General molecular biology techniques
Genomic DNA extractions were carried out using the standard phenol chloroform procedure72. Genotyping PCRs were performed using GoTaq Green Master Mix (Promega, Madison, WI, USA). The oligonucleotides used in this study (Supplementary Table 9) were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). Escherichia coli DH5α was used for routine transformations. All of the constructed plasmids were verified by DNA sequencing (GENEWIZ, South Plainfield, NJ, USA).
Growth media
Unless otherwise specified, yeast cells were grown at 30 °C on either YPD medium (10 g/L yeast extract, 20 g/L peptone, 0.15 g/L tryptophan and 20 g/L glucose) or synthetic complete (SC) drop out medium (20 g/L glucose, 1.5 g/L yeast nitrogen base without amino acids or ammonium sulfate, 5 g/L ammonium sulfate, 36 mg/L inositol, and 2 g/L amino acid drop out mixture) supplemented with 2% glucose, or non-fermentable carbon sources such as 2% galactose, or the mixture of 3% glycerol and 2.5% ethanol. 2% Bacto™-agar (BD, Franklin Lakes, NJ, USA) was added to make agar plates.
Assembly of DNA constructs
DNA construction was performed using standard restriction-enzyme digestion and ligation cloning and isothermal assembly (Gibson Assembly)73. Endogenous S. cerevisiae genes (ILV6, LEU4, and AFT1) were amplified from genomic DNA of CEN.PK2-1C by PCR using a forward primer containing an NheI restriction recognition site and a reverse primer containing an XhoI restriction recognition site (Supplementary Table 9). This enabled subcloning of PCR-amplified genes into pJLA vectors29 and pJLA vector-compatible plasmids. Genes from other organisms, including Bs_alsS, Ec_ilvCP2D1-A1 and Ll_ilvD were codon optimized for S. cerevisiae and synthesized by Bio Basic Inc. (Amherst, NY, USA). These genes were designed with flanking NheI and XhoI sites at the 5′ and 3′ ends, respectively. All plasmids constructed or used in this study are listed in Supplementary Table 2.
Constructing the isobutanol configuration of the biosensor
The reporter for the biosensor in its isobutanol configuration was constructed by placing the S. cerevisiae LEU1 promoter (PLEU1), in front of the yeast-enhanced green fluorescent protein (yEGFP) fused to a PEST tag. The LEU1 promoter and yEGFP-PEST fragments amplified via PCR were inserted by Gibson assembly into the vector JLAb131 to make an intermediate plasmid (pYZ13) containing the fragment PLEU1-yEGFP_PEST-TADH1, which was then subcloned into a HIS3 locus integration (His3INT) vector pYZ12B46, yielding the intermediate plasmid pYZ14. The truncated LEU41–410 was amplified from genomic DNA of CEN.PK2-1C and subcloned into plasmid JLAb23, which contains the constitutive promoter PTPI1 and terminator TPGK1. The PTPI1–LEU41–410-TPGK1 cassette from the resulting plasmid pYZ2 was then subcloned into pYZ14 using sequential gene insertion cloning29 to form the plasmid pYZ16 (Supplementary Table 2).
Constructing the isopentanol configuration of the biosensor
To construct the biosensor in its isopentanol configuration, we removed the PEST-tag of the yEGFP reporter in plasmid pYZ14 by annealed oligo cloning. The two single-stranded overlapping oligonucleotides (Yfz_Oli59 and Yfz_Oli60) were annealed and cloned directly into the overhangs generated by restriction digest of pYZ14 at SalI and BsrGI sites. The resulting plasmid (pYZ24) contains cassette PLEU1-yEGFP-TADH1. Next, we added a catalytically active and leucine-insensitive LEU4 mutant (LEU4∆S547, a deletion in Ser547)28,37 to pYZ24 to form the isopentanol configuration of the biosensor, pYZ25. The deletion in Ser547 was achieved by site-directed mutagenesis using a plasmid containing wild-type LEU4 (LEU4WT). LEU4∆S547 was then subcloned into JLAb23 to form plasmid pYZ1. The PTPI1–LEU4∆S547-TPGK1 cassette from pYZ1 was then subcloned into pYZ24 to form the plasmid pYZ25.
