Strains and media
The yeast strain used in this study was Saccharomyces cerevisiae BY4741 (MATa, his3Δ1, leu2Δ0, met17Δ0, ura3Δ0) [26]. Cultivation was carried out in liquid or solid (20 g L-1 w/v agar) YPD (10 g L-1 w/v yeast extract, 20 g L-1 w/v peptone, 20 g L-1 w/v glucose). Selection medium was 1.7 g L-1 yeast nitrogen base without amino acids and ammonium sulfate (Millipore), 1 g L-1 L-Glutamic acid monosodium salt monohydrate (Sigma-Aldrich), 20 g L-1 w/v glucose, 120 mg L-1 L-Leucine (Sigma-Aldrich), 20 mg L-1 Uracil (Sigma-Aldrich), 200 µg ml-1 Hygromycin B (Invivogen) and 20 g L-1 w/v agar. All yeast cultivation was done at 30 °C.
Cellulase control strain construction
Standard protocols were used for expression vector preparation [27]. Restriction endonucleases, T4 DNA ligase and Phusion DNA polymerase were purchased from Thermo Scientific and used as directed by the manufacturer. The Zymoclean Gel DNA Recovery kit (Zymo Research) was used according to the manufacturer’s instructions to elute digested DNA from agarose gels. For the construction of the episomal cellulase expression plasmids, the hygromycin B resistance (hphMX4) cassette was obtained from the pBHD1_SOD1 plasmid [28] by digesting with BamHI and SpeI and subsequent ligation into the pMU1531 [29], creating pHK112. The Saccharomycopsis fibuligera CEL3A and Trichoderma reesei CEL5A ORFs were amplified from the ySFI vector [30] and pLEGII vector [31] respectively, using primers containing restriction sites for PacI and AscI. These PCR fragments were separated on an agarose gel, purified, digested with PacI and AscI, and cloned into the corresponding sites of the pHK112 vector, yielding pHK112_S.f.CEL3A and pHK112_T.r.CEL5A. The integrity of the constructs were verified with Sanger sequencing (Central Analytical Facility, Stellenbosch, South Africa). The pHK112_S.f.CEL3A and pHK112_T.r.CEL5A. vectors were transformed into BY4742 strains [26] using the LiOAc/SS carrier DNA/PEG method as described by Gietz and Schiestl [32] and recovered for 4-6 hours in liquid YPD medium at 30°C and subsequently plated out on YPD agar plates containing 300 µg/mL hygromycin B. The presence of the expression plasmid in putative transformants was confirmed with polymerase chain reaction (PCR) analysis using the following primers; 5'-GGATCCACTAGTCTTCTAGGCGGGTTATC-3' and 5'-GACTGGCGCGCCTTACAAACATTGAGAGTAGTATGGG-3'. Confirmed transformants harbouring the pHK112_S.f.CEL3A and pHK112_T.r.CEL5A vectors were referred to as BY4242[Cel3A] and BY4742[Cel5A] respectively.
SCRaMbLE acceptor plasmid construction
Detailed diagrams of all plasmid maps are provided in the supplementary material. The acceptor plasmid (pAcceptor) was constructed by replacing the cellulase expression cassette with two loxPsym sites in the backbone pHK112 vector. The loxPsym pair was PCR amplified from S. cerevisiae synthetic chromosome XIV (unpublished, from our laboratory). The 862 bp between the loxPsym sites contains no known coding sequences. A PCR was performed to amplify this sequence, with 200 µM dNTPs, 0.5 µM of “loxP pair SynXIV F” primer and 0.5 µM of “loxP pair SynXIV R” primer (Table 1), 50 ng of template DNA (gDNA from a yeast strain containing synthetic chromosome XIV), 1x Phusion® HF buffer (NEB) and 1 unit/50 µl Phusion® High-Fidelity DNA Polymerase (NEB) in a final volume of 50 µl, with the following program: 1 cycle of 98 °C for 30 sec, 30 cycles of 98 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec and 1 cycle of 72 °C for 5 min. The primers were designed to produce a PCR product with ends homologous to the plasmid backbone, as such, pAcceptor (Fig. S1) was constructed using Gibson assembly with the NEBuilder® HiFi DNA Assembly Master Mix (NEB) [33] according to the manufacturer’s instructions.
SCRaMbLE donor plasmid construction
The two donor DNA plasmids were chemically synthesized by GenScript, USA. pCEL3A-loxP contained the 2717 bp S. fibuligera CEL3A ORF encoding a β-glucosidase I, flanked by the constitutive TEF1 promoter and the homologous HXT7 terminator for transcriptional control, followed by HIS3 as an auxotrophic marker (Fig. S2). pCEL5A-loxP contained a S. cerevisiae codon-optimized 1194 bp T. reesei CEL5A encoding endoglucanase with an upstream 57 bp T. reesei XYN2 signal sequence. CEL5A was placed under the transcriptional control of the PGK1 promoter and the homologous HXT1 terminator followed by MET17 as an auxotrophic marker (Fig. S3). The entire cassettes on both pCEL3A-loxP and pCEL5A-loxP plasmids were flanked by loxPsym sites and reside in the multiple cloning site of a pUC57 cloning plasmid.
