Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae
- Fenghua Chai†1, 2,
- Ying Wang†1, 2,
- Xueang Mei†1, 2,
- Mingdong Yao1, 2,
- Yan Chen1, 2,
- Hong Liu1, 2,
- Wenhai Xiao1, 2Email authorView ORCID ID profile and
- Yingjin Yuan1, 2
© The Author(s) 2017
Received: 2 December 2016
Accepted: 15 March 2017
Published: 29 March 2017
Due to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.
By introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.
Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.
KeywordsMetabolic engineering Crocetin Saccharomyces cerevisiae Synthetic biology Enzyme sources
Screening enzymes sources and regulating protein expression level have been proved to be efficient strategies for manipulating the key enzymes for balancing metabolic flux, consequently improving production [11–13]. Cao et al.  once improved odd-chain fatty alcohols production in Escherichia coli through balancing the expression level of TesA, αDOX, AHRs and the genes involved in fatty acids metabolism pathway. Meanwhile, through combinatorially screening the carotenogenic enzymes (CrtE, CrtB and CrtI) from diverse organisms and fine-tuning the expression level of CrtI, an optimal enzymes combination with the highest lycopene yield was obtained in Saccharomyces cerevisiae . In crocetin biosynthesis fields, CrtZ, CCD and ALD have been characterized separately in the last decades. Li et al.  once achieved zeaxanthin titer as 43.46 mg/L in a recombinant E. coli strain by integrating Pantoea ananatis CrtZ into a β-carotene producing strain. Meanwhile, Crocus ZCD was firstly annotated as 7, 8 (7′, 8′)-zeaxanthin cleavage dioxygenase in 2003 . However, Frusciante et al.  demonstrated this enzyme could not achieve crocetin synthesis in E. coli and corn. Another two Crocus CCDs, CCD2  and ZCD1 , have been proved to cleavage of zeaxanthin at the 7, 8- and 7′, 8′-positions for forming crocetin dialdehyde in E. coli and Chlorella vulgaris, respectively. Moreover, even though EVOVA Inc.  and Lou et al.  realized crocetin synthesis by using endogenous ALD in yeast and algae, respectively, there was no titer data uncovered yet. It could guess that the complexity of fine-tuning CrtZ, CCD and ALD was the main obstacle. Therefore, it is urgent to explore CrtZ, CCD and ALD systematically for crocetin higher production.
S. cerevisiae strains and plasmids used in this study
MATa, ura3-52, trp1-289, leu2-3,112, his3Δ1, MAL2-8C, SUC2
Δgal1 Δgal7 Δgal10::HIS3, Δypl062w::KanMX, trp1::TRP1_T CYC1 -BtCrtI-P GAL10 -P GAL1 -PaCrtB-T PGK1 , leu2::LEU2_T CYC1 -BtCrtI-P GAL7 -T ACT1 -tHMG1-P GAL10 -P GAL1 -TmCrtE-T GPM1 , Δymrwdelta15::P UAS-GAL1 -PaCrtY-T ADH1 , Δynrcdelta9:: P UAS-GAL1 -PaCrtY-T ADH1
SyBE_SC0014CY06, Δho::P GAL1 -Aa_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -As_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -Eu_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -Pa_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -Ps_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -Ss_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -B.SD_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -B.DC_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -Hp_CrtZ-T HIS5 -URA3
SyBE_SC0014CY06, Δho::P GAL1 -Aa_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -As_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -Eu_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -Pa_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -Ps_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -Ss_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -B.SD_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -B.DC_CrtZ-T HIS5
SyBE_SC0014CY06, Δho::P GAL1 -Hp_CrtZ-T HIS5