Constructing template plasmids for error-prone PCR
Plasmid pYZ125, used to prepare the random mutagenesis libraries, was constructed by subcloning the region of JLAb131 spanning from the TDH3 promoter (PTDH3) to the ADH1 terminator (TADH1) into the CEN plasmid pRS416 to generate pYZ12574. ILV6, LEU4, and Ll_ilvD were subcloned into pYZ125, after linearization with NheI and XhoI, to create pYZ127, pYZ149, and pYZ126, respectively. The ILV6V90D/L91F mutant was generated using QuikChange site-directed mutagenesis and subcloned into pYZ125 to create pYZ14835. Leucine inhibition insensitive LEU4 mutants LEU4∆S547 and LEU41–410 were subcloned into pYZ125 to form pYZ154 and pYZ155, respectively (Supplementary Table 2).
Constructing δ-integration cassettes
We used a previously developed δ-integration (δ-INT) vector, pYZ2346, to integrate multiple copies of gene cassettes into genomic YARCdelta5 δ-sites, the 337 bp long-terminal-repeat of S. cerevisiae Ty1 retrotransposons (YARCTy1-1, SGD ID: S000006792). The selection marker in pYZ23 is the shBleMX6 gene, which encodes a protein conferring resistance to zeocin and allows selection of different numbers of integration events by varying zeocin concentrations75. Resistance to higher concentrations of zeocin correlates with a higher number of gene cassettes integrated into δ-sites. To construct δ-integration plasmids pYZ33, pYZ113, and pYZ34, we used restriction site pairs XmaI/AscI (to extract gene cassettes) and MreI/AscI (to open pYZ23)29,76. Plasmid pYZ417 was constructed by sequentially inserting cassettes PTDH3– Ec_ilvCP2D1-A1-TCYC1 (from pYZ196), PTEF1–Ll_ilvDI433V-TTPS1 (from pYZ383), and PGal1-S–Bs_alsS-TACT1 (from pYZ384) into the δ-integration plasmid EZ-L23546, which contains the OptoEXP-PDC1 cassette (PC120–PDC1-TACT1). All of the δ-integration plasmids were linearized with PmeI prior to yeast transformation.
Error-prone PCR and random mutagenesis library construction
The random mutagenesis libraries of ILV6, LEU4 and Ll_ilvD were generated by error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The CEN plasmids harboring wild-type ILV6 (pYZ127), LEU4 (pYZ149), Ll_ilvD (pYZ126) or the ILV6V90D/L91F mutant (pYZ148) were used as templates for error-prone PCR. Various amounts of DNA template were used in the amplification reactions to obtain low (0–4.5 mutations/kb), medium (4.5–9 mutations/kb), and high (9–16 mutations/kb) mutation frequencies as described in the product manual. Primers Yfz_Oli198 and Yfz_Oli242, which contain NheI and XhoI restriction sites at their 5′ ends, respectively, were used to amplify and introduce random mutations into the region between the start codon and stop codon of each gene. The PCR fragments were incubated with DpnI for 2 h at 37 °C to degrade the template plasmids before purification with a the QIAquick PCR purification kit (Qiagen). The purified PCR fragments were digested overnight at 37 °C using NheI and XhoI, and ligated overnight at 16 °C with pYZ125. The same process was used to create an ep_library of the leucine regulatory domain of Leu4p, except the forward primer contained a BglII restriction site at its 5′ end, rather than an NheI site. The plasmid libraries that resulted were transformed into ultra-competent E. coli DH5α and plated onto LB-agar plates (five 150 mm petri dishes per library) containing 100 µg/ml of ampicillin. After incubating the plates overnight at 37 °C, the resulting lawns (with a library size of ~109) were scraped off the agar plates and the plasmid libraries were extracted using a QIAprep Spin Miniprep Kit (QIAGEN). The plasmid libraries were subsequently used for yeast transformation. Based on yeast transformation efficiency with these plasmid libraries, and the number of transformants collected for FACS, we estimate the size of our pre-sorted libraries to be ~106–107 variants.
