Strains, media, and culture conditions
The industrial Saccharomyces cerevisiae strain Still Spirits (Classic) Turbo Distiller’s Yeast was purchased from Homebrew Heaven (Everett, WA). Yeast strains were cultivated in YP media (1% yeast extract, 2% peptone) with 2% glucose (YPD) or 2-8% cellobiose (YPC). YPC with 8% cellobiose was used in fermentation analysis while 2% cellobiose was used for plate screening. S. cerevisiae strains were cultured at 30°C with orbital shaking at 250 rpm for aerobic growth or 100 rpm for oxygen-limited conditions in non-baffled flasks. As needed, 200 μg/mL G418 (KSE Scientific, Durham, NC) supplemented the YPD for pRS-KanMX plasmid selection. Escherichia coli DH5α (Cell Media Facility, University of Illinois at Urbana-Champaign, Urbana, IL) was used for recombinant DNA manipulations. E. coli strains were cultured in Luria broth (LB) (Fischer Scientific, Pittsburgh, PA) at 37°C and 250 rpm, supplemented with 100 μg/mL ampicillin. Yeast and bacterial strains were stored in 15% glycerol at −80°C. All chemicals were purchased from Sigma Aldrich or Fisher Scientific.
Plasmid and strain construction
Restriction enzymes were purchased from New England Biolabs (Ipswich, MA). All cloning work was performed via DNA Assembler, which relies on the in vivo homologous recombination mechanism in yeast . Yeast plasmids were isolated using Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research, Irvine, CA), then transformed into E. coli for isolation of high purity DNA. E. coli plasmids were isolated using Qiagen Spin Plasmid Mini-Prep Kit (Qiagen, Valencia, CA). PCR fragments were purified by Qiagen QIAQuick Gel Purification Kit.
The β-glucosidase gene gh1-1 [GenBank Accession number XM_951090] from Neurospora crassa was expressed using a PYK1 promoter and an ADH1 terminator. The cellobiose transporter gene cdt-1 [GenBank Accession number XM_958708] from N. crassa was expressed using a TEF1 promoter and a PGK1 terminator as previously constructed . To transfer the pathway to the pRS-KanMX plasmid , primers kanMX-PYKp-F and PGKt-kanMX-R (Additional file 1: Table S1) were designed to amplify the full cellobiose utilization pathway. To facilitate the creation of a library of cellobiose utilization pathways, a pRS-KanMX helper plasmid was constructed. The helper plasmid contained the PYK1 promoter and the PGK1 terminator, separated by a unique restriction enzyme recognition site BamHI for plasmid linearization. The helper plasmid was later linearized and used as a backbone for the library assembly. The PYK1 promoter was amplified using primers kanMX-PYKp-F and PYKp-BamHI-PGKt-R (Additional file 1: Table S1). The PGK1 terminator was amplified using primers PYKp-BamHI-PGKt-F and PGKt-kanMX-R (Additional file 1: Table S1).
The ADH1 terminator and TEF1 promoter were not subjected to random mutagenesis, thus this cassette was amplified independently using primers internal-BGL-ADHt-F and internal-TEFp-CDT-R (Additional file 1: Table S1) by standard PCR amplification. The gh1-1 and cdt-1 genes were subjected to error prone PCR with error rates of 2–3 basepair mutations per gene, resulting in one amino acid mutations per protein on average. Each fragment was amplified with 60–80 basepair homologies to upstream and downstream DNA sequences. The three fragments were co-transformed with the linearized helper plasmid into S. cerevisiae. The library size obtained was 104, consistent with libraries previously generated by this method [15, 36]. To confirm diversity of the library, ten colonies were randomly picked from the YPD + G418 plate and their plasmids were isolated and sequenced.
The library was screened on YPC plates for large colonies [15, 36] (Additional file 1: Figure S1). The growth rate of the largest colonies was quantified by small-scale fermentation. The colonies were seeded overnight in YPD + G418, washed twice with sterile water, and then inoculated into 10 mL YPC unbaffled flasks at 100 rpm and 30°C (Additional file 1: Figure S2). After confirmation of the top 5 clones with the fastest growth rate, the plasmids were isolated and retransformed. After retransformation, the strains were seeded overnight in YPD + G418, washed twice with sterile water, and then inoculated into 50 mL YPC in 250 mL unbaffled flasks at 100 rpm and 30°C. The cultures were sampled and analyzed for growth and metabolite production as described previously [15, 36]. After confirmation of the strains with improved phenotype, the plasmids were isolated and sequenced to identify the mutations within the genes of the pathway. The genes were used as a template for a second round of directed evolution.
