RESEARCH ARTICLE

Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

  • Ruizhao Wang 1,2 ,
  • Xiaoli Gu 1,2 ,
  • Mingdong Yao 1,2 ,
  • Caihui Pan 1,2 ,
  • Hong Liu 1,2 ,
  • Wenhai Xiao , 1,2 ,
  • Ying Wang , 1,2 ,
  • Yingjin Yuan 1,2
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  • 1. Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China
  • 2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Received date: 21 Aug 2016

Accepted date: 12 Nov 2016

Published date: 17 Mar 2017

Copyright

2017 Higher Education Press and Springer-Verlag Berlin Heidelberg

Abstract

The conversion of β-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high β-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from Brevundimonas vesicularis DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtW were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both crtZ and crtW were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes.

Cite this article

Ruizhao Wang , Xiaoli Gu , Mingdong Yao , Caihui Pan , Hong Liu , Wenhai Xiao , Ying Wang , Yingjin Yuan . Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae[J]. Frontiers of Chemical Science and Engineering, 2017 , 11(1) : 89 -99 . DOI: 10.1007/s11705-017-1628-0

Introduction

Astaxanthin, a natural carotenoid pigment, is regarded as the fourth generation antioxidant and widely used in aquaculture, food, cosmetic and pharmaceutical industries [ 1]. Compared with traditional methods including chemical transformation and extraction from natural producers, de novo synthesis by engineered microbes, especially by Saccharomyces cerevisiae, is a promising choice for astaxanthin manufacture, concerning the safety and economy issue [ 2, 3]. The final reaction in astaxanthin biosynthesis pathway (i.e., from β-carotene to astaxanthin) is actually a metabolic web, which requires two steps of hydroxylation and two steps of ketolation at the 3,3- and 4,4-positions of the β-ionone rings, respectively [ 4]. According to Ukibe et al. [ 3] and Chang et al. [ 5], combined expression CrtZ (encoding β-carotene hydroxylase) and CrtW (β-carotene ketolase) from bacteria/ algae would achieve higher astaxanthin production in yeast than co-expression the P450 oxidoreductase system CrtS-CrtR from Xanthophyllomyces dendrorhous. Herein, we construct a yeast to produce astaxanthin in a high yield by employing two key heterologous enzymes, CrtZ and CrtW.
It was reported that enzymes from different species show distinct catalysis property, indicating that combination of enzymes from different organisms is an efficient way to enhance limited steps [ 58]. In our case, the combination between CrtZ and CrtW with particular substrate specificity and enzymatic activity [ 911] decides the catalysis order and transformation rate of hydroxylation and ketolation, leading to different astaxanthin proportion [ 12]. Choi et al. [ 13] have reported that a combination of CrtW from Brevundimonas sp. SD212 (BSD212_CrtW) and CrtZ from Erwinia uredovora (Eu_CrtZ) generated more astaxanthin and fewer hydroxylated intermediates than the combination of CrtW from Paracoccus sp. strain N81106 (PN81106_CrtW) and Eu_CrtZ, probably due to substrate preference for nonketolated carotenoids [ 9, 14]. Meanwhile, it has also been reported that by integrating CrtZ and CrtW from Haematococcus pluvialis into a β-carotene hyper producer, higher astaxanthin content was achieved in S. cerevisiaevia via ketolation first and hydroxylation subsequently [ 2]. Thus, further searching for a more optimal CrtZ/CrtW combination would be critical for higher astaxanthin accumulation.
On the other hand, according to a two enzyme multi-step metabolic pathway model set up by Zelcbuch et al. [ 15], low flux, protein burden and intermediates accumulation are the main outcomes for imbalanced enzymes concentrations, which impairs the desired molecules accumulation. Thus, balancing the key enzymes expression levels is also a crucial factor affecting heterologous product output. Ajikumar et al. [ 16] pointed out that taxadiene titer was increased 15000-fold via fine-tuning the expression level of two key modules involved in the isoprenoid pathway. Cao et al. [ 7, 17] increased the titer of odd-chain fatty alcohols by 12.7-fold through adjusting the expression level of genes associated with the fatty acid metabolism pathway. Therefore, it is vital to rebalance the expression level of CrtZ and CrtW for astaxanthin heterologous biosynthesis. Zhou et al. [ 2] once reported a 2.2-fold increase on astaxanthin accumulation achieved by adjusting the copy numbers of CrtZ and CrtW in the carotenogenic yeast. Lemuth et al. [ 18] reduced the intermediate, zeaxanthin accumulation (from 25% to less than 5% of the total carotenoid product) by using inducible promoters to make the expression of both CrtZ and CrtW responding to culture condition. Thus, fine-tuning the expression ratio of CrtZ to CrtW would be a helpful strategy to promote astaxanthin production.
In this study, CrtZ and CrtW were introduced into an existing β-carotene producer [ 19] to achieve astaxanthin production. Combined screening enzymes sources and manipulation of the expression levels were the two strategies for improving astaxanthin accumulation. Finally, the strain processing CrtZ from Alcaligenes sp. strain PC-1 (controlled by promoter TEF1p) and CrtW from Brevundimonas vesicularis DC263 (controlled by promoter TDH3p) achieved the highest astaxanthin production in a 5-L bioreactor via high cell density fermentation using a carbon restriction strategy. This work highlighted that combinatorial expression of key genes in terms of enzymes sources screening and genes expression manipulation is a productive strategy to unlock metabolic web, consequently improving desired molecules production.

