Experiments

    In this project, we attempted to engineer the nonconventional oleaginous yeast Yarrowia lipolytica (Y. lipolytica) as a competitive platform host to produce 2-PE. As a result, the heterologous production of 2-PE from Y. lipolytica is considered as an economically viable alternative to plant extraction.

    All engineering strains were performed the optimized screening before shake flask cultivations. For performing shake flask cultivations, seed culture was carried out in the shaking tube with 2 mL seed culture medium at 30 oC and 250 r.p.m. for 48 h. Then, 0.8 mL of seed culture was inoculated into the 250 mL flask containing 35 mL of fermentation medium and grown under the conditions of 30 oC and 250 r.p.m. for 144 h. One milliliter of cell suspension was sampled every 24h for OD600, glucose, 2-PE, L-phenylalanine, and penylacetate measurements.

    Seed culture medium used in this study was the yeast complete synthetic media regular media CSM containing: glucose 20.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, ammonium sulfate 5.0 g/L, and CSM-Leu or CSM-Ura 0.74 g/L. Two types of fermentation medium were used in our work includes nitrogen-limited media CSM and YNB. The nitrogen-limited media CSM contained: glucose 40.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, ammonium sulfate 1.1 g/L, CSM-Leu 0.74 g/L, and appropriate L-phenylalanine. The nitrogen-limited media YNB contained: glucose 40.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, leucine or uracil 0.2 g/L, and appropriate L-phenylalanine.

    For prepare the whole-cell biocatalyst, cells were harvested during the exponential growth phase (48 h) from the shake flask cultivation. Then, cells were washed twice with 100 mM phosphate buffer (pH 7.0), and resuspended to an OD600 of 4 in the same buffer. Next, whole-cell biocatalytic conversion of phenylacetaldehyde was performed in 20-ml glass tube containing 1 mL of cell suspension (OD600=4) and 1 mL phenylacetaldehyde-water solution (2 g/L phenylacetaldehyde) at 30 oC and 250 r.p.m. for 4 h. One hundred microliter of cell suspension was sampled every 1 h for 2-PE and penylacetate measurements.

    The standard protocols of Y. lipolytica transformation by the lithium acetate method were described as previously reported (Liu et al., 2019; Lv et al., 2019). In brief, one milliliter cells was harvested during the exponential growth phase (16-24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) in the 14-mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Then, cells were resuspended in 105 uL transformation solution, containing 90 uL 50% PEG4000, 5 uL lithium acetate (2M), 5 uL boiled single stand DNA (salmon sperm, denatured) and 5 uL DNA products (including 200-500 ng of plasmids, lined plasmids or DNA fragments), and incubated at 39 oC for 1 h, then spread on selected plates. It should be noted that the transformation mixtures needed to be vortexed for 15 seconds every 15 minutes during the process of 39 oC incubation. The selected markers, including leucine and uracil, were used in this study. All engineering strains after genetic manipulations were performed optimized screening by the shaking tube cultivations, and the optimal strain was used to perform shaking flask (these data have been shown in Supplementary Materials).

    The YaliBrick plasmid pYLXP’ was used as the expression vector in this study(Wong et al., 2017). Plasmid constructions were performed by using preciously described methods(Lv et al., 2019). In brief, recombinant plasmids of pYLXP’-XX (a single gene expression) were obtained by Golden Gate method using linearized pYLXP’ and the appropriate PCR-amplified DNA fragment. Multi-genes assembly was achieved by restriction enzyme digestion subcloning based on the application of isocaudamers AvrII and NheI(Wong et al., 2017). All genes were respectively expressed by the TEF promoter with intron sequence and XPR terminator. The modified DNA fragments and plasmids were sequenced by Quintarabio. The endonucleases used in this research were purchased from Thermo Fisher Scientific or NEB.

    A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported(Fickers et al., 2003). For performing gene knockout, the upstream and downstream sequences (both 1000 bp) flanking the deletion targets were PCR-amplified. These two fragments, the loxP-URA-loxP cassette (digested from plasmid pYLXP’-loxp-URA by by BasI), and the residual plasmid backbone of pYLXP’-loxp-URA were ligase, obtaining the gene knockout plasmids pYLXP’-loxP-URA-XX. The obtained plasmids were sequenced by Sangon Biotech Co., Ltd (Shanghai, China). Next, the gene knockout cassettes were PCR-amplified from construction plasmids pYLXP’-loxp-URA-XX, and further transformed into Y. lipolytica. The positive transformants were determined by colony PCR. Next, plasmid pYLXP’-Cre was introduced into the positive transformants and promoted the recombination of loxP sites, which recycle the selected marker. Finally, the intracellular plasmid pYXLXP’-Cre was evicted by incubation at 30 oC for 48h.

    Cell densities were monitored by measuring the optical density at 600 nm (OD600). The concentrations of 2-PE, penylacetate, and L-phenylalanine were measured by high-performance liquid chromatography (HPLC) through Agilent HPLC 1220 equipped with a ZORBAX Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm, Agilent) and a VWD detector. The analysis was performed at 215 nm under 40 oC column temperature with a mobile phase comprising 50% (v/v) methanol in water at a flow rate of 0.5 mL/min.

    Fickers, P., Le Dall, M.T., Gaillardin, C., Thonart, P., Nicaud, J.M. 2003. New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods, 55(3), 727-37.

    Liu, H., Marsafari, M., Wang, F., Deng, L., Xu, P. 2019. Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica. Metab Eng, 56, 60-68.

    Lv, Y., Edwards, H., Zhou, J., Xu, P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol.

    Wong, L., Engel, J., Jin, E., Holdridge, B., Xu, P. 2017. YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metab Eng Commun, 5, 68-77.

    Xu, P., Li, L.Y., Zhang, F.M., Stephanopoulos, G., Koffas, M. 2014. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. PNAs, 111(31), 11299-11304.