Yeast strains and yeast transformations
S. cerevisiae strain CEN.PK2-1C (MATa ura3–52 trp1-289 leu2-3,112 his3-1 MAL2-8c SUC2) and its derivatives were used in this study (Supplementary Table 1). Deletions of BAT1, BAT2, ILV6, LEU4, LEU9, ILV3, TMA29, PDC1, PDC5, PDC6, and GAL80 were obtained using PCR-based homologous recombination. DNA fragments containing lox71- and lox66-flanked antibiotic resistance cassettes were amplified with PCR from plasmids containing an antibiotic resistance gene, using primers with 50–70 base pairs of homology to regions upstream and downstream of the ORF targeted for deletion30. Transformation of the gel-purified PCR fragments was performed using the lithium acetate method77. Cells transformed using antibiotic resistance markers, were first plated onto nonselective YPD plates for overnight growth, then replica plated onto YPD plates containing 300 µg/mL hygromycin (Invitrogen, Carlsbad, CA), 200 µg/mL nourseothricin (WERNER BioAgents, Jena, Germany), or 200 µg/mL Geneticin (G-418 sulfate, Gibco, Life Technologies, Grand Island, NY, USA). Chromosomal integrations and plasmid transformations were also performed using the lithium acetate method77. Cells transformed for δ-integration were first incubated in YPD liquid medium for six hours and then plated onto nonselective YPD agar plates for overnight growth. The next day, cells were replica plated onto YPD agar plates containing 300 µg/mL (for FACS) or higher concentrations (500, 800, 1200, and 1500 µg/mL for titrating higher copy numbers of integrations) of Zeocin (Invitrogen, Carlsbad, CA), and incubated at 30 °C until colonies appeared. Cells transformed with plasmid libraries or 2µ plasmids (used for FACS) were plated on three 150 mm petri plates per library. The resulting lawns (containing ~6.5 × 104 individual transformants in each plate, for a total of ~2 × 105 transformants) were scraped off the agar plates, small aliquots of the collected cells were then grown to exponential phase in fresh medium, and used for flow cytometry and FACS (see below). All strains with gene deletions or chromosomal integrations (expect δ-integrations) were genotyped with positive and negative controls to confirm the removal of the ORF of interest or the presence of integrated DNA cassette.
Strains with cytosolic isobutanol pathway
We first constructed a baseline strain for cytosolic isobutanol production (YZy449). We used constitutive promoters to express Bs_alsS, Ec_ilvCP2D1-A1, and AFT1, and the galactose-inducible and glucose-repressible promoter PGAL10 to express Ll_ilvD. This approach allows us to use the same strain to screen for Ec_ilvCP2D1-A1 and Ll_ilvD mutants using different carbon sources (galactose and glucose, respectively). We first deleted ILV3 and TMA29 in strain YZy121, which contains the isobutanol-configured biosensor, resulting in strain YZy443. We then transformed strain YZy443 with the URA3 integration cassette from plasmid pYZ196 (loxP-URA3-loxP-PTDH3–AFT1-TADH1-[PTEF1–Bs_alsS-TACT1-PTDH3–Ec_ilvCP2D1-A1-TADH1]-PGAL10–Ll_ilvD-TACT1), resulting in strain YZy447. Next, we recycled the URA3 marker using the Cre-loxP site-specific recombination system78 and counter selected on YPD plates with 1 mg/mL of 5-FOA (see below), resulting in the final strain YZy449. Both YZy447 and YZy449 make ~170 mg/L of isobutanol in fermentations using galactose as carbon source but are unable to make isobutanol from glucose (Supplementary Fig. 16).