Materials and methods

Strains and media

All the S. cerevisiae strains used in this study were summarized in Table 1. S. cerevisiae strains were cultivated at 30 °C in YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose). The engineered yeast cells were selected and grown on synthetic complete drop-out medium (SD medium, 0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose with the appropriate amino acid drop out mix supplemented). E. coli DH5α, which was used for plasmids construction and replication, was cultivated at 37 °C in Luria-Bertani (LB) complete medium (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1 % (w/v) NaCl, pH 7.0) with 50 µg/mL kanamycin or 100 µg/mL ampicillin for selection.
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

All the plasmids used in this study were provided in Table S3. Primers used for PCR amplification were listed in Table S1. The detail procedure to construct CrtW-CrtZ expression cassette plasmids were presented in Fig. S1. To be specific, the cassette ADH1t-FBA1p-TDH3p-TDH2t, which processed two back-to-back cassettes FBA1p-ADH1t and TDH3p-TDH2t with opposite direction, was synthesized by Genewiz (Beijing, China) and inserted into the PstI/BamHI sites of pLD2, obtaining pLD2-01 (Fig. S1). Genes encoding CrtW and CrtZ from different organisms were codon optimized (Table S2), synthesized by Genscript Inc. (NJ, China) and cloned into plasmid pUC57-simple, producing pUC57-Simple series plasmids (pUC57-Simple-01~17, Table S3 and Fig. S1). Gene crtW was recovered from plasmids pUC57-Simple-10–17 by BsaI digestion and inserted into pLD2-01 treated with the same enzyme. Then the products (pLD2-02–09, Table S3) were digested by BsmBI for sub-cloning those crtZ genes, which were recovered by BsaI digestion from plasmids pUC57-Simple-01–09, obtaining pLD2 series plasmids (pLD2-10–39, Table S3). Then the CrtW-CrtZ expression cassettes ADH1t-crtZ-FBA1p-TDH3p-crtW-TDH2t were digested with PstI/BamHI from plasmids pLD2-10–39, and inserted into the same sites of plasmid pRS425k, producing CrtW-CrtZ expression cassette plasmids pWRZ01–30 (Table S3).
For fine-turning CrtZ expression level, cassette ADH1t-TEF1p-TDH3p-TDH2t was synthesized by Genewiz (Beijing, China) and inserted into the PstI/BamHI sites of pRS425K to obtain pLD2-40. Gene BDC263crtW (crtW from Brevundimonas sp. DC263) was digested from pUC57-Simple-14 by BsaI and inserted into the same site of pLD2-40 to produce pLD2-41. Then Gene AspcrtZ (crtZ from Alcaligenes sp. strain PC-1) was cut from pUC57-Simple-02 by BsaI and inserted into the BsmBI site of pLD2-41 to obtain pLD2-41. The cassette ADH1t-AspcrtZ-TEF1p-TDH3p-BDC263crtW-TDH2t was digested from pLD2-42 by PstI/BamHI and inserted into the same site of pLD2-01 to produce pWRZ31.

