Additional file 1 of In vitro and in vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast
Creators: name: Qi, Dan-Dan
name: Jin, Jin
name: Liu, Duo
name: Jia, Bin
name: Ying-Jin Yuan
Publisher: name: figshare
Year: 2020
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. HPLC analysis of the yQDD000 and yQDD001. a Beta-carotene producing strain yQDD000 showed an onefold β-carotene peak at 20.2 min. b Strain yQDD001 showed astaxanthin peak at 6.4 min along with other peaks for the identified intermediates, such as zeaxanthin (IV) at 7.5 min, canthaxanthin (III) at 10.5 min and lycopene (II) at 18.3 min. Figure S2. Principle of the in vitro recombination. Marker for Hygromycin, Ura3 and His3 were used to screen the acceptor vector, crtZ and crtW donor vector respectively. Then acceptor fragments and donor fragments with LoxPSym sites were exposed by restriction enzyme digestion, producing the in vitro recombination reaction pool. Next the pool of diverse plasmids was produced by the action of Cre recombinase. Reaction pool was transformed to target yeast and screening for the yeast library with different astaxanthin yield. The darker red colonies were selected for genotype and phenotype assay. Figure S3. Copy number analysis of crtZ and crtW in strains that selected from in vitro recombination. a The crtZ copies number analysis of yQDD002, yQDD003, yQDD004, yQDD005, yQDD009, yQDD010. b The crtW copies number analysis of yQDD006, yQDD007, yQDD008, yQDD009, yQDD010. The results indicated that there have only one copy of crtZ or crtW in the yQDD002-yQDD010. Figure S4. Homologous arm design of in vivo recombination. Ty1 retrotransposon site is consisted of TyA and TyB. And there are two δ sites on the each side of Ty1 site. Two homologous arms of Ty1 (Ty-1 and Ty-2) were integrated into the flank of the crtZ/crtW fragment. Figure S5. Principle of the in vivo recombination. The DNA fragments of in vivo recombination were linearized by the digestion of NotI. Then the pool of all the DNA fragments was transformed into yQDD001, generating the yeast library with different color. The darker red colonies were selected for genotype and phenotype assay. Figure S6. Copy number analysis of crtZ and crtW in strains that selected from in vivo recombination. a The copies number analysis of crtZ in yQDD011, yQDD012, yQDD013, yQDD017, yQDD018, yQDD019, yQDD020, yQDD021 and yQDD022. b The copy number analysis of crtW in yQDD014, yQDD015, yQDD016, yQDD017, yQDD018, yQDD019, yQDD020, yQDD021, yQDD022. Figure S7. Transformation efficiency comparison between the in vitro and the in vivo recombination. The colony’s number after yeast transformation with three biological repeats was used to assess the efficiency of the in vitro and the in vivo recombination. All the experimental conditions remained consistent, including the concentration of DNA fragment of crtZ and crtW, the biomass of host strain yQDD001 and other operating environments. Photograph of the in vitro and the in vivo screening was attached. The colony’s number was listed in Fig. S6. Compared with in vitro recombination, the much more colonies of in vivo screening indicating the high transformation efficiency of the in vivo recombination. Figure S8. Color stability assay of yQDD008 and yQDD022. The yQDD008 and yQDD022 were serially subcultured in YPD for 6 days and screened on SD agar each day. Figure S9. Profile of glucose titer during fermentation in 250-mL flasks with strain yQDD008 and yQDD022. The profile of glucose titer during fermentation was similar between yQDD008 and yQDD022. Table S1. PCRTag used in this study. Table S2. Primers used in this study.