(A) Overview of astaxanthin biosynthesis pathway. The pathway before β-carotene has been optimized in former study and boxed by blue line here. Meanwhile, the pathway from β-carotene to astaxanthin was boxed by red line and engineered in this study. (B) Sketch map of CrtW-CrtZ expression cassette plasmids (pWRZ01~30). CrtW-CrtZ expression cassette was carried by a multiple copy plasmid pRS425k. Expression modules for CrtW (TDH3p-crtW-TDH2t) and CrtZ (FBA1p-crtZ-ADH1t) were arranged back-to-back with opposite transcriptional direction. Promoters, enzymes recoding sequences and terminators were presented as triangles, arrows and boxes, respectively. (C) HPLC analysis of the parent strain S. cerevisiae SyBE_Sc118030 (blue) and astaxanthin producing strain SyBE_Sc118040 (red). Strain SyBE_Sc118030 showed a significant β-carotene peak (I) at 21.2 min, while strain SyBE_Sc118040 showed astaxanthin peak (V) at 8.0 min along with other peaks for the identified intermediates, such as zeaxanthin (IV) at 8.0 min, canthaxanthin (III) at 9.9 min and lycopene (II) at 19.3 min

(A) Phylogenetic analysis of CrtZs and CrtWs protein sequences. Phylogenetic trees were constructed based on the protein sequences of CrtZ and CrtW, respectively. The particular CrtZ and CrtW in one tested group were connected by solid lines. The CrtW/CrtZ combinations, which were evaluated in shake flask, were highlighted in dark blue lines. (B) Visual color screening of CrtZ/CrtW combinations on solid SD medium. Thirty astaxanthin producing strains were constructed by introducing heterologous CrtW and CrtZ from various sources and tested for astaxanthin production primarily by their colors. The yellow stars indicated the control strain SyBE_Sc118031 without CrtW or CrtZ. (C) Determination of astaxanthin production in shake flasks. Strains processing intense pigment were picked up visually and cultured in shake flasks to measure their carotenoids levels by HPLC. β-Carotene, zeaxanthin, canthaxanthin and astaxanthin were separated on a BDS HYPERSIL C18 column (150 mm×4.6 mm, 5 mm, Thermo Scientific) and detected by a UV/VIS detector (Waters 2489) at 470 nm. The error bars represent standard deviations calculated from duplicate experiments. Aa, A. aurantiacum; Asp, Alcaligenes sp. strain PC-1; BDC263, Brevundimonas sp. DC263; BSD212, Brevundimonas sp. SD212; Eu, E. uredovora; Gv, G. violaceus PCC 7421; Pa, P. agglomerans; SsP2, S. solfataricus P2. (D) The correlation of the evolutionary distance between CrtZs and the corresponding astaxanthin yield. (E) The correlation of the evolutionary distance between CrtWs and the corresponding astaxanthin yield

(A) The relative expression level of CrtZ to CrtW was adjusted by changing the promoter of CrtZ from FBA1p to TEF1p. Strains were cultured in YPD medium with 2%, 4%, 5% and 10% (w/v) glucose. (B) The production of astaxanthin along with other carotenoids intermediates were analyzed by HPLC. (C) Meanwhile, the transcription level of genes crtZ and (D) crW in the engineered strains under 2% and 4% (w/v) glucose concentration were analyzed by Real-Time PCR. Cells were harvested after 10 h (early exponential phase), 14 h (middle exponential phase) and 30 h (late exponential phase). The relative transcription level for each gene (C,D) was determined as 2^−ΔΔCt using gene ACT1 for normalization. The relative ratio of crtZ to crtW (E) was calculated as 2^{−ΔCt(crtZ)}/2^{−ΔCt(crtW)}. All data are from duplicate experiments. Significant levels of t-test: * P<0.05, ** P<0.01

(A) Astaxanthin producing strain SyBE_Sc118076 was fed-batch cultured in a 5-L bioreactor. (B) The glucose concentration (blue line, B) was maintained at lower than 1 g/L by controlling the feeding rate. Biomass (OD600) and astaxanthin production were indicated by red line and green line, respectively. The error bar here represented two batches of independent bioreactor experiments

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