Applying the biosensor in optogenetically controlled strains
To combine the biosensor (in its isobutanol configuration) with optogenetic controls of the cytosolic isobutanol pathway, we integrated OptoINVRT749 (using EZ-L439) into the HIS3 locus of YZy90, a gal80Δ, triple pdcΔ (pdc1Δ pdc5Δ pdc6Δ) strain containing a constitutively expressed copy of PDC1 in a 2μ plasmid, resulting in YZy480. We removed the 2μ-PDC1 plasmid using 5-FOA (see below) to generate strain YZy481, which is able to grow in medium supplemented with glycerol and ethanol, but not in medium supplemented with glucose. We next introduced the biosensor in its isobutanol configuration into the GAL80 locus of YZy481 to yield YZy487, and used GFP fluorescence intensity measurements and genotyping to confirm the biosensor was successfully integrated. A dark-inducible cytosolic isobutanol pathway and the light-inducible PDC1 were then introduced to strain YZy487 via δ-integration. After two rounds of FACS, we analyzed the GFP fluorescence and isobutanol titers of ten colonies in dark fermentations with 2% glucose (see below). The colony with the highest GFP fluorescence intensity, corresponding to the highest isobutanol titer, was chosen as the host strain (YZy502) for further enhancement of the cytosolic isobutanol production. To achieve a strain with even higher metabolic flux through the cytosolic isobutanol pathway, we introduced a 2µ plasmid pYZ350, containing partial cytosolic isobutanol pathway genes, and applied FACS to isolate high-producing transformants.
Flow cytometry/FACS
A BD LSRII Multi-Laser flow cytometer equipped with FACSDiva software V.8.0.2 (BD Biosciences, San Jose, CA) was used to quantify yEGFP fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 510 nm (525/50 nm bandpass filter). Cells were gated on forward scatter (FSC) and side scatter (SSC) signals to discard debris and probable cell aggregates (Supplementary Fig. 17). The typical sample size was 50,000 events per measurement. A BD FACSAria Fusion flow cytometer with FACSDiva software was used for fluorescence activated cell sorting (FACS) with a 488 nm excitation wavelength and a bandpass filter of 530/30 for yEGFP detection. Cells exhibiting high levels of GFP fluorescence (top ~1%) were sorted. Sorted cells were collected into 1 mL of medium (see below), and 50 µL of each sample of collected sorted were streaked on a corresponding agar plate and incubated at 30 °C to obtain 24 random colonies. For each of the single colonies isolated, MFI and BCHA production were measured. The rest of the sorted cells (~950 µL) were incubated at 30 °C and at 200 rpm agitation to reach the stationary growth phase, followed by subculturing (1:100 dilution) in the same medium for the next round of sorting. FlowJo X software (BD Biosciences, San Jose, CA) was used to analyze the flow cytometry data.
Sample preparation for flow cytometry assays and FACS high-throughput screens
Fluorescence measurements and FACS were performed on samples in mid-exponential growth phase. Single colonies from agar plates or yeast transformation libraries diluted 1:100 were first cultured overnight until stationary phase in synthetic complete (SC), or synthetic complete minus uracil (SC-ura) medium, supplemented with 2% glucose or galactose (for screening the library of strains containing varying copies of Ec_ilvCP2D1-A1). Overnight cultures were diluted 1:100 in the same fresh medium and grown to mid-exponential growth phase (12–13 h after inoculation). The growth media used for flow cytometry assays and FACS of ILV6-samples contain four times more valine (2.4 mM) than the synthetic defined medium described above. For strains with PDC1 and Bs_alsS controlled by optogenetic circuits (OptoEXP46 and OptoINVRT749, respectively), cultures were incubated for 8 h under constant blue light after inoculation with cells in stationary phase, followed by 10 h of incubation in the dark (see below) before flow cytometry or FACS. For all flow cytometry assays, cultures were diluted to an OD600 of approximately 0.1 with PBS. All samples used for FACS were diluted in fresh medium identical to their growth medium to OD600 of approximately 0.8.