Seed cultures were inoculated from plates of freshly streaked frozen culture stocks. YPD + G418 seed cultures were used for the individual and combined mutant pathway fermentation tests. YPC seed cultures were used for final mutant pathway analysis. The seed cultures were grown at 30°C and 250 rpm overnight, then washed with sterile water twice before inoculating 50 mL YPC in 250 mL un-baffled flasks to an initial OD of 0.2. The cultures were incubated at 30°C and 100 rpm orbital shaking for oxygen-limited growth . The cultures were sampled and analyzed for growth and metabolite production as previously described [15, 36]. For the metabolite analysis, end-point values at the time required to consume more than 95% of total sugar was used to determine rate, yield, and productivity.
Construction of single mutants
The single mutant genes were constructed through site-directed mutagenesis and the mega primer PCR method with primers listed in Additional file 1: Table S1. After PCR amplification, the genes were transformed into the Classic strain along with the ADHt1/TEFp1 cassette into a linearized pRS-KanMX helper plasmid. The final plasmid was purified and sequenced for confirmation.
β-Glucosidase enzyme activity assays
Classic strains harboring an empty vector, the wild-type, and mutant pathways were grown to mid-exponential phase and washed three times with potassium phosphate buffer (pH 7). A final cell mass equivalent to an OD600 of 20 was harvested. Cell-free extracts were prepared through YPER Extraction Reagent (Thermo Scientific, Rockport, IL), 125 μL of YPER was used to lyse the cells for 20 minutes at 25°C with vigorous shaking at 700 rpm in a thermomixer (Eppendorf, Germany). After lysing, the cell debris was pelleted for 10 minutes with 15,000 rpm at 4°C. The total protein concentration was determined via the BCA protein assay kit (Pierce, Rockford, IL), the standard manufacturer protocol was followed.
The lysate was tested for p-NPG activity with 1 mM p-NPG in 100 mM potassium phosphate buffer at pH 7. The colorimetric change was monitored at 405 nm in a 96-well Biotech Synergy 2 plate-reader (Winooski, VT) for 30 minutes at 30°C. The amount of p-nitrophenol (p-NP) released was quantified by a p-NP standard in100 mM potassium phosphate buffer pH 7. One unit of activity is equivalent to 1 μmol of p-NP released per min. For cellobiose-based enzymatic assay, the linear range of the β-glucosidase was determined. The reactions were carried out at 60 mM cellobiose in 100 mM potassium phosphate buffer pH 7. After addition of lysate, the reaction was allowed to react for 15, 30, 45, and 60 minutes at 30°C. The reaction was stopped by boiling at 100°C for 10 minutes. The samples were then centrifuged at 15,000 rpm for 5 minutes before being stored on ice. The amount of glucose which had been produced in the allotted time frame was then measured using the D-glucose kit (R-Biopharm, Germany). Standard manufacturer’s instructions were followed. To determine the β-glucosidase protein abundance in each strain, the wild-type and mutant proteins were fused to GFP. There was no statistically significant difference in β-glucosidase abundance of each strain (Additional file 1: Figure S4). The total protein concentration, as determined through BCA assay, was comparable in each lysate. Therefore, the ratio of β-glucosidase to total protein was assumed constant, and hence the activity was normalized to total protein concentration. One unit (U) is defined as one micromole of glucose produced per minute.
CDT activity assay
The cellodextrin transporter was assayed using the oil-stop protocol previously reported [12, 38]. Cultures of the wild-type and mutant strains were grown in YPC to an OD of 15–20, washed three times with ice-cold assay buffer (30 mM MES-NaOH + 50 mM ethanol), and then normalized to an OD of 20. 50 μL of cells were added to 50 μL of [3H]-cellobiose (Movarek Biochemicals, Brea, CA) at 30°C and layered over 100 μL of silicone oil (Sigma 85419), incubated for 10, 20, 40, and 80 seconds. The cells were then centrifuged through the oil at 15,000 rpm for 30 seconds. After being placed in ethanol/dry-ice bath, the cell pellets were solubilized in NaOH overnight. The amount of [3H]-cellobiose present in the cells was then quantified by a Beckman Coulter LS 6500 liquid scintillation counter (Brea, CA). The amount of labeled cellobiose that was transported into the cell was plotted against the time of reaction. One unit (U) is defined as one micromole of cellobiose taken up by the cell per min. The rate of the cellobiose uptake was normalized by the transporter abundance determined through GFP fluorescence measurements (Additional file 1: Figure S4) and the gram cell dry weight (gcdw).
A homology model of the β-glucosidase enzyme was constructed to identify the structure-function relationships of the mutations discovered. The gene encoding the β-glucosidase from Trichoderma reesei [PDB accession code 3AHY] afforded the highest homology to the gh1-1 gene from N. crassa with 73% sequence identity and was used as a template for homology modeling. The structure model of the β-glucosidase from N. crassa was constructed using the modeling program Molecular Operating Environment (Chemical Computing Group, Montreal, Canada). After constructing the homology model, the substrate in the co-crystal structure of Neotermes koshunensis β-glucosidase [PDB accession code 3VIK] was docked into the model . The model was energy minimized before ligand interactions were investigated. To identify the effects of the mutations, the mutations were introduced to the model and the energy was minimized again before investigating the ligand interactions.