SyBE_Sc0123Cz10 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz10 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz10 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz10 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz11 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz11 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz11 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz11 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz12 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz12 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz12 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz12 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz13 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz13 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz13 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz13 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz14 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz14 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz14 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz14 with pRS416-C-04(pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz15 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz15 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz15 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz15 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz16 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz16 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz16 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz16 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz17 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz17 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz17 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz17 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz18 with pRS416-C-01 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 )
SyBE_Sc0123Cz18 with pRS416-C-02 (pRS416-T HIS5 -P GAL10 -CCD3-T TEF2 )
SyBE_Sc0123Cz18 with pRS416-C-03 (pRS416-T HIS5 -P GAL10 -ZCD-T TEF2 )
SyBE_Sc0123Cz18 with pRS416-C-04 (pRS416-T HIS5 -P GAL10 -ZCD1-T TEF2 )
SyBE_Sc0123Cz14 with pRS416-A-02 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 -P GAL7 -ALD6-T PGI1 )
SyBE_Sc0123Cz14 with pRS416-A-03 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 -P GAL7 - Bix_ALD-T PGI1 )
SyBE_Sc0123Cz14 with pRS416-A-04 (pRS416-T HIS5 -P GAL10 -CCD2-T TEF2 -P GAL7 - Syn_ALD-T PGI1 )
SyBE_Sc0123Cz14 with pRS426-A-02 (pRS426-T HIS5 -P GAL10 -CCD2-T TEF2 -P GAL7 - Syn_ALD-T PGI1 )
Blunt Cloning vector, resistant to ampicillin
Blunt Cloning vector, resistant to ampicillin
Single copy plasmid in S.cerevisiae with URA3 and Ampr marker
Multiple copy plasmid in S.cerevisiae with URA3 and Ampr marker
Multiple copy plasmid in S.cerevisiae with LEU2 and KanMX marker
CrtZ from Agrobacterium aurantiacum (Aa_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Alcaligenes sp. PC-1 (As_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Erwinia uredovora (Eu_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Pantoea agglomerans (Pa_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Pantoea stewartii (Ps_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Sulfolobus solfataricus P2 (Ss_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Brevundimonas sp. SD212 (B.SD_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Brevundimonas vesicularis DC263 (B.DC_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CrtZ from Haematococcus pluvialis (Hp_CrtZ) was codon optimized, synthesized and cloned into pUC57-Simple
CCD2 from Crocus was codon optimized, synthesized and cloned into pUC57-Simple
CCD3 from Crocus was codon optimized, synthesized and cloned into pUC57-Simple
ZCD from Crocus was codon optimized, synthesized and cloned into pUC57-Simple
ZCD1 from Crocus was codon optimized, synthesized and cloned into pUC57-Simple
ALD6 from S. cerevisiae was cloned into pUC57-Simple
ALD from Bixa orellana (Bix_ALD) was codon optimized, synthesized and cloned into pUC57-Simple
ALD from Synechocystis sp. PCC6803 (Syn_ALD) was codon optimized, synthesized and cloned into pUC57-Simple
The cassette ho_F-P GAL1 -T HIS5 -URA3-ho_R was cloned and inserted into the pJET1.2
Aa_CrtZ was digested from pUC57-Simple-01 by BsaI and inserted into the same site of pJET1.2-Z-01
As_CrtZ was digested from pUC57-Simple-02 by BsaI and inserted into the same site of pJET1.2-Z-01
Eu_CrtZ was digested from pUC57-Simple-03 by BsaI and inserted into the same site of pJET1.2-Z-01
Pa_CrtZ was digested from pUC57-Simple-04 by BsaI and inserted into the same site of pJET1.2-Z-01
Ps_CrtZ was digested from pUC57-Simple-05 by BsaI and inserted into the same site of pJET1.2-Z-01
Ss_CrtZ was digested from pUC57-Simple-06 by BsaI and inserted into the Same site of pJET1.2-Z-01
B.SD_CrtZ was digested from pUC57-Simple-07 by BsaI and inserted into the Same site of pJET1.2-Z-01
