Strains and reagents

Y. lipolytica strains and plasmids used are listed in Supplementary Tables 2 and 3. KAPA HiFi DNA Polymerase with high-fidelity was purchased from KapaBiosystems. GoTaq Green Master Mix was purchased from Promega Corp and used for colony PCR. Gibson assembly, chemical competent E. coli cell (DH 5α), and restriction enzymes were purchased from New England Biolabs. DNA gel recovery and plasmid extraction kits were purchased from Qiagen. All heterologous genes were codon-optimized and synthesized by ThermoFisher Scientific. All primers were synthesized by MilliporeSigma. Hexane, methanol, H₂SO₄, NaOH, and dodecane were purchased from MilliporeSigma. Yeast extract and peptone were from Bacto. The nitrogen content of the yeast extract was 11% (w/w). Yeast nitrogen base without amino acids and ammonium sulfate (YNB-AA^-AS^-) was from VWR Life Science. Alkane standards were purchased from VWR with purity higher than 98%.

Wheat straw was pretreated according to the previous work⁴². Briefly, 50 g wheat straw was cut to pieces with the average length of roughly 0.5 cm and added to 500 mL of 2% NaOH aqueous solution to a final solid concentration of 10%. The mixture was subsequently subjected to 121 °C for 1 h and filtered through a 20 mesh (0.85 mm) filter to collect the solids. The solids were then washed to reach neutral pH, concentrated by pressing water out, and yielded around 140 g pretreated wheat straw with water content of approximately 75%. This mixture was directly hydrolyzed by adding 10 mL of citrate buffer (pH 5.5) and 2 mL Cellic CTec2 (Sigma-Aldrich) for 4 days at 50 °C and 250 rpm. The final glucose concentration was 83.8 g/L in the hydrolysate.

Bjour Curtain String Lights 300 LED Window Curtain Icicle Blue Light manufactured by Twinkle Star was purchased from Amazon (Light source 1). Water-Resistance IP65, 12 V Waterproof Flexible LED Strip Light, 16.4 ft/5 m Cuttable LED Light Strips, 300 Units 2835 LEDs Lighting String manufactured by Tasodin was purchased from Amazon (Light source 2). The LED power was measured with a PD300-3W photodiode sensor (Ophir Photonics, https://www.ophiropt.com/laser–measurement/laser-power-energy-meters/products/Laser-Photodiode-Sensors/Standard-Photodiode-Sensors/PD300-3W) set at the LED peak wavelength connected to a Vega power meter (Ophir Photonics, https://www.ophiropt.com/laser–measurement/laser-power-energy-meters/products/smart-displays/vega).

Genetic manipulation

Gene fragments were either synthesized as shown in Supplementary Data 1 or amplified with primers listed in Supplementary Table 5. Gibson Assembly was used to ligate the fragments into ring form plasmids. Transformation of the resultant mixture into E. coli DH 5α by heat shock at 42 °C for 1 min, followed by plating cells on Petri dishes with LB agar containing corresponding antibiotics were performed. Colonies were checked with colony PCR using primers on promoter and structural gene to select positive ones. All plasmids harboring one cassette were constructed by the above-described procedures. Plasmids harboring repeated pTef-in-CvFAP-XPR2t cassette were constructed by the single restriction enzyme digestion, followed by Gibson Assembly. Plasmids were extracted from the cultivated positive strains and sequenced. All plasmids used in this work are listed in Supplementary Table 2.

All plasmids were linearized by restriction enzyme NotI or AseI before transformation into auxotrophic Y. lipolytica (URA3^-). The transformation medium was composed of 2.25 mL of 50% PEG 3350 in water, 0.125 mL 2 M lithium acetate, and 0.125 mL 2 M DTT in water. Transformation medium of 100 μL, 5 μL of single-strand DNA (10 g/L), and plasmid DNA of interest (1 μg) were mixed together with the auxotrophic parent strain, vortexed for 15 s, and placed in 28 °C for 30 min, followed by incubating at 39 °C for 30 min. The resultant mixture was spread on YNB-Uracil^- agar plate (see below for the composition) and allowed to be in 30 °C incubator for 2–3 days. Recombinants were checked by colony PCR and the positive ones were selected for the first-round screening fermentation.

Gene knockouts were performed according to the developed CRISPR method⁴³. Briefly, gRNA sequences were designed by Benchling with NGG as PAM. Homologous arms with 100 bps composed of 50 bps from the upstream and 50 bps from downstream were used to delete the part in between. The positive knockouts were examined by PCR with genomic DNA as templates. All primers, homologous arms, and gRNA sequences related to gene knockouts are listed in Supplementary Table 4.

