Strains, plasmids and growth conditions
The yeast and bacterial strains, as well as the plasmids used in this study, are listed in Table 1. The parent industrial strain BY14 was used to create the high-leavening haploid BY14a strain, which was used to create all of the other strains, including the overexpression and deletion strains.
The Escherichia coli DH5a and yeast strains were cultured as described previously [15]. Briefly, yeast cells in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30℃ on a rotor with a speed of 180 rpm. 0.08% of G418 (final concentration) (Thermo Fisher, MA, US) was added to YPD plates to select G418-resistant transformants. After growing in YPD for 24 h, 20 mL of cultured cells were inoculated into 200 mL of cane molasses medium (0.5% yeast extract, 0.05% (NH4)2SO4, and 12° Brix cane molasses) at the initial OD600 = 0.4. Cells were cultured at 30°C to OD600 = 1.8 (about 24 h). Cells were centrifuged at 4°C, 5,000 rpm for 5 min and then washed twice with sterile water. A modified low sugar model liquid dough (LSMLD) medium[17]was used for measurement of trehalose, intracellular glycerol and the cell viability during prefermentation and after prefermentation freezing.
RNA sequencing (RNA-seq)
RNA-seq based transcriptome analysis was performed to identify the differentially expressed genes after MAL62 overexpression. Cells constitutively overexpressing MAL62 (B+MAL62) and its control (BY14a+K) were harvested from cane molasses medium. RNA isolation and cDNA synthesis were performed as previously described [18]. Briefly, total RNA was isolated using the hot acid phenol method, followed by DNase treatment. The RNA concentration was measured using a Qubit fluorometer and a Qubit RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The RNA integrity was assessed using a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) and an RNA Nano 6000 Assay Kit.
The RNA-seq libraries were generated from 1 μg of RNA from each sample using a NEBNext Ultra RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. The clustering of samples, which was index coded to attribute individual sample sequences, was performed using a cBot Cluster Generation System (Illumina, San Diego, CA, USA). The RNA libraries were sequenced using an Illumina Hiseq 2500 system (Illumina). Paired-end reads of 125 bp/150 bp were generated and analyzed.
RNA-seq data analysis
The differential expression of two different groups was analyzed using DESeq R software (https://www.bioconductor.org, version 1.18.0). Genes with a false-discovery rate-adjusted p-value < 0.05 were considered as differentially expressed.
Volcano plots and hierarchical clustering were used to screen the differentially expressed genes and to analyze the clusters of differentially expressed genes. GOseq[19] was used for gene ontology (GO) term enrichment analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to enrich the pathways. STRING (www.string-db.org/) and Cytoscape (https://cytoscape.org) software programs were used for protein-protein interaction (PPI) network analysis.
Validation of gene expression levels
Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was used to detect the expression levels of the target genes. qRT-PCR was conducted using the THUNDERBIRD probe one-step qRT-PCR kit (TOYOBO, Osaka, Japan). The yeast UBC6 gene, which encodes a ubiquitin-conjugating enzyme involved in endoplasmic reticulum-resident proteins for degradation, was used as a reference gene[20]. The PCR primers are listed in Table 2. Yeast cDNA was extracted using an RNAiso kit (Takara Biotech, Dalian, China) and a PrimeScript RT reagent kit with gDNA eraser (Perfect Real Time, Takara Biotech). The PCR was conducted using a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows: 95℃ for 30 s; 61℃ for 20 min; 95℃ for 30 s; 43 cycles of 95℃ for 5 s, 55℃ for 10 s, and 74℃ for 15 s; and 72℃ for 5 min. Quantitative analysis of the qRT-PCR was conducted using the 2−ΔΔCT method.
Measurement of enzymatic activities
Tps1 activity was measured as previously described [21]. One unit of Tps1 activity was defined as the production of 1.0 μM of trehalose-6-phosphate per minute. The final activity was calculated based on cell dry weight (CDW). Data were expressed as mean ± SD from three independent experiments.
For α-glucosidase activity, crude extracts were prepared using the Salema-Oom method[22] and the α-glucosidase activity was measured as previously described [23]. Data were expressed as mean ± SD from three independent experiments.