Measurement of the response of the biosensor to α-IPM, α-KIV, and α-K3MV
Measurements of the responses of the biosensor to α-IPM, α-KIV and α-K3MV were carried out in a CEN.PK2-1C background strain, with the biosensor in its isobutanol configuration integrated at the HIS3 locus (YZy121). A single colony from an agar plate was cultured overnight in SC medium supplemented with 2% glucose. The overnight culture was diluted 1:100 in fresh SC medium supplemented with 2% glucose, and 1 mL of the diluted culture was added to each well of a 24-well cell culture plate (Cat. 229524, CELLTREAT Scientific Products, Pepperell, MA, USA). Then, 10 µL of freshly made α-IPM (pH 7.0), α-KIV (pH 7.0), or α-K3MV (pH 7.0) solutions were added to each well to reach different final concentrations ranging from 0 µM to 75 µM. The 24-well plate was shaken for 12 h in an orbital shaker (Eppendorf, New Brunswick, USA) at 30 °C and at 200 rpm agitation. The GFP fluorescence of each sample in exponential phase (OD600 = 0.8–1.2) was measured using flow cytometry, with samples diluted in PBS in plates (50 μL of culture into 200 μL of buffer) for α-IPM and α-KIV, or in tubes (1 mL into 1 mL) for α-K3MV. The diluted samples were then kept on ice and until flow cytometry measurements were performed.
Yeast fermentations for isobutanol or isopentanol production
High-cell-density fermentations were carried out in sterile 24-well microtiter plates (Cat. 229524, CELLTREAT Scientific Products, Pepperell, MA, USA) in an orbital shaker (Eppendorf, New Brunswick, USA) at 30 °C and at 200 rpm agitation. Single colonies were grown overnight in 1 mL of synthetic complete (SC), or synthetic complete minus uracil (SC-ura) medium, supplemented with 2% glucose. The next day, 10 µL of the overnight culture were used to inoculate 1 mL of SC (or SC-ura) medium supplemented with 2% glucose in a new 24-well plate. After 20 h, the plates were centrifuged at 234 g for 5 min, the supernatant was discarded, and cells were resuspended in 1 mL of SC (or SC-ura) supplemented with 15% glucose (or galactose). The plates were covered with sterile adhesive SealPlate® films (Cat. # STR-SEAL-PLT; Excel Scientific, Victorville, CA) and incubated for 48 h at 30 °C with 200 rpm shaking. The SealPlate® films were used in all 24-well plate fermentations to maintain semi-aerobic conditions in each well, and to prevent evaporation and cross-contamination between wells. At the end of the fermentations, the OD600 of the culture in each well was measured in a TECAN infinite M200PRO plate reader (Tecan Group Ltd., Männedorf, Switzerland). Plates were then centrifuged for 5 min at 234 g, and the supernatant from each well was analyzed using HPLC as described below.