In vitro SCRaMbLE
The bottom-up in vitro SCRaMbLE strategy, described by [19], specifies that loxPsym-flanked donor DNA is SCRaMbLEd into the loxPsym sites in an acceptor plasmid, facilitated by Cre recombinase. In vitro SCRaMbLE was performed as previously described, with adjustments. Briefly, the 50 μl reaction was set up with 1 unit of Cre recombinase (NEB), 400 ng acceptor plasmid, 800 ng pCEL5A-loxP and 800 ng pCEL3A-loxP. Following 1 h incubation at 37 °C, the Cre recombinase enzyme was deactivated at 70 °C for 10 min.
Transformation and of plasmid library
The library of in vitro SCRaMbLEd plasmids was transformed into BY4741 S. cerevisiae [26] as described above. Following transformation, cells were recovered in YPD medium for 4 h, shaking at 200 rpm. Cells were selected on SC -met -his agar to isolate putative transformants which contained pAcceptor and at least one copy of CEL3A and CEL5A.
Enzyme assays
Supernatants from 160 S. cerevisiae cultures grown for 48 h in YPD supplemented with 200 μg mL-1 Hygromycin B were collected for measurement of enzymatic activity. CEL5A activity of culture supernatants was determined by DNS (dinitrosalicylic acid) assay. 10 µl of culture supernatant was incubated with 70 µl of 10 g L-1 w/v carboxymethyl cellulose (CMC) in 0.05 M sodium acetate buffer pH 5.0 at 50 °C for 10 min. The addition of 120 µl of DNS reagent [34] was added for determination of reducing sugars. Reactions were boiled and absorbance was measured at 540 nm. For CMC assays, glucose was used to draw a standard curve in the range of 3–50 mM from which the amount of enzymatic Units of each sample was calculated. One unit of enzyme activity was defined as the amount of enzyme releasing 1 μM of reducing sugar per min. CEL3A activity was determined by the release of p-nitrophenol from p-nitrophenyl-β-glucoside (pNPG). 10 µl of culture supernatant was incubated with 1 µl of 1 mM pNPG and 89 µl of 0.05 M Sodium Acetate Buffer pH 5.0, at 50 °C for 10 min. The reaction was stopped with 100 µl of 1 M sodium carbonate and absorbance was measured at 400 nm. One unit of enzyme activity was defined as the amount of enzyme required for producing 1 μM of p-nitrophenol from the substrate per min. The synergistic activity of CEL5A and CEL3A was determined by the release of p-nitrophenol from BPNPG5 (4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside) obtained from Megazyme. The Cellulase Assay Kit (CellG5 Method) (Megazyme® K-CellG5-4V) was used per the manufacturer’s instructions, except that the addition of β-glucosidase was substituted for 0.05 M acetate buffer. Culture supernatants were incubated with BPNPG5 for 10 min at 37 °C and absorbance was recorded at 405 nm. One unit of enzyme activity was defined as the amount of enzyme required for producing 1 μM of p-nitrophenol from the substrate per min. For pNPG and BPNPG5 assays a pNP standard curve in the range of 1.5–25 mM was used.
Synergy evaluation and modelling
To evaluate the suitability of BPNPG5 as a substrate to reflect synergistic enzyme activity of Cel3A and Cel5A, supernatants of BY4242[Cel3A] and BY4742[Cel5A] cultures containing either Cel3A or Cel5A, were prepared and the respective enzymatic activities determined using pNPG and DNS assays. Supernatants were mixed in different ratios and BPNPG5 hydrolysis evaluated, as described above. Keeping the total enzymatic units constant (at 100 U), the combined action of different ratios (increments of 10%) of Cel3A and Cel5A were determined. To model this synergistic relationship between Cel3A and Cel5A activity a multiple linear regression analysis (least squares) was performed using GraphPad Prism version 8.01 for Windows, GraphPad Software, La Jolla California USA (www.graphpad.com). Based on this model, theoretical BPNPG5 activity were predicted for 160 yeast strains with in vitro SCRaMbLEd plasmids, and compared with measured BPNPG5 activity.
Extraction of DNA from yeast
Total DNA from yeast transformants was extracted using the dilute sodium hydroxide lysis method. In short, yeast cells 100 μl of an overnight yeast culture, grown in YPD supplemented with 200 µg ml-1 Hygromycin B, were pelleted by centrifugation at 4000 rpm for 2 min. The cell pellet was resuspended in 20 mM NaOH. Cell suspensions were incubated at 95 °C for 10 min. After pelleting cell debris by centrifugation at 4000 rpm for 2 min, cell lysates were directly used for quantitative PCR.
Determination of gene copy number in plasmids
Quantitative real time PCR (qPCR) was carried out using the Roche LightCycler® 480 instrument. Each reaction contained 5 μl of Agilent Technologies Brilliant II SYBR® Green QPCR Master Mix, 200 nM forward primer, 200 nM reverse primer, 1 µl of DNA template (supernatant from NaOH extractions) and nuclease-free water to obtain a final volume of 10 μl. Four sets of primers were designed to specifically amplify ~150 bp of the CEL3A, CEL5A and hphMX4 cassettes on plasmids and the native TAF10 from the genome (primers are listed in Table 2). Primer amplification efficiencies were determined using serial dilutions of total extracted yeast DNA. The cycling protocol used an initial denaturing step of 95 °C for 1 min, followed by 40 cycles of 95 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec. Fluorescence was recorded following each 72 °C cycling step. Crossing point (Cp) values were calculated using the Absolute Quantification software modules of the LightCycler® 480 Software. The number of plasmids per cell was determined as the relative hphMX4 copies per sample compared to the relative number of TAF10 copies of the same sample.