Visual color screening for astaxanthin production strains

S. cerevisiae SyBE_Sc118030 with high β-carotene production was employed as the host cell to construct an astaxanthin biosynthetic pathway. CrtW-CrtZ expression cassette plasmids carrying enzymes from diverse species were transformed into S. cerevisiae SyBE_Sc118030 by the lithium acetate method [ 20] to obtain astaxanthin producing strains (Table 1). Then the colony of the engineered yeast was picked up from SD plate and grown in the same liquid medium for 24 h until the cell density of OD600 reached 6‒9. Then each culture was diluted by sterile distilled water to OD600 of 0.1, and the cells were harvested, washed and suspended with 20 µL sterile distilled water before being spotted on SD plate. After incubation at 30 °C for 3 d, the clones with intense red color were selected and cultured in a shake flask to measure astaxanthin production.

Shake flask and fed-batch cultivation for astaxanthin production

For shake flask culture, recombinant yeast colonies were inoculated into 3 mL SD medium and grown at 30 °C, 250 r/min for 24 h to exponential phase (OD600 ≈ 8.0). Then the preculture was inoculated into the corresponding fresh SD medium (3 mL) with an initial OD600 of 0.2 for further 14 h cultivation (to OD600 ≈ 6.0). After that, the seed culture was transferred into 100 mL fresh YPD medium at an initial OD600 of 0.05 and grown until harvest.
For an experiment using a 5-L stirred-tank bioreactor (BLBIO-5GJG-2, Shanghai, China), a 10% (v/v) seed culture was transferred to 2.5 L of YP medium supplemented with 2% (w/v) glucose. A glucose solution (50%, w/v) was fed periodically to keep the glucose concentration under 1.0 g/L. The temperature, pH, dissolved oxygen and air flow were controlled at 30 °C, 6,>30% and 1.5 vvm, respectively. A yeast extract (1%, w/v) was added into the medium every 24 h for providing efficient nitrogen source. Duplicate samples were collected to determine the cell density, glucose concentration and the astaxanthin production. To avoid the spontaneous oxidation of astaxanthin, cultures were covered with foils.

Analysis of glucose concentration and carotenoid production

The glucose concentration was measured by the enzyme-coupled glucose assay kit (Rsbio, China). To determine carotenoid accumulation, the standard lycopene, β-carotene, zeaxanthin, canthaxanthin, and astaxanthin were purchased from Sigma Aldrich, USA. Carotenoids were extracted from the HCl-heat-treated cells with acetone and analyzed by reverse-phase high-performance liquid chromatography (HPLC, Waterse2695, Waters Corp., USA), according to the method described before [ 2]. β-Carotene, zeaxanthin, canthaxanthin and astaxanthin were separated on a BDS HYPERSIL C18 column (150 mm × 4.6 mm, 5 µm, Thermo Scientific) and detected by a UV/VIS detector (Waters 2489) at 470 nm. The flow rate of the mobile phase was 1 mL·min–1 and the column temperature was set at 25 °C.

Bioinformatics identification of CrtWs and CrtZs

The protein identified sequences of the target CrtWs and CrtZs from different taxa were queried from the GenBank public database and subjected to a brief bioinformatics investigation to ensure suitable diversity. Phylogenetic analyses were conducted in MEGA7 [ 21] and inferred by Neighbor-Joining method [ 22]. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed [ 23]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were computed using the Poisson correction method [ 24].

Transcriptional analysis of genes in the engineered strains

The transcription level of genes crtW and crtZ in the engineered strains was analyzed by Real-Time PCR. Strains were cultured in shake flasks for 10, 14 and 30 h respectively, and then harvested. Total RNA extraction and reverse transcription were conducted with Yeast Total RNA Extraction Kit (APEXBIO (Beijing, China) and GoScript™ Reverse Transcription System (Promega, USA), respectively. The PCR procedure was carried out on a CFX96 real time PCR system (Bio-Rad, USA) in white-walled PCR plates (96 wells). A ready-to-use master-mix containing a fast proof-reading polymerase, dNTPs, stabilizers, MgCl2 and SYBR® Green dye was used according to the manufacturer’s instructions (Bio-Rad, USA). Reactions were performed in a total volume of 18 µL containing 400 nmol/L each primer, 2×SsoAdvanced™ SYBR® Green Supermix (Bio-Rad, USA) and 2 µL reverse transcription product. The cycle conditions were set as follows: initial template denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and combined primer annealing/elongation at 57 °C for 20 s. The amount of fluorescence for each sample, given by the incorporation of SYBR® Green into dsDNA, was measured at the end of each cycle and analyzed via CFX Manager™ software 2.1 (Bio-Rad, USA). The relative transcription level for each gene was determined by 2−ΔΔCt [ 25] using gene ACT1 for normalization [ 26]. T-Tests were performed by software SPSS 19.0 with significance levels set at P<0.05.