B.DC_CrtZ was digested from pUC57-Simple-08 by BsaI and inserted into the Same site of pJET1.2-Z-01
Hp_CrtZ was digested from pUC57-Simple-09 by BsaI and inserted into the Same site of pJET1.2-Z-01
The cassette T HIS5 -P GAL10 -CCD2-T TEF2 was cloned and inserted into the NotI site of pRS416
The cassette T HIS5 -P GAL10 -CCD3-T TEF2 was cloned and inserted into the NotI site of pRS416
The cassette T HIS5 -P GAL10 -ZCD-T TEF2 was cloned and inserted into the NotI site of pRS416
The cassette T HIS5 -P GAL10 -ZCD1-T TEF2 was cloned and inserted into the NotI site of pRS416
The cassette T TEF2 -P GAL7 -T PGI1 was cloned and inserted into the PstI/BamHI site of pRS425 K
ALD6 was digested from pUC57-Simple-14 by BsaI and inserted into the same site of pRS425 K-A-01
Bix_ALD was digested from pUC57-Simple-15 by BsaI and inserted into the same site of pRS425 K-A-01
Syn_ALD was digested from pUC57-Simple-16 by BsaI and inserted into the same site of pRS425 K-A-01
The cassette T HIS5 –T PGI1 was cloned and inserted into the XhoI/SacI site of pRS416
The cassette T HIS5 -P GAL10 -CCD2-T TEF2 (digested from pRS416-C-01 by NotI), the cassette T TEF2 -P GAL7 -ALD6-T PGI1 (digested from pRS425 K-A-02 by PstI/BamHI) and plasmid pRS416-A-01 (digested by BamHI) were assembled based on RADOM method
The cassette T HIS5 -P GAL10 -CCD2-T TEF2 (digested from pRS416-C-01 by NotI), the cassette T TEF2 -P GAL7 -Bix_ALD-T PGI1 (digested from pRS425 K-A-03 by PstI/BamHI) and plasmid pRS416-A-01 (digested by BamHI) were assembled based on RADOM method
The cassette T HIS5 -P GAL10 -CCD2-T TEF2 (digested from pRS416-C-01 by NotI), the cassette T TEF2 -P GAL7 -Syn_ALD-T PGI1 (digested from pRS425 K-A-04 by PstI/BamHI) and plasmid pRS416-A-01 (digested by BamHI) were assembled based on RADOM method
The cassette T HIS5 –T PGI1 was cloned and inserted into the XhoI/SacI site of pRS426
The cassette T HIS5 -P GAL10 -CCD2-T TEF2 (digested from pRS416-C-01 by NotI), the cassette T TEF2 -P GAL7 -Syn_ALD-T PGI1 (digested from pRS425 K-A-04 by PstI/BamHI) and plasmid pRS426-A-01 (digested by BamHI) were assembled based on RADOM method
Construction of plasmids and strains
Primers and plasmids used in this study were listed in Additional file 1: Table S1; Table 1, respectively. All the heterologous genes including crtZ, ccd, and ald were codon optimized (Additional file 1: Table S2) and synthesized by GENEWIZ (Suzhou, China). All these genes were delivered as pUC57-simple serious plasmids (Table 1). Promoters (P GAL1 , P GAL7 and P GAL10 ), terminators (T HIS5 , T TEF2 , and T PGI1 ) and integration homologous arms (ho_L and ho_R) were amplified from the genomic DNA of S. cerevisiae CEN.PK2-1C, as well as the auxotroph marker URA3 was amplified from the plasmid pRS416. Cassette ho_L-P GAL1 -T HIS5 -URA3-ho_R was assembled by overlap extension PCR (OE-PCR) and cloned into pJET1.2, obtaining the plasmid pJET1.2-Z-01 (Table 1; Additional file 1: Figure S1). Genes crtZ were recovered by BsaI digestion from pUC57-Simple-01–09 and inserted into the same site of pJET1.2-Z-01, generating pJET1.2-Z series plasmids (CrtZ expression cassette plasmids pJET1.2-Z-02–10, Table 1; Additional file 1: Figure S1). Then the CrtZ expression cassette ho_L-P GAL1 -CrtZ-T HIS5 -URA3-ho_R were cut from pJET1.2-Z series plasmids by PmeI and transformed into S. cerevisiae SyBE_SC0014CY06 for genomic integration (Fig. 1b) via the lithium acetate method . Marker URA3 was deleted according to Boeke et al. , obtaining zeaxanthin producing strains SyBE_Sc0123Cz10-18 (Table 1) as the host cell in our study.
For constructing the initial crocetin producing strain and screening CrtZ/CCD combination, only heterologous CCDs were carried by single copy plasmid pRS416 and introduced into zeaxanthin producing strains (Fig. 1b). Genes ccd were amplified from the plasmid pUC57-Simple-10–13 and assembled together with promoter P GAL10 , terminators T HIS5 and T TEF2 into CCD expression cassette T HIS5 -P GAL10 -CCD-T TEF2 by OE-PCR. The products were inserted into the NotI site of plasmid pRS416, obtaining pRS416-C serious plasmids (CCD expression plasmids pRS416-C-01-04, Table 1; Additional file 1: Figure S2). These plasmids were transferred into zeaxanthin producing strains according to Table 1, producing crocetin producing strains (Table 1).