After each transformation or knockout, colony PCR-positive Y. lipolytica strains were further screened by microplate fermentation. Briefly, 12 positive strains were inoculated to 12-well microplate containing 2 mL YEM medium in each well and cultivated in dark at 30 °C, 225 rpm for 2 days, followed by cultivation in blue light at 25 °C, 150 rpm for 1 day prior to alka(e)ne analyses. Strains with the highest alka(e)ne titer were selected for further studies.

Counterselection against 5 -Fluoroorotic acid (5-FOA) was performed to recover URA3 marker. Briefly, gene fragment of 1000 bp containing both upstream and downstream of URA3 was transformed into the Y. lipolytica strains of interest, followed by spreading on yeast nitrogen base medium (YNB-FOA) containing 1 mg/mL 5-FOA and 5 mg/mL uracil. The colonies that grew over 3 days were transferred onto an uracil-minus plate and a corresponding YPD plate to confirm the auxotroph.

Media and conditions

Luria–Bertani (LB) medium containing 10 g/L peptone (Bacto), 5 g/L yeast extract (Bacto), 10 g/L NaCl (Sigma), and corresponding antibiotics (carbenicillin 100 μg/mL, kanamycin 50 μg/mL, or chloramphenicol 34 μg/mL) was used to culture E. coli harboring plasmid. Culturing tubes with the inoculated medium was placed on a rotatory drum at 37 °C for around 15 h for plasmid proliferation.

Different solid media were prepared as follows. YNB-Uracil^- medium composed of 1.7 g/L YNB-AA^-SA^-, 20 g/L glucose (Sigma), 5 g/L ammonium sulfate, 15 g/L agar (Bacto), with an appropriate supplement of 0.77 g/L complete supplement mixture minus uracil (Sunrise science products) was used for selecting transformed Y. lipolytica strains. YNB-FOA medium containing 1.7 g/L YNB-AA^-SA^-, 5 g/L ammonium sulfate, 20 g/L glucose, 0.77 g/L complete supplement mixture and 1 g/L 5-fluoroorotic acid (Zymo Research) was used to counterselect the URA3-disrupted strains. YPD medium containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose was used to culture auxotrophic strains.

For alka(e)ne fermentation before process optimization, YEM medium composed of 1 g/L yeast extract, 6.9 g/L YNB-AA^-SA^-, and 20 g/L glucose was used unless stated otherwise.

Batch fermentation

Batch fermentations for alka(e)ne production were performed in either 50 mL conical glass flasks or Falcon polystyrene 12 well microplates for either 3 days or 2 days in dark followed by 3, 2, or 1 day in blue light. The temperature was controlled at 30 °C in dark while 25 °C in light. The light sources with a maximum peak wavelength of 454 and 458 nm (Supplementary Fig. 8a) were applied during light cultivation phase. The working volume for 50 mL conical glass flasks and 12 well plates was 13 mL and 2 mL, respectively with inoculum of the initial OD₆₀₀ of 0.1 and 0.5, respectively. The self-made instrument for cultivation in blue light is shown in Supplementary Fig. 16.

Batch fermentations with supplementation of palmitic acid-d₃₁

Fermentations were carried out by using strains YLjbl-2 and YLjbl-2-ΔFAA1. Medium containing 2 g/L yeast extract, 6.9 g/L YNB-AA^-SA^-, and 20 g/L glucose was used. Palmitic acid-d₃₁ (dissolved in absolute ethanol) with a final concentration of 5 g/L was added before switching to blue light cultivation. In control experiments, the same amount of absolute ethanol was added.

Fed-batch fermentation with glucose

Medium containing 2 g/L yeast extract, 6.9 g/L YNB-AA^-SA^-, and 20 g/L glucose was used for cell growth for 2 days in dark. Thereafter, glucose or glucose with different amounts of yeast extract composed of 200 g/L glucose and 5, 10, 15 g/L yeast extract, equivalent to a C/N ratio of 170, 85, and 57 was fed every 24 h until the total glucose reached 80, 120, and 160 g/L. From day 2 to day 7, the feeding medium was supplemented to increase the glucose concentration by 10 g/L each day. pH was not controlled. All fed-batch fermentations were carried out in 50 mL conical flasks with a working volume of 13 mL for 2 days in dark followed by culture in blue light generated by light source 2 at 25 °C (Supplementary Fig. 16). The initial OD₆₀₀ was 0.1 and feeding was conducted when the increase in glucose/acetate could be observed in Figs. 4 and 5.

