2. Experimental Section

2.1. Strains, plasmid, primers, and chemicals

This study lists all stains, recombinant plasmids, and primers in Supplementary Table1 and 2, respectively. Herein, strain po1g[20] was chosen as the starting strain. Chemicals used in this study, including glucose, (NH4)2SO4, and YNB (yeast nitrogen base without (NH4)2SO4 and amino acids), were all purchased from Sangon Biotech Co., Ltd (Shanghai, China). The Nano-Glo® Luciferase Assay System kit was purchased from Promega (Catalog: #N1120), and CSM-Leu powder was purchased from Sunrise Science Products (Catalog: #1001-100).

2.2. Construction of the luciferase reporter vectors

The reporter vectors were constructed based on the plasmid pYLXP’, and NanoLuc luciferase is encoded by the geneNluc .[19] Firstly, the chassis plasmid pYLXP’-Nluc was obtained by the Gibson Assembly method, using the gene Nluc fragment (PCR-amplified by primers Nluc-F and Nluc-R from pYLXP’-PTEF-Nluc ) and linearized pYLXP’ (digested by SnaBI and KpnI ). Then, plasmid pYLXP’-Nluc was further digested by SnaBI andAvrII , giving linearized pYLXP’-Nluc . Next, the promoter sequences of Pxx were obtained by PCR-amplified from the genome of Y. lipolytica using appropriate primers. Finally, the promoter sequences and linearized pYLXP’-Nluc were assembled to reporter plasmids pYLXP’-Pxx-Nluc by the Gibson Assembly method. The constructed plasmids were all sequenced by Sangon Biotech Co., Ltd (Shanghai, China).

2.3. Yeast transformation by the lithium acetate method

The standard protocol of the lithium acetate yeast transformation has been described in the previous report.[3] Briefly, cells were harvested from 0.5 ml culture solution at 24 h using the YPD medium by the shaking tube. Then, cells were washed twice using the phosphate buffer (PBS, 100 mM, pH 7.0) and resuspended by the transformation solution (105 uL), containing the lithium acetate (2M, 5 uL), PEG4000 (50%, 90 uL), boiled single strand DNA (salmon sperm, denatured, 5 uL), and reporter plasmids (5 uL). Next, the mixture was incubated at 39 °C for one hour, which needed to be vortexed for 15 seconds every 15 minutes. Finally, the mixture was spread on the CMS-leu selected plates. YPD medium used in this study included glucose 20 g/L, peptone 20 g/L, and yeast extract 10 g/L.

2.4. Shaking flask cultivations

For this, seed culture was carried out in the seed culture medium (2 mL) at 30 oC and 250 r.p.m for 48 h, using the shaking tube. Then, seed culture (0.8 mL) was inoculated into the CSM medium (C/N=80, 30 mL) in the 250 mL flask and grown at 30 oC and 250 r.p.m for 120 h. During the process of fermentation, 1 ml culture solution was sampled every 12 h for luciferase and OD600 measurements. The seed culture medium contains yeast nitrogen base without ammonium sulfate (YNB) 1.7 g/L, glucose 20.0 g/L, (NH4)2SO4 5.0 g/L, and CSM-Leu 0.74 g/L. Moreover, the CSM medium (C/N=80) contains YNB 1.7 g/L, glucose 40.0 g/L, (NH4)2SO4 5.0 g/L, and CSM-Leu 0.74 g/L.

2.5. Quantification of cell densities and the promoter strength

Cell densities of Y. lipolytica were monitored by measuring the optical density at 600 nm (OD600). The promoter strength was determined by performing the luciferase whole-cell assay analysis. In detail, 0.5 ml culture solution was centrifuged at 8,000 r.p.m for 3 min to collect the cell pellet. Then, the collected cell pellets were washed twice using the phosphate buffer (PBS, 100 mM, pH 7.0), and resuspended by the same buffer (1 ml). It should be noted that OD600 of cell pellet suspension solution needed to be measured and recorded. Next, the reaction mixture of the luciferase whole-cell assay was prepared for the luciferase activity assay by the microplate system (following the protocol of Nano-Glo® Luciferase Assay System kit), which contained the luciferase buffer 100 ul, substrate 2ul, cell pellet suspension solution 10 ul, and sterile water 88 ul. As a result, the promoter strength was obtained by dividing the luciferase activity data by the recorded OD600 of the cell pellet suspension solution.