Fermentations of strains with optogenetic controls were also carried out in sterile 24-well microtiter plates, as described above, but with modifications as previously described46 (see Supplementary Note 7). The parameter ρ, the cell density at which we switched cells from growing in blue light to dark and θ, the incubation time in the dark before fermentation used in the initial screening fermentations are an OD600 of 6 and 6 h, respectively, which were estimated based on the characterization and modeling of the optogenetic circuits OptoEXP46 and INVRT749. The optimal ρ (OD600 of 5) and θ (6 h) of the best isobutanol-producing strains (YZy502 and YZy505) were determined experimentally by measuring the isobutanol titers from fermentations using different ρ and θ values. To vary these parameters, a single colony of each strain was first grown to stationary phase in SC (for YZy502) or SC-ura (for YZy505) medium supplemented with 2% glucose under constant blue light at 30 °C. The overnight cultures were diluted to an OD600 of 0.1 in the same medium. We began incubations (at 30 °C and 200 rpm) of the diluted cultures at different times and under pulsed blue light to achieve cultures with different OD600 values, which correspond to variations in ρ, ranging from 1 to 9. After measuring the OD600 of each culture, we switched them from light to dark and incubated for 6 h at 30 °C and 200 rpm. After the dark incubation period, the cells were centrifuged and resuspended in 1 mL of the same medium supplemented with 2% glucose. The plates were covered with sterile adhesive SealPlate® films (Cat. # STR-SEAL-PLT; Excel Scientific, Victorville, CA) and incubated in the dark (wrapped in aluminum foil) for 48 h at 30 °C and 200 rpm. Subsequently, the cultures were prepared for HPLC analysis (see below). To determine the optimal θ, we diluted the overnight culture to an OD600 of 0.1 and incubated it at 30 °C and 200 rpm, and under pulsed blue light to reach an OD600 of 5, which was the optimal ρ determined in the previous experiment. Next, we incubated the cells in the dark (30 °C and 200 rpm) for different numbers of hours, ranging from 1 to 10, which correspond to variations in θ. After the dark incubation period, cells were centrifuged and resuspended in 1 mL of the same medium supplemented with 2% glucose, followed by 48 h of incubation in the dark (30 °C and 200 rpm) and HPLC analysis as described below.
Analysis of biosensor fluorescence, intracellular α-IPM, and BCHA production throughout low-cell density fermentations
Low and high-isobutanol (YZy121, YZy235) or isopentanol producers (SHy187, SHy159) were grown overnight in 1 mL of SC (or SC-ura) medium supplemented with 2% glucose in 24-well plates at 30 °C. 100 µL of overnight culture was used to inoculate 100 mL of SC (or SC-ura) medium supplemented with 15% glucose at 30 °C, and agitated at 200 rpm in an orbital shaker for a time course of 30 h. Timepoints were taken at 5 h, 12 h, and 30 h. For each time point, samples were collected for analysis of either cellular florescence, intracellular α-IPM, or extracellular BCHA concentration. Cellular florescence was measured via flow cytometry as described above. Intracellular α-IPM concentration was measured with Thermo Fisher Q Exactive HPLC-Orbitrap MS equipment. The protocol for metabolite extraction from cells was based on previously described methods60 with minor modifications. At each time point, an extraction solution containing 40:40:20 (v/v/v) methanol:acetonitrile:ddH2O with 0.5% formic acid stored at −20 °C, was used to extract metabolites from a volume of cells equivalent to 15 mL of culture at an OD600 of 1. Although volumes collected for each time point differ, equal cell masses were pelleted by centrifuging at 2107 g for 10 min at 4 °C. The cell pellet was vortexed with 1 mL of extraction solution and incubated for 1 min at room temperature. A total of 88 µL of 15.8% (w/v) NH4HCO3 was added immediately after, and mixed by vortex to neutralize the extraction solution. The resulting mixture was incubated at −20 °C for 15 min and centrifuged at 17,000 g for 8 min at 4 °C. A total of 150 µL of supernatant was dried via vacufuge for 2 h. The dried metabolites were resuspended in 50 µL of ddH2O and analyzed using an Atlantis T3 3 µm 2.1 × 150 mm reversed-phase column (Waters, Part No. 186003719, Milford, MA, USA). Gas chromatography (GC/MS) was used to measure extracellular product concentration of isobutanol or isopentanol. A total of 800 µL of the fermentation broth was centrifuged at 17,000 g for 40 min at 4 °C to remove cells and residual debris. The supernatant was subjected to an alcohol extraction with hexane, in which hexane and supernatant were mixed at a 1:1 ratio, vortexed for 15 min, and then centrifuged at 17,000 g for 10 min at 4 °C. The organic phase was analyzed using a DB-WAX UI 0.5 µm gas chromatography column (Agilent, Part No. 122-7033UI, Santa Clara, CA, USA).