For screening ALD sources, heterologous CCD and ALD were carried by centromeric plasmid pRS416 and introduced into zeaxanthin producing strain (Fig. 1c). Cassette T TEF2 -P GAL7 -T PGI1 was also assembled by OE-PCR and cloned into pRS425 K, obtaining the plasmid pRS425 K-A-01 at first (Table 1; Additional file 1: Figure S3). Genes ald were recovered by BsaI digestion from pUC57-Simple-14–16 and inserted into the same site of pRS425 K-A-01, generating pRS425 K-A series plasmids (pRS425 K-A-02–04, Table 1; Additional file 1: Figure S3). Meanwhile, cassette T TEF2 -T PGI1 was assembled by OE-PCR. The product was incubated with XhoI/SacI and inserted into the same sites of pRS416, producing pRS416-A-01. Then cassettes T HIS5 -P GAL10 -ccd2-T TEF2 (digested from pRS416-C-01 by NotI), T TEF2 -P GAL7 -ald-T PGI1 (digested from pRS425 K-A-02–04 by PstI/BamHI) and linearized vector pRS416-A-01 (digested by BamHI) were assembled based on RADOM method in the particular zeaxanthin producing strain (producing strains SyBE_Sc0123C048–50 harboring plasmids pRS416-A-02–04 respectively, Table 1; Additional file 1: Figure S3) . For adjusting the expression level of CCD and ALD, multiple plasmid pRS426, instead of pRS416, was employed to carry CCD and ALD expression cassettes. Similar procedures were taken as motioned above, which were presented in Additional file 1: Figure S3.
Strains and culture conditions
Escherichia coli DH5α or TransT1 was used for plasmid construction, which was cultured at 37 °C in Luria–Bertani medium  supplemented with 50 μg/mL kanamycin or 100 μg/mL ampicillin for selection. Meanwhile, all the engineered yeast strains summarized in Table 1 were based on an existing β-carotene producing strain, S. cerevisiae SyBE_SC0014CY06. Engineered yeast strains were cultured on YPD medium or synthetic complete (SC) medium lacking appropriate nutrient component for selection . When needed, 1% (w/v) d-(+)-galactose were used as the inducer in fermentations and supplied into YPD medium (generating YPDG medium).
For shake-flask cultivation, colonies on solid plates were picked up and cultured in 3 mL SC medium for overnight growth at 30 °C. Then the preculture was transferred into 25 mL fresh SC medium and grew until reaching to mid-log phase. After that, the seed culture was inoculated into 50 mL YPD medium with an initial OD600 of 0.1 and cultivated at 30 °C for 72 h or 20 °C for 96 h. All the fermentation experiments were performed in triplicate.
The strain SyBE_Sc0123C053 was used for fed-batch fermentation. 100 µL glycerol-stock was inoculated into 25 mL SC medium and cultured at 30 °C, 250 rpm for overnight growth. Then the preculture was transferred to 200 mL fresh SC medium and grew until entering mid-exponential phase. Seed cultures were transferred to 1.8 L YPD medium (20 g/L glucose) in a 5 L bioreactor (BLBIO-5GJG-2, Shanghai, China) at a 10% (v/v) inoculum. The pH was automatically controlled at 5.5 with ammonia hydroxide (6 M). And the dissolved oxygen was kept at 40% by agitation cascade from 400 to 600 rpm, while the air flow was set at 2.5 vvm.
As the crocetin production modules were controlled by employed galactose-inducible system, the fed-batch fermentation should be divided into two stages: cell growth stage and crocetin accumulation stage. During the period of the cell growth stage, fermentation was carried out at 30 °C. The glucose concentration was monitored every 2 h and the glucose consumption rate was obtained accordingly. Based on this data, the glucose concentration was maintained less than 1 g/L by adding an appropriate volume of concentrated glucose solution (500 g/L) continuously. And 5 g yeast extract was added into the bioreactor every 12 h by feeding 400 g/L yeast extract stock solution. When the cell growth fell into stable phase, fermentation entered the second stage: crocetin accumulation stage. Then after fermentation temperature reduced to 20 °C, 10 g/L of d-(+)-galactose was fed to induce crocetin biosynthesis. As glucose was exhausted, cells begun to use ethanol as carbon source. The ethanol concentration was controlled below 5 g/L through adjusting the feeding rate of ethanol until harvest. Duplicate samples were collected to determine the cell density, glucose concentration, ethanol concentration and crocetin production. To avoid the spontaneous degradation from light, bioreactor should be covered with foils.
