Introduction
Materials and methods
Strains and media
Tab.1 Strains used in this study |
S. cerevisiae strains | Description | Sources |
---|---|---|
SyBE_Sc118030 | BY4741, Delta::URA3-TEF1p-crtE-PDX1t-TDH3p-crtI-MPE1t-FBA1p-crtYB-TDH2t, Dypl062w::HIS3_TDH3p-BTS1-ERG20-PGI1t-TEF1p-tHMG1-TEF2t | This lab |
SyBE_Sc118031 | SyBE_Sc118030 with pRS425k | This study |
SyBE_Sc118040 | SyBE_Sc118030 with pWRZ01 (pRS425k-ADH1t-AacrtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118041 | SyBE_Sc118030 with pWRZ02 (pRS425k-ADH1t-AspcrtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118042 | SyBE_Sc118030 with pWRZ03 (pRS425k-ADH1t-BDC263crtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118043 | SyBE_Sc118030 with pWRZ04 (pRS425k-ADH1t-BSD212crtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118045 | SyBE_Sc118030 with pWRZ05 (pRS425k-ADH1t-EucrtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118046 | SyBE_Sc118030 with pWRZ06 (pRS425k-ADH1t-PacrtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118047 | SyBE_Sc118030 with pWRZ21 (pRS425k-ADH1t-PscrtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118048 | SyBE_Sc118030 with pWRZ07 (pRS425k-ADH1t-SsP2crtZ-FBA1p-TDH3p-AacrtW-TDH2t) | This study |
SyBE_Sc118051 | SyBE_Sc118030 with pWRZ08 (pRS425k-ADH1t-Hpchyb-FBA1p-TDH3p-AspcrtW-TDH2t) | This study |
SyBE_Sc118053 | SyBE_Sc118030 with pWRZ09 (pRS425k-ADH1t-AspcrtZ-FBA1p-TDH3p-AspcrtW-TDH2t) | This study |
SyBE_Sc118054 | SyBE_Sc118030 with pWRZ22 (pRS425k-ADH1t-Ssp2crtZ-FBA1p-TDH3p-BSD212crtW-TDH2t) | This study |
SyBE_Sc118055 | SyBE_Sc118030 with pWRZ23 (pRS425k-ADH1t-BSD212crtZ-FBA1p-TDH3p-BSD212crtW-TDH2t) | This study |
SyBE_Sc118056 | SyBE_Sc118030 with pWRZ24 (pRS425k-ADH1t-EucrtZ-FBA1p-TDH3p-BSD212crtW-TDH2t) | This study |
SyBE_Sc118057 | SyBE_Sc118030 with pWRZ10 (pRS425k-ADH1t-Hpchyb-FBA1p-TDH3p-BSD212crtW-TDH2t) | This study |
SyBE_Sc118058 | SyBE_Sc118030 with pWRZ30 (pRS425k-ADH1t-PscrtZ-FBA1p-TDH3p-BSD212crtW-TDH2t) | This study |
SyBE_Sc118060 | SyBE_Sc118030 with pWRZ11 (pRS425k-ADH1t-AspcrtZ-FBA1p-TDH3p-BDC263crtW-TDH2t) | This study |
SyBE_Sc118062 | SyBE_Sc118030 with pWRZ25 (pRS425k-ADH1t-HpChyb-FBA1p-TDH3p-BDC263crtW-TDH2t) | This study |
SyBE_Sc118063 | SyBE_Sc118030 with pWRZ12 (pRS425k-ADH1t-SsP2crtZ-FBA1p-TDH3p-BDC263crtW-TDH2t) | This study |
SyBE_Sc118064 | SyBE_Sc118030 with pWRZ13 (pRS425k-ADH1t-PscrtZ-FBA1p-TDH3p-BDC263crtW-TDH2t) | This study |
SyBE_Sc118065 | SyBE_Sc118030 with pWRZ26 (pRS425k-ADH1t-EucrtZ-FBA1p-TDH3p-BDC263crtW-TDH2t) | This study |
SyBE_Sc118066 | SyBE_Sc118030 with pWRZ14 (pRS425k-ADH1t-Hpchyb-FBA1p-TDH3p-GvcrtW-TDH2t) | This study |
SyBE_Sc118067 | SyBE_Sc118030 with pWRZ15 (pRS425k-ADH1t-EucrtZ-FBA1p-TDH3p-GvcrtW-TDH2t) | This study |
SyBE_Sc118068 | SyBE_Sc118030 with pWRZ16 (pRS425k-ADH1t-PacrtZ-FBA1p-TDH3p-GvcrtW-TDH2t) | This study |
SyBE_Sc118069 | SyBE_Sc118030 with pWRZ17 (pRS425k-ADH1t-BDC263crtZ-FBA1p-TDH3p-GvcrtW-TDH2t) | This study |
SyBE_Sc118071 | SyBE_Sc118030 with pWRZ27 (pRS425k-ADH1t-BSD212crtZ-FBA1p-TDH3p-GvcrtW-TDH2t) | This study |
SyBE_Sc118072 | SyBE_Sc118030 with pWRZ28 (pRS425k-ADH1t-BDC263crtZ-FBA1p-TDH3p-SDC18crtW-TDH2t) | This study |
SyBE_Sc118073 | SyBE_Sc118030 with pWRZ18 (pRS425k-ADH1t-Hpchyb-FBA1p-TDH3p-CrBKT-TDH2t) | This study |
SyBE_Sc118074 | SyBE_Sc118030 with pWRZ29 (pRS425k-ADH1t-AspcrtZ-FBA1p-TDH3p-NpcrtW-TDH2t) | This study |
SyBE_Sc118082 | SyBE_Sc118030 with pWRZ19 (pRS425k-ADH1t-PacrtZ-FBA1p-TDH3p-BDC263crtW-TDH2t | This study |
SyBE_Sc118083 | SyBE_Sc118030 with pWRZ20 (pRS425k-ADH1t-EucrtZ-FBA1p-TDH3p-CrBKT-TDH2t) | This study |
SyBE_Sc118076 | SyBE_Sc118030 with pWRZ31 (pRS425k-ADH1t-AspcrtZ-TEF1p-TDH3p-BDC263crtW-TDH2t) | This study |
Construction of functional modules
Visual color screening for astaxanthin production strains
Shake flask and fed-batch cultivation for astaxanthin production
Analysis of glucose concentration and carotenoid production
Bioinformatics identification of CrtWs and CrtZs
Transcriptional analysis of genes in the engineered strains
Results and discussions
Construction of astaxanthin synthetic pathway in β-carotene producing strain
Fig.1 Construction of astaxanthin biosynthesis pathway in β-carotene producing S. cerevisiae strain.(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 |
Combinatorial optimization of CrtW and CrtZ from diverse species
Fig.2 Combinatorial optimization of CrtZ and CrtW from diverse species.(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 |
Fine-tuning the expression ratio of CrtZ to CrtW
Fig.3 Fine-turning the ratio of CrtZ to CrtW for higher astaxanthin production.(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 |
Optimization of astaxanthin production in bioreactor
Fig.4 Bioreactor fermentation under carbon source restriction strategy.(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 |