Fed-batch fermentation with acetate

For fed-batch fermentation with acetate, medium containing 2 g/L yeast extract, 6.9 g/L YNB-AA^-SA^-, and 27.3 g/L sodium acetate was used for cell growth for 3 days in dark. Acetic acid was added at 4.5 days and 5 days to adjust pH to 6.8. Sodium acetate (273 g/L with 5 g/L yeast extract) was used as a feeding medium. The acetic acid added was 2.31 g/L and in total 57.3 g/L acetic acid equivalence was added. Yeast extract of 2.75 g/L was added during the whole fermentation process. For stabilizing pH, 5 mM PBS (pH 6.0) was used at the initial stage and HCl (6 M) was used to control pH around 6.8 every 24 h.

Fed-batch fermentation with wheat straw hydrolysate

For fed-batch fermentation with wheat straw hydrolysate, medium containing 1 g/L yeast extract (optimized), 6.9 g/L YNB-AA^-SA^-, and wheat straw hydrolysate with final glucose of 25.74 g/L was used for cell growth for 2 days in dark. The hydrolysate with a glucose concentration of 83.8 g/L without yeast extract supplement was used as feeding medium for 6 times. The total glucose and yeast extract added were 50.89 g/L and 1 g/L yeast extract, respectively, during the whole fermentation process without controlling the pH.

Analytical methods for metabolites

OD₆₀₀ was measured by a spectrophotometer against corresponding blanks. Dilutions were carried out to make sure the OD₆₀₀ value ranging from 0.1 to 0.9.

Glucose and acetic acid in the supernatant of cultures were analyzed by high-performance liquid chromatography (HPLC, Agilent Technologies, 1260 Infinity) equipped with a BioRad HPX-87H column (BioRad) and a refractive index detector (Agilent Technologies, 1260 Infinity). External standard curves using corresponding compounds with analytical grades were prepared for quantification purposes. Agilent OpenLab Software 7890B was used to collect and analyze data.

For alkane/alkene quantification, cell pellets from 0.05 to 1 mL cell culture depending on the content were collected by centrifuge at 18,406.75 × g for 5 min. Sodium hydroxide-methanol solution (0.5 mol/L) of 0.5 mL was mixed with cell pellets, followed by adding internal standards, glyceryl triheptadecanoate (Sigma, 2 g/L) and dodecane (Sigma, 37.5 mg/L) in hexane. The mixture was sonicated for 15 min and vortexed (Vortex-Genie 2, Scientific Industries) for 1 h at room temperature (24 ± 2 °C) prior to being acidified by adding 60 μL condensed H₂SO₄ (Sigma) with caution. Hexane (0.5 mL) was added and vortexed for 30 min to extract fatty acid methyl esters and alkanes/alkenes. External standard curves were prepared by running the same procedure using corresponding standard compounds of analytical grade. The upper phase after centrifugation was analyzed by GC-MS (Agilent Technologies, 7890B Series GC and a 5977B MS) or GC-FID (Agilent 7890B). For GC-MS, 3 μL of extract in hexane was injected without split into an HP-5MS UI column (Agilent Technologies). Whereas for GC-FID, 1 μL of extract in hexane was injected with the split ratio of 2:1 into an HP-INNOWAX column (Agilent Technologies). For GC-FID, the inlet and FID temperature was held at 260 °C using the following temperature program: initial 2 min at 40 °C, then ramped to 200 °C at a rate of 20 °C/min, held for 2 min, and ramped to 250 °C at a rate of 40 °C/min, and held for 5 min. For GC-MS, the inlet, transfer tube, and MS source temperatures were set at 320, 300, and 280 °C, respectively. The MS was operated in scan mode from 100 to 450 m/z. The temperature program was: initial 2 min at 40 °C, then ramped to 270 °C at a rate of 12 °C/min, and ramped to 310 °C at a rate of 6 °C/min, and held for 10 min. The standard curves of different alka(e)nes were shown in Supplementary Fig. 17. Agilent OpenLab Software 7890B was used to collect data.

For FFA quantification, 0.5 mL culture was mixed with 80 μL glass beads, 150 μL methanol, and 150 μL hexane containing pentadecanoic acid as internal standard. The mixture was rigorously vortexed for 2 h, followed by centrifugation to facilitate phase separation. The upper hexane phase was taken for GC-MS analysis. The inlet, transfer tube, and MS source temperatures were set at 320, 300, and 280 °C, respectively. The MS was operated in scan mode from 150 to 500 m/z. The temperature program was: initial at 100 °C, then ramped to 270 °C at a rate of 12 °C/min, and ramped to 310 °C at a rate of 6 °C/min, and held for 10 min. Helium was used as a carrier gas.