Extraction and analysis of carotenoids
To determine carotenoids accumulation, standards of lycopene, β-carotene and zeaxanthin were purchased from Sigma (Sigma-Aldrich, MO, USA), and standard of crocetin was purchased from Yuanye Bio-Technology (Shanghai, China). The procedures for extracting and analyzing carotenoids were modified according to Xie et al. . To be specific, after harvested cells were washed with distilled water, the cell pellet was re-suspended in 3 N HCl and boiled for 2 min, and then immediately cooled in ice for 3 min. Then cells debris were harvested and resuspended in acetone containing 1% (w/v) butylated hydroxytoluene. The above mixture was vortexed until colorless. After centrifugation, the acetone phase containing the extracted carotenoid was collected and evaporated by nitrogen blow. The products were analyzed by high-performance liquid chromatography system (HPLC, Waterse2695, Waters Corp, USA) equipped with a BDS HYPERSIL C18 column (150 mm × 4.6 mm, 5 μm, Thermo Scientific) and a UV/VIS detector (Waters 2489). To characterize lycopene, β-carotene and zeaxanthin, the product was dissolved in acetone and the signals were detected at 450 nm. The mobile phase consisting of acetonitrile-methanol (65:35 v/v) was chosen with a flow rate of 0.8 mL/min and the column temperature was set at 25 °C. In the meanwhile, for crocetin analysis, sample was dissolved in methanol-dimethylformamide (7:1 v/v) and crocetin was detected at 430 nm. 70% (v/v) methanol–water (containing 2% formic acid) was utilized as the mobile phase with a flow rate of 1 mL/min at 40 °C. Notably, considering that carotenoids are extremely unstable and susceptible to light, brown centrifugal tubes were used in the above procedures to avoid exposure to light.
Bioinformatics and structural analysis of CCD
The protein identified sequences of the target CCD from different taxa were queried from protein knowledgebase (UniProtKB) available at http://www.uniprot.org/, using the key term “carotenoid cleavage dioxygenase”, and subjected to a brief bioinformatics analysis to guarantee suitable diversity. Initially the CCD protein sequences were aligned by means of clustal W with default settings . Phylogenetic tree of CCD gene family was conducted in MEGA7  and inferred by Neighbor-Joining method . The bootstrap consensus tree deduced from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed .
The structures of the CCD2 and CCD3 were both modeled based on the target-template (PDB ID: 2biw) alignment using SWISS-MODEL [30, 31]. And the Coordinates which are conserved between the targets and the template are copied from the template to the model. Insertions and deletions are remodeled using a fragment library. Side chains are then rebuilt. Finally, the geometry of the resulting model is regularized by using a force field. The modeled structures of target proteins were resolved with PyMol software .
Results and discussion
Construction of inducible crocetin biosynthesis pathway
To realize crocetin biosynthesis, heterologous crtZ and ccd were codon optimized and introduced into an existing β-carotene producer (S. cerevisiae SyBE_SC0014CY06), which processed endogenous ALDs to catalyze the final step in crocetin synthesis pathway (Fig. 1a) . At first, crtZ was integrated into the ho locus of the chromosome, while ccd was carried by centromeric plasmid pRS416. The expression of CrtZ and CCD were under the control of galactose-regulated GAL promoters GAL1 and GAL10, respectively (Fig. 1b). Because a highest zeaxanthin production was once achieved in yeast strain harboring CrtZ from Erwinia uredovora (Eu_CrtZ) among nine selected CrtZ species , Eu_CrtZ were also selected and intergraded into the chromosome of strain SyBE_SC0014CY06, generating strain SyBE_Sc0123Cz12 as a host cell in our study. In the meanwhile, CCD2 from Crocus was also selected to convert zeaxanthin to crocetin dialdehyde, obtaining strain SyBE_Sc0123C009. Strains SyBE_Sc0123C009 and SyBE_Sc0123Cz12 together with the parent strain SyBE_SC0014CY06 were cultured in shake-flask with YPDG medium at 30 °C and their products were analyzed by HPLC after 72 h incubation. As shown in Fig. 1d, crocetin (peak III) was successfully detected with a titer as 9.42 μg/L in strain SyBE_Sc0123C009, indicating that a functional crocetin biosynthesis pathway succeeded here. To be notably, there was no distinct β-carotene accumulation in zeaxanthin producing strain SyBE_Sc0123Cz12, while an amount of β-carotene (peak II), zeaxanthin (peak I), as well as other unidentified byproducts or intermediates were observed in crocetin producing strain SyBE_Sc0123C009 (Fig. 1e), suggesting that the step catalyzed by CCD was rate-limiting here and the selected CrtZ/CCD combination did not match well, which needed to be optimized further.
