Comparison of Yarrowia lipolytica growth and rProt synthesis when cultured in different vessels

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1. Introduction The aim of this experiment was to quantitatively describe the costs of culturing Y. lipolytica strain synthesizing a fluorescent reporter protein (rProt) in different volumes / different vessels. 2. Methodology The Yarrowia lipolytica strain used in this experiment was JMY2810 (genotype: MATa, ura3::pTEF-RedStar2-LEU2-Zeta-URA3ex-pTEF-empty, leu2-270, xpr2-322; phenotype: ΔAEP, ΔAXP, suc+, ura+, leu+, intracellular RedStarII, Zeta platform). The YPG medium was composed as follows [g L-1]: yeast extract, 10 (BTL, Lodz, Poland); peptone 20 (BTL); glycerol, 35 (POCH). Precultures were developed in standard YPG medium. All the media were autoclaved. Technical parameters of the culturing vessels used and shaking parameters are presented in Table 1 (pdf). The main cultures were continued for 48 h and samples were collected at the end of the cultivation time. Precultures were developed for 18h at 28 °C in the 300mL shake flasks. The main cultures were inoculated at 5% (v/v). Samples were analyzed for growth and fluorescence from the reporter protein (RedStar2) following dilution in 0.75% NaCl (POCH) to match a linear range of the methods. Absorbance was measured at 600 nm in transparent 96-well plates (Costar; Merck). FL was determined under at ex/em 550/595 nm in black opaque plates (Thermo Fisher Scientific). Both measurements were done using a Tecan Spark automatic plate reader (Tecan Group Ltd., Mannedorf, Switzerland). pH level at the end of cultures was determined in the supernatant using indicatory strips (POCh, Poland). The concentration of the carbon source and the main metabolites was analyzed through HPLC, under the following conditions: Agilent Technologies 1200 series (Agilent Technologies, Santa Clara, USA), equipped with a refractive-index detector (G1362A) and a Rezex ROA-Organic Acid H+ column (Phenomenex, Torrance, USA), and 0.005 N H2SO4 as eluent at a flow rate of 0.6 [mL/min], at 40 °C. The 96-well plate cultures were pooled to obtain sufficient amounts of the supernatant. The analysis was conducted in technical duplicate. Statistical significance of the difference in a given measure was assessed by analysis of variance (ANOVA) test, with a significance level set at p-value < 0.05 (RStudio and Visual Studio Code, Microsoft), and equality of variances was checked with the Levene test. The homogenous groups were calculated using Tukey HSD post-hoc analysis (RStudio with relevant packages). The results were calculated based on 5 – 48 biological repetitions. 3. Results The raw results from fluorescence and optical density measurements, as well as the HPLC data, are presented in the Table S1 (xlsx) excel file. A graphical presentation of the data processed using statistical analysis is given in the main manuscript as Figures 1 to 4.

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