Digital droplet PCR to estimate library and sorted strain genotypes
The Digital Droplet PCR (ddPCR) experiment was performed on a Bio-Rad QX200 Droplet Digital PCR system with an Automated Droplet Generator using QX200 ddPCR Evagreen Supermix according to the manufacturer’s instructions. Four PCR primers were used (Supplementary Table 9) to amplify a genomic single copy reference, ILV2, ARO10, and the Zeocin resistance cassette on genomic DNA templates from the pre-sorted library (PSL) as well as from the high-producers isolated from the second round of FACS in Fig. 2 (Y436-439, Y442 and Y443). The copy numbers of each amplification target were normalized to the counts of the single copy genome reference. As the upstream (A) and downstream pathway (B) share homology with the complete pathway (C), the following linear equations were solved to determine the number of integrations of each cassette, where X is the copy number of the zeocin resistance cassette, Y is the copy number of ILV2, and Z is the copy number of ARO10:
$${{{{{rm{A}}}}}}+{{{{{rm{B}}}}}}+{{{{{rm{C}}}}}}={{{{{rm{X}}}}}}$$
(1)
$${{{{{rm{A}}}}}}+{{{{{rm{C}}}}}}={{{{{rm{Y}}}}}}$$
(2)
$${{{{{rm{B}}}}}}+{{{{{rm{C}}}}}}+1={{{{{rm{Z}}}}}}$$
(3)
As ARO10 has a single integration in wild-type yeast, 1 copy is added to Eq. (3). The copies of unique strains are rounded to the nearest integer, while copies in PSL, are reported as the number of average integrations normalized per genome in the diverse library population.
Removal of plasmids from Saccharomyces cerevisiae
URA3 plasmids in yeast strains were removed by 5-fluoroorotic acid (5-FOA) selection79. Cells were first grown in YPD overnight and then streaked on SC agar plates containing 1 mg/mL 5-FOA (Zymo Research, Orange, CA, USA). A single colony from an SC/5-FOA agar plate was streaked again on a new SC/5-FOA agar plate. To confirm that strains were cured of the URA3-containing plasmid, they were inoculated into SC-ura medium supplemented with 2% glucose; strains lacking the URA3-containing plasmid were not able to grow in SC-ura medium.
Yeast plasmid isolation
Plasmid isolation from yeast was performed according to a user-developed protocol from Michael Jones (protocol PR04, Isolation of plasmid DNA from yeast, QIAGEN) using a QIAprep Spin Miniprep kit (QIAGEN, Valencia, CA, USA)80. The isolated plasmids were retransformed into E. coli DH5α to produce plasmids at higher titer for subsequent sequencing and retransformation into the parental yeast strain.
Analysis of BCHA production
The concentrations of isobutanol, and isopentanol were determined with high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity instrument (Agilent Technologies, Santa Clara, CA, USA). Samples were centrifuged at 17,000 g for 40 min at 4 °C to remove residual cells and other solid debris, and analyzed using an Aminex HPX-87H ion-exchange column (Bio-Rad, Hercules, CA USA). The column was eluted with a mobile phase of 5 mM sulfuric acid at 55 °C and with a flow rate of 0.6 mL/min for 50 min. The chemical concentrations were monitored with a refractive index detector (RID) and quantified by comparing the peak areas to those of standards with known concentrations.
Statistical analysis
Two-sided Student’s t tests were performed using GraphPad Prism (version 8.0 for Mac OS, GraphPad Software, San Diego, California USA, www.graphpad.com) to determine the statistical significance of differences observed in product titers between strains. Probabilities (P-values) less than (or equal to) 0.05 are considered sufficient to reject the null hypothesis (that the means of the two samples are the same) and accept the alternative hypothesis (that the means of the two samples are different).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
