetrad dissection were carried out in heterozygous JZ1C transformants. The expected 2:2 pattern of segregation of SUC2 and SUC2::kanMX genes was observed on yeast agar-peptone-dextrose (YPD) and YPD plus G418 plates. The functional kanMX marker was looped out in both JZ1C and BY4741 transformants by transformation with the pSH65 plasmid, expressing the inducible Cre recombinase and carrying the phleomycin resistance gene ble^r (15). Loss of pSH65 was achieved by growing cells in liquid YPD medium at 30°C for 2 days. Growth assays showed that the complete deletion of the SUC2 genes significantly decreased the utilization of inulin and sucrose in JZ1C and BY4741 (Fig. 3). Also, no enzyme activity toward inulin and sucrose was detected in cultures of the deletion strains. This suggested the importance of SUC2 in inulin utilization for the two strains.

Fig 3.

Growth array of SUC2 deletion variants and complementation transformants in yeast nitrogen base medium (YNB) supplemented with inulin (A and B) or sucrose (C and D), respectively. The growth dynamics was determined in a Bioscreen C MBR reader (Oy Growth Curves Ab Ltd., Helsinki, Finland). The initial optical density at 600 nm (OD₆₀₀) was 0.2. JZ1C and BY4741 are the original strains. JZ1C-D and BY4741-D are the SUC2 deletion strains. JZ1C-C and BY4741-C denote randomly selected complementation transformants. All tests were performed in triplicate, and error bars indicated standard deviations.

For the complementation assay, the SUC2 gene in BY4741 or JZ1C was inserted into an episomal plasmid, pYC230, under the control of a PGK1 promoter to produce the plasmid pYC230-SUC2 (see Table S2 and Fig. S1 in the supplemental material). The vector pYC230 was a kind gift from Kjeld Olesen at Carlsberg Laboratory, Denmark (18). Complementation of the respective SUC2 genes in the JZ1C and BY4741 deletion strains restored the growth phenotypes on inulin and sucrose (Fig. 3). These results demonstrated that the invertase SUC2 was the key enzyme not only for sucrose metabolism but also for inulin metabolism in the two S. cerevisiae strains.

Inulin metabolism in both the JZ1C and BY4741 strains was attributed to the identical invertase SUC2, but the two strains showed significantly distinct inulin utilization (see Fig. S2 in the supplemental material). In the present study, the enzyme activities of fermentation supernatants and cell extracts toward inulin and sucrose were determined for JZ1C and BY4741. The strain JZ1C displayed both extracellular and intracellular enzyme activities toward inulin that were more than 7-fold those of strain BY4741 at the stationary growth stage (Fig. 4A). Secreted inulinase activity accounted for 60 to 70% of total inulinase activity for both strains after growth for 24 h, which suggested that secretion was not the major cause of the inulin utilization discrepancy between the two strains (Fig. 4A). Although the sucrose/inulin (S/I) activity ratios of the secreted enzymes from the two strains were comparable and were in the range between 8.4 and 10.2 after 30 h, the specific activities of SUC2 might be different between the two yeast strains (Fig. 4B). The influence of gene variation on specific activity of SUC2 was estimated by comparing mutation sites with published data. Invertases (EC 3.2.1.26) belong to the CAZy family GH32, which contains eight well-conserved domains predicted to be involved either in substrate binding or in catalysis (19, 20). Four of the eleven nucleotide substitutions in the SUC2 gene in JZ1C led to amino acid changes (N84H, Q88E, A409P, and V431A), while none of the mutations were located in either the substrate binding or catalytic domain. The invertase SUC2 is a glycosylation protein and contains 14 Asn-X-Thr/Ser glycosylation sites (21). Variation of glycosylation sites might also alter specific enzyme activity. However, the SUC2 mutations in JZ1C were found to cause no glycosylation site changes. Additionally, reciprocal expression of the SUC2 gene from JZ1C and BY4741 in the SUC2 deletion variants of the two strains did not change their growth on inulin compared to that with expression of the respective SUC2 gene (data not shown). Thus, sequence variation in SUC2 did not lead to variations of specific enzyme activity. When the glycosylation site is not modified, glycosylation heterogeneity can still occur, which may influence the specific enzyme activity. The heterogeneity associated with a glycosylation site in a protein depends on multiple factors, such as the rate of mRNA translation and the rate of protein folding (22). However, glycosylation levels of SUC2 and gene expression variations among S. cerevisiae strains are currently unknown. Continued investigations of SUC2 glycosylation and regulation of gene expression are needed to elucidate the mechanisms of the inulin utilization discrepancy between S. cerevisiae strains.

Fig 4.

Enzyme activities of crude enzyme solutions and extracts during the growth of strains JZC and BY4741 in YPI liquid medium. Intracellular proteins were extracted by using a yeast active protein extraction kit (Sangon Biotech, Shanghai, China). All tests were performed in triplicate, and error bars indicate standard deviations. (A) Extracellular and intracellular enzyme activity toward inulin. (B) Sucrose/inulin (S/I) activity ratio of the secreted enzymes from both strains. The S/I value of strain BY4741 at 8 h was not calculated because the extracellular enzyme activity toward inulin was not detected at this time point.