Optimization of cultivation temperature
Optimal CrtZ/CCD combination by screening enzymes from diverse sources
In this study, ZCD, ZCD1 and CCD3 could not achieve crocetin production in yeast, which required sequential cleavage at C7–C8 and C7′–C8′ double bonds adjacent to the 3-OH-β-ionone ring . Even though there was no crocetin detected in strains carrying these three enzymes separately, zeaxanthin accumulations were consumed at varying degrees in these strains, suggesting their cleave activities in yeast might at other position or only at one side of the molecules. Among the five subfamilies of plant CCDs, the CCD1 and CCD4 families were the only two involved in the cleave activities at 7, 8/7′, 8′ positions [44, 45]. A phylogenetic analysis of CCD sequences from diversity sources belonging to CCD1 and CCD4 families, illustrated that CCD2 and CCD3 belonged to CCD1 subfamily, while ZCD and ZCD1 were members of CCD4 subfamily (Additional file 1: Figure S5).
For ZCD and ZCD1, they share 96% identities (Additional file 1: Figure S6), and both truncated at the N-terminal as lacking a blade of β-propeller and part of the dome in classic CCD4 subfamily members. The truncation was once proved to lead to loss on any cleavage activity for ZCD in E. coli . ZCD1 was reported to once achieve crocetin production in C. vulgaris . However, in our study, both these two enzymes could not sequentially cleave zeaxanthin on 7, 8/(7′, 8′) positions in yeast (Fig. 3b). These conflicting dates highlight the importance of host cell compatibility on the performance of heterologous enzymes, which were also corroborated by the reports from Greene et al. .
Screening ALD sources and fine-tuning of CCD/ALD
Optimization of crocetin production in bioreactor
In our study, crocetin biosynthesis pathway was successfully established in S. cerevisiae through incorporating heterologous CrtZ and CCD in an existing β-carotene producing strain. Then the effects of culture temperature, combination of CrtZ/CCD, ALD from different species, as well as the expression level of CCD and ALD on crocetin were investigated respectively. Compared to culturing at 30 °C, the crocetin accumulation performed much better at 20 °C. The accumulation of crocetin was further promoted by 2.8-fold by screening of CrtZ/CCD combination and ALD sources. Moreover, the crocetin titer was reached to 1219 μg/L by overexpression of ccd and ald. Consequently, the highest reported crocetin titer of 6278 μg/L was obtained in 5-L bioreactors. This study promotes the opportunities for industrialization of crocetin and crocin. This study also sets a good reference for microbial production of pharmaceuticals and chemicals in complex structure by fine-tuning multiple enzymes systematically.
carotenoid cleavage dioxygenase
- Crocus :
Crocus sativus L.
Sulfolobus solfataricus P2
Brevundimonas sp. SD212
Brevundimonas vesicularis DC263
Alcaligenes sp. PC-1
Synechocystis sp. PCC6803
FC, WX and YY conceived of the study and drafted the manuscript. FC and XM carried out the molecular genetic studies. FC and WX carried out the fed-batch fermentation experiments. YW participated in design and coordination of the study and helped to draft the manuscript. MY carried out the protein analysis. YC and HL participated in strain construction and HPLC analysis respectively. WX supervised the whole research and revised the manuscript. All authors read and approved the final manuscript.
The authors are grateful for the financial support from the International S&T Cooperation Program of China (2015DFA00960), the National Natural Science Foundation of China (31600052 and 21676192) and Innovative Talents and Platform Program of Tianjin (16PTSYJC00050).
Availability of data and materials
The material and data supporting their findings can be found in the main paper and the additional file.
The authors declare that they have no competing interests.
The International S&T Cooperation Program of China (2015DFA00960), the National Natural Science Foundation of China (31600052 and 21676192) and Innovative Talents and Platform Program of Tianjin (16PTSYJC00050) supported this work.
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