Substrate docking and structural analysis

Crystal structure images were produced by using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR‐01081)⁴⁴. Substrate docking was performed by using AutoDock Vina 1.1.2, with implementation through Chimera 1.13.1rc⁴⁵. Protein structure (with fatty acid substrate and FAD deleted) and fatty acyl-CoA substrate were prepared by using the DockPrep tool of Chimera. Analysis of the resulting docking modes was performed through the ViewDock tool, which listed 10 docking modes according to their energy scores. The best docking model was decided by a combination of criteria: visual inspection (correct orientation of the fatty acid and nucleotide ends of acyl-CoA, match of the nucleotide binding site) and energy scores.

Quantitative real-time PCR and reverse transcription–qPCR analyses

For quantitative real-time PCR, total genomic DNA extracted from each individual strains of interest by using the Phenol-Chloroform protocol was used as template. For reverse transcription–qPCR, mRNA extracted by MasterPure^TM Yeast RNA purification kit (Lucigen, Wisconsin, USA) was used as template. PCRs were performed using iScript^TM one-step RT-PCR kit with SYBR^® green (Bio-Rad) with the designed primers of CvFAP and internal reference ACT (Supplementary Table 5) on an iCycler (Bio-Rad), according to the manufacturer’s instructions. Relative gene expression was performed using the comparative 2^-∆∆Ct or 2^-∆Ct method.

CvFAP protein expression and purification

The construct consisting of the CvFAP gene with an optimized codon for Y. lipolytica and a sequentially 6×His tag on 3′-terminus were cloned into a pET28 vector backbone using Gibson Assembly. The plasmid was then transformed in E. coli DH5α and the positive colonies were identified by colony-PCR. Three plasmids were extracted and sequenced to select the correct one for protein expression.

The selected plasmid was transformed in E. coli BL21 (DE3) (purchased from NEB). The recombinant was precultured in 5 mL LB medium containing 50 μg/L kanamycin overnight. The preculture was then inoculated to 500 mL medium with the same composition as the preculturing medium. IPTG was added with the final concentration of 0.5 mM when the OD₆₀₀ reached 0.8–0.9. The culture was then moved to an incubator at 16 °C and 160 rpm for 20 h. Cells were harvested by centrifugation at 4 °C and the cell pellet was washed with Tris-HCl buffer (50 mM, pH 8.5) once.

The cell pellet was resuspended by NPI-10 for cell disruption. The suspended cells were disrupted by a sonic disruptor on ice for 30 min. Protein purification was carried out according to the protocol (https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Protino/UM_Protino96NTA.pdf). The resultant protein fractions were examined by SDS-PAGE as shown in Supplementary Fig. 18. Fractions from NPI-125 and NPI-250 were combined for following buffer change. Buffer change was performed by washing NPI-125 and NPI-250 fractions through 10 kD cut-off using Tris-HCl buffer (50 mM, pH 8.5) for 3 times. The obtained protein was used for in vitro photodecarboxylation experiments.

In vitro photocatalytic reactions

The reactions using stearoyl-CoA lithium salt (Sigma) and stearic acid lithium salt as substrates were performed at 25 °C in a total volume of 100 μL. Different volumes of substrates in DMSO (0.97 mM), a fixed amount of purified enzyme (20 μL, 3.30 mg/mL), pure DMSO, and Tris-HCl buffer (50 mM, pH 8.5) were added in transparent glass vials (2 mL) with final DMSO of 30 vol%. The vials were screw-capped and placed in the shaker with blue light for 20 h at 150 rpm. The product was extracted with ethyl acetate and analyzed by the early-mentioned GC-FID method.

Cell viability measurements

To measure cell viability, Trypan blue (Sigma-Aldrich, 0.4% in water) was added directly to aliquots of undiluted cultures with a final concentration of 0.2% and visualized immediately at 1000× magnification on a ZEISS Axioskop (West Germany) by bright field microscope. Images were recorded using a Nikon color camera.

Staining measurements of alka(e)nes and cells containing alka(e)nes

An artificial mixture of 10 mg of alka(e)nes containing C17:0, C17:1 (terminal double bond), and C15:0 was mixed with 0.5 mL water and 100 μL Tween 80. The mixture was vigorously vortexed at 1000 rpm for 5 min before being stained by Nile red (Ethanol solution, 0.1 μg/ml). Images were taken by using a DeltaVision2-TIRF Microscope.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.