Overview
In this project, we constructed xylose reductase-expressing strains with different enzyme sources to catalyze xylose for xylitol production in Escherichia coli. The strategy of promoter engineering was utilized to improve the efficiency of conversion. We overexpressed the erythritol synthesis pathway enzyme in Yarrowia lipolytica. Based on a high-throughput screening strategy, we constructed fluorescent reporter system for characterizing erythritol production and improving erythritol production in Y.lipolytica.
Shake flask culture of E. coli
All engineering strains were optimized and screened before shake flask culture. For shake flask culture, 500 uL of seed solution was transferred to 30 mL LB+kana+Cm medium, incubated at 37℃ and 220 rpm for 3 h, added 0.1 mmol/L inducer IPTG, incubated at 28℃ and 220 rpm under shaking bed for 20 h. 1.5/3 mL of 100 g/L xylose precursor was added to 30 mL of fermentation broth, transformed at 30℃ and 220 rpm for 36 h. Samples were taken every 12 h, OD600 was measured, and samples were retained for HPLC to determine the xylitol yield. The seed medium used in this study was LB+kana medium: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, and kana 50 mg/L. The fermentation medium was TB+kana+Cm medium: tryptone 12 g/L, yeast extract 24 g/L, glycerol 4 ml/L, KH2PO4 2.31 g/L and K2HPO4 12.54 g/L.
Shake flask culture of yeast
The engineered strain was selected from the plate po1g-pylxp- ylER-EcYida, and the control strain po1g was selected from the po1g plate, both of which were transferred to CSM-URA seed solution, and the seed solution was obtained by shaking bed culture at 30℃ for 48 h. Transfer 500 uL seed solution in 30 mL YNB-URA fermentation medium, 30℃ and 220 rpm shaker culture 120 h, during the period of every 24 h sampling, and detection of OD600. Preparation of YNB-Leu fermentation broth: glucose 40 g/L, YNB (no ammonium sulfate and no amino acid yeast nitrogen source) 1.7 g/L, CSM-Leu (leu-free) 0.74 g/L, (NH4)2SO4 1.1 g/L. Preparation of YNB-URA fermentation broth: glucose 40 g/L, YNB (no ammonium sulfate and no amino acid yeast nitrogen source) 1.7 g/L, CSM-URA (URA -free) 0.74 g/L, (NH4)2SO4 1.1 g/L.
E.coli transformation
The pYLXP' recombinant plasmid was transformed into E. coli DH5α, incubated on ice for 30 min and then heat-excited at 42°C for 1 min. The bacterial solution was spread on LB+amp plates and incubated at 37°C in an incubator for 14-16h. The pET-28a recombinant plasmid transformation procedure is the same, only the antibiotic (chloramphenicol) needs to be changed.
Yeast transformation
The standard protocol for transformation of defatted yeast by lithium acetate method was as previously reported (Islam, Zhisheng et al. 2015, Xu, Wu et al. 2022). Briefly, during the exponential growth period (16-24 h), 1 mL of the bacterium was extracted from 2 mL of YPD medium (yeast extract 10 g/L, peptone 20 g/L, and dextrose 20 g/L) in a 14 mL shaker tube and washed twice with 100 mM phosphate buffer (pH 7.0). Then, the cell were resuspended in 105 µL transformation solution, containing 90 uL of 50% PEG 4000 solution, 5 μL lithium acetate (2 mol/L), 5 uL boiled single strand DNA (salmon spermatozoa, denatured), and 5 μL DNA products (including 200-500 ng of plasmid, liner plasmid, or DNA fragment), which were mixed well and then heated and incubated for 1 h at 37°C in a water bath, and finally coated on selected solid plates. It should be noted that the transformation mixtures needed to be oscillated every 15 min for 15s during the process at 37°C incubation. The selected markers chosen in this study were leucine or uracil.
Expression vector construction and plasmid assembly
The YaliBrick plasmid pYLXP' and plasmid pET-28a were used as the expression vectors in this study (Wong, Engel et al. 2017). Plasmid constructions were performed by using preciously described methods (Lv, Edwards et al. 2019). In brief, utilized linearized pYLXP' and the corresponding PCR-amplified DNA fragments, and the recombinant plasmid of pYLXP ' -XX (single-gene expression) was obtained by golden gate assembly. The recombinant plasmid of pYLXP ' -XX-XX was obtained by restriction endonuclease and T4 ligase for multi-gene assembly (Wong, Engel et al. 2017). The modified DNA fragments and plasmids were sequenced by Sangon Biotech (Shanghai, China). The endonucleases used in this study were purchased from Thermo Fisher Scientific or Takara.
The relevant plasmid construction steps for pET-28a were as follows: linearized pET-28a and the corresponding PCR-amplified DNA fragments were obtained by PCR amplification, and the recombinant plasmid of pET-28a -XX (single gene expression) was obtained by Gibson assembly method. Sequencing was performed by Sangon Biotech (Shanghai, China).
Fluorescence intensity detection
The reaction mixture for luciferase whole-cell assay was prepared according to the operating procedure of Nano-Glo Luciferase Assay System Kit, including 100 μL of luciferase buffer, 2 μL of substrate, 10 μL of cell suspension and 88 μL of sterile water. The program of multifunctional microtiter plate detector was set up for 60 cycles of 90s each, in which the plate was vibrated for 10s, the detection was performed for 80s, and the total time of each detection was 90min. The total time of each assay was 90min. The data of each assay were integrated to obtain the luciferase activity data, and then divided by OD600 to obtain the unit fluorescence intensity (RUL/OD600).
GFP fluorescence intensity measurement
The samples of 200 μL were transferred into the 96-well transparent microplate for GFP fluorescence intensity. The fluorescence intensity was measured at 485 nm excitation wavelength and 520 nm emission wavelength. Measured the fluorescence intensity to screening high-yield strains.
HPLC detection of xylitol and erythritol content
1ml of the sample was centrifuged at 12000 rpm for 15 min, and the liquid was passed through an water-based filter membrane and HPLC was performed to detect the content of xylitol/erythritol in the sample. Gradient concentrations (1g, 2g, 3g, 4g, 5g) of xylitol and erythritol were prepared and the standard curve was plotted. The concentrations of xylitol and erythritol were measured by Agilent HPLC 1220 equipped with a SupelcogelTM Carbohydrate column (Sigma, USA) and a refractive index detector. H2SO4 (5 mM) was used as the mobile phase at a flow rate of 0.6 mL/min at 35°C.
Reference
Islam, Z. U., Y. Zhisheng, B. Hassan el, C. Dongdong and Z. Hongxun (2015). "Microbial conversion of pyrolytic products to biofuels: a novel and sustainable approach toward second-generation biofuels." J Ind Microbiol Biotechnol 42(12): 1557-1579.
Lv, Y., H. Edwards, J. Zhou and P. Xu (2019). "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica." ACS Synth Biol 8(3): 568-576.
Wong, L., J. Engel, E. Jin, B. Holdridge and P. Xu (2017). "YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica." Metab Eng Commun 5: 68-77.
Xu, Y., Y. Wu, Y. Liu, J. Li, G. Du, J. Chen, X. Lv and L. Liu (2022). "Sustainable bioproduction of natural sugar substitutes: Strategies and challenges." Trends in Food Science & Technology 129: 512-527.