For the activity of other enzymes, including hexokinase, phophoglucomutase, UGPase and glycerol-3-phosphate dehydrogenase (G3PDH), cells were grown in cane molasses or LSMLD medium to late-log phase. Cells were centrifuged at 5,000 rpm for 5 min at 4℃, then washed twice with cold sterile water. Activities of hexokinase [24], phosphoglucomutase [25], UGPase [26] and G3PDH [27] were assayed as described. Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Richmond, USA) following the manufacturer’s instruction. Data were expressed as mean ± SD from three independent experiments.
Measurement of intracellular trehalose contents
A total of 0.1 g of freshly cultured cells were washed twice with water. Trehalose was extracted using 4 mL of 0.5 M trichloroacetic acid then measured as previously described[28]. Data were expressed as mean ± SD from three independent experiments.
Measurement of extracellular maltose
Cultured cells were filtered through a 0.45-µm-pore-size cellulose acetate filter (Millipore, Danvers, MA, USA). The extracellular maltose was measured by high-pressure liquid chromatography (HPLC) analysis using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and an HPLC pump (Waters 515). The column was eluted at 65℃ with 5 mM H2SO4 at a flow rate of 0.6 ml/min [29]. Maltose was detected with a differential refractometer detector (Waters 410 RI). Data were expressed as mean ± SD from three independent experiments.
Yeast strain construction
Yeast genomic DNA was extracted using a yeast DNA isolation kit (Omega Bio-Tek, Norcross, GA, USA). The tps1D (B-T) strain (Table 1) was constructed as follows: the TPS1U fragment containing the TPS1 upstream homologue sequence and the TPS1D fragment containing the TPS1 downstream homologue sequence were amplified from the BY14a yeast genome with the primers TU1-F/TU1-R and TD1-F/TD1-R, respectively. The fragment KanMX was amplified from the plasmid pUG6 using the primers KAN1-F/KAN1-R [30]. Then, the fragments of TPS1U, TPS1D, and loxP-KanMX-loxP were transferred into BY14a using the lithium acetate/polyethylene glycol method [25]. G418 (300 μg/mL) was used to select the positive recombinants, which were further verified by PCR with the primers UUK-F/UUK-R and KDD1-F/KDD1-R. The tps1D plus MAL62-overexpression (B-T+M) strain (Table 1) was constructed as follows: The MAL62 gene was amplified from the BY14a genome with the primers TU1-F/TU2-R, TD1-F/TD1-R, and MAL-F/MAL-R. The fragments containing the yeast phosphoglycerate kinase gene promoter (PGK1p) and terminator (PGK1t) were amplified from the BY14a genome with the primers PGKP-F/PGKP-R and PGKT-F/PGKT-R, respectively. The fragment loxP-KanMX-loxP was amplified from the plasmid pUG6 using the primer pair KAN2-F/KAN1-R. Six fragments (TPS1U, PGK1P, MAL62, PGK1T, KanMX, and TPS1D) were transferred into the BY14a strain, and the recombinant B-T+M strain was verified via PCR using the primer pairs of UUP-F/UUP-R, PPM-F/PPM-R, MPT-F/MPT-R, PTK-F/PTK-R, and KDD2-F/KDD2-R. B+MAL62, the MAL62-overexpression strain, was constructed as described previously[15].
Measurement of intracellular glycerol content
To measure the intracellular glycerol levels, approximately 25 mg (wet weight) of cells was washed, resuspended in 1 mL of deionized water, and boiled twice (30 min, with occasional shaking). The supernatants were then centrifuged for 10 min at 15,000 × g. The level of glycerol was measured as described previously [31]. Data were expressed as the mean ± standard deviation (SD) from three independent measurements.
Measurement of cell viability and leavening ability
Yeast cells were cultured in the cane molasses medium [5 g/L yeast extract, 0.5 g/L (NH4)2SO4, and 12° Brix cane molasses] for 24 h at 30 ℃ and then transferred to LSMLD medium. The cells were prefermented for 5, 10, 15, 20, and 25 min, then moved to a −20 ℃ freezer for 7 days. The cell viability and leavening ability were measured after freezing, as described previously [15]. Data were expressed as the mean ± SD from three independent experiments.
Statistical analysis
Data were represented as mean ± SD from three independent experiments. Differences among the various strains were analyzed using analysis of variance. Differences between the parent and the MAL62-overexpression strains were analyzed using the Student’s t-test. For all analyses, p < 0.05 was considered statistically significant.