2.1 Construction of plasmids and strains
The plasmids, strains, and primers used and constructed in this study are listed in Dataset S1. All yeast strains were constructed using GTR-CRISPR system as described in [8]. For gene integration and deletion, donor DNA containing 50 bp of homology arms and Cas9 plasmid containing gRNA targeting sequence were co-transformed using the electroporation method as described in [42].
Six genes for PDH pathway from Enterococcus faecalis [16] were synthesized by Sangon Company (Shanghai, China) and then cloned in pUC57 vectors, yielding plasmids pPdhB, pPdhA, pLpd, pAceF, pLplA and pLplA2, repectively. lplA and lplA2 were cloned using Golden Gate into the vector pAMPori with primers PP1-5, yielding pLplA_A2. These six genes were integrated into two sites XI-3 and XII-5 as described in [28]. lplA and lplA2 were integrated at XI-3 using the Cas9 plasmid pCas9_XI-3.3 and donor DNA amplified from pLplA_A2 with primers PP6 and PP7. pdhB, pdhA, lpd and aceF were integrated at the XII-5 site simultaneously using pCas9_XII-5 and donor DNA amplified from the corresponding plasmids with primers PP8-15.
Codon-optimized GapN from Streptococcus mutans [39] was synthesized by Sangon Company (Shanghai, China) and cloned in pAMPori vector using Golden Gate assembly with primers PP16-23, yield pGapN. GapN was then integrated into XI-1 using pCas9_XI-1 and donor DNA amplified with primers PP24 and PP25 from pGapN.
GPD2 was deleted using pCas9_gpd2 and donor DNA amplified with primers PP26 and PP27. GPD1 was deleted using pCas9_gpd1 and donor DNA amplified with primers PP28 and PP29. PAH1, DPP1, LPP1 and ARE1 were simultaneously deleted using pCas9_PDLA and donor DNA fragments with primers PP30-PP37. ZWF1 was deleted using pCas9_zwf1 and donor DNA amplified with primers PP38 and PP39. ALD6 was deleted using pCas9_ald6 and donor DNA amplified with primers PP40 and PP41. LPD was deleted using pCas9_lpd and donor DNA amplified with primer PP42 and PP43.
All Cas9 plasmids containing gRNAs for target genes and were constructed using Golden Gate assembly. Take pCas9_gpd2 as an example, PCR fragments amplified from pScURA with primer PP47, containing 20 bp gRNA targeting sequence, were assembled into pCas plasmid [8].
2.2 Media and cultivation conditions
E. coli strains were cultured at 37 °C in LB medium, composed of 5 g/L yeast extract,10 g/L peptone, and 10 g/L NaCl, and 80 mg/ml ampicillin was added when needed.
Yeast strains were cultured either in YPD medium composed of 10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose, or SC-URA medium for the selection of strains containing URA3-based plasmids, composed of 5 g/L (NH4)2SO4, 1.7 g/L yeast nitrogen base without amino acids, 1.914 g/L amino acid mixture without uracil, and 20 g/L glucose. 5-FOA plates were used to lose URA3-based plasmid, composed of 1.7 g/L yeast nitrogen base without amino acids, 5 g/L (NH4)2SO4, 1.914 g/L amino acid mixture without uracil, 0.05 g/L uracil, 1 g/L 5-FOA, 25 g/L agar. All solid plates contained 15 g/L agar except for 5-FOA plates.
Minimal medium was used for cultivation in shake flasks for FFA production, composed of 5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, trace metal solution and vitamin solution and 20 g/L glucose, and 40 mg/L histidine or 60 mg/L uracil was added when needed. For all PDH strains, minimal medium was supplemented with 100 mg/mL lipoic acid.
Yeast strains were cultured at 30 °C. For shake flask cultivation, yeast cells were precultured in falcon tubes with 5 ml minimal medium for 24 h, and then inoculated into 100 mL shake flasks with 30 mL medium with initial OD600 of 0.1.
2.3 Measurement of biomass and extracellular metabolites
Biomass was measured by optical density at 600 nm (OD600) with a GENESYS 30 Visible Spectrophotometer (Thermo Electron Scientific, Madison, USA).
Extracellular metabolites including glucose, ethanol and glycerol were measured by HPLC (Shimadzu LC-20AT, Japan) equipped with RID and UV detectors, using 5 mM H2SO4 as eluent and Aminex HPX-87H column (Bio-Rad) at 65 °C with a flow rate of 0.6 ml/min.
2.4 Extraction, measurement and analysis of FFAs
FFAs in the broth were extracted and measured as described in [6]. Specifically, 200 µl of cell culture or diluted cell culture was transferred into a glass vial, and 10 µl 40% tetrabutylammonium hydroxide and 200 µl methylation reagent was added immediately. Methylation reagent contained 200 mM methyl iodide as methyl donor and 12.5 mg/L pentadecanoic acid as an internal standard. The mixture was shaken for 30 min, and then centrifuged at 5000 g for 3 minutes. Then, 100 µl of dichloromethane layer with extracted methyl esters was transferred into a GC vial, evaporated 3 h, and resuspended in 100 µl hexane. Samples were analyzed by GC-MS (QP2020, Shimadzu, Japan) with a DB-5MS column (30m × 0.250mm × 0.25 µm, Agilent). The program was set as follows: 40 °C 2 min; ramp to 130 °C at 5 °C/min; raise to 280 °C at 10 °C/min; 280 °C 3 min. The temperatures of the inlet, mass transfer line and ion source were set at 280 °C, 300 °C, 230 °C, respectively. Flow rate of carrier gas was 3.0 mL/min and the data were collected in the full inspection mode (50–650 m/z) and analyzed using GCMS solution 4.4 software.
Samples for FFA analysis were harvested at different time points due to varied strain growth. The highest FFA titers were achieved when ethanol was completely consumed.