Results and discussions

Construction of astaxanthin synthetic pathway in β-carotene producing strain

The conversion of β-carotene to astaxanthin required two hydroxyl- and two keto- groups added to each β-ionone ring by CrtZ and CrtW, respectively [ 4]. To achieve astaxanthin biosynthesis in S. cerevisiae, heterologous CrtZ and CrtW were codon optimized and introduced into an existing high β-carotene producer (S. cerevisiae SyBE_Sc118030) [ 19]. As shown in Fig. 1(A), tHMGR, fused ERG20-BTS1, CrtE, CrtI and CrtBY were overexpressed respectively in strain SyBE_Sc118030, achieving a titer of β-carotene as 162.1 mg/L [ 19]. In this study, the expression of genes crtZ and crtW were initially controlled by promoters FBA1p and TDH3p, respectively (Fig. 1(B)). At first, CrtW and CrtZ from Agrobacterium aurantiacum (AaCrtZ and AaCrtW) were chose to generate astaxanthin producing strain SyBE_Sc118040. Both the strain SyBE_Sc118030 and SyBE_Sc118040 were cultivated in shake flasks with YPD medium and the products were analyzed by HPLC after 82 h incubation. Compared to strain SyBE_Sc118030, astaxanthin (peak V) was successfully detected with a yield of 1.8 mg/g DCW in strain SyBE_Sc118040 (Fig. 1(C)), suggesting the constructed astaxanthin biosynthesis pathway does function here. However, an amount of lycopene (peak II) were detected in strain SyBE_Sc118040, whereas no distinct lycopene accumulation was found in the parent strain (Fig. 3(C)), indicating the reaction catalyzed by AaCrtZ and AaCrtW might be the rate-limiting steps in astaxanthin production. Meanwhile, zeaxanthin (peak IV) and canthaxanthin (peak III) along with other unidentified intermediates were obviously accumulated in strain SyBE_Sc118040 (Fig. 1(C)), revealing that the substrates selectivity and catalytic capability of AaCrtZ and AaCrtW did not matched well, and thus needed to be optimized further.
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

Full size|PPT slide

Combinatorial optimization of CrtW and CrtZ from diverse species

It has been proved in many cases that screening enzymes from diverse organism is a conventional and efficient strategy to identify appropriate enzyme(s) for improving heterologous metabolism in a specific host [ 58]. Even though the reactions catalyzed by hydroxylase CrtZ and ketolase CrtW constitute a multi-step reticular metabolic pathway, it may enhance the pathway output and thus simplify metabolic network to screen different CrtZ/CrtW combinations. To date, only three combinations of CrtZs and CrtWs both from Paracoccus sp., Pantoea ananatis and H. pluvialis respectively have been employed in S. cerevisiae for astaxanthin biosynthesis [ 2, 3]. Here, nine CrtZs from A. aurantiacum (AaCrtZ), Alcaligenes sp. strain PC-1 (AspCrtZ), Brevundimonas sp. SD212 (BSD212CrtZ), Brevundimonas sp. DC263 (BDC263CrtZ), Pantoea agglomerans (PaCrtZ), Pantoea stewartii (PsCrtZ), E. uredovora (EuCrtZ), Sulfolobus solfataricus P2 (SsP2CrtZ) and H. pluvialis (HpChyb), and eight CrtWs from Gloeobacter violaceusPCC 7421 (GvCrtW), A. aurantiacum (AaCrtW), Alcaligenes sp. strain PC-1 (AspCrtW), Brevundimonas sp. DC263 (BDC263CrtW), Brevundimonas sp. SD212 (BSD212CrtW), Chlamydomonas reinhardtii (CrBKT), Nostoc punctiform PCC 73102 (NpCrtW), Sphingomonas sp. DC18 (SDC18CrtW) were selected and combinatorially expressed in strain SyBE_Sc118030. And thirty strains with different CrtZ/CrtW combination have been successfully constructed (Fig. 2(A) and Table 1). These constructed strains were cultivated on solid SD medium, and strain SyBE_Sc118031 without CrtZ/CrtW was employed as the negative control. As shown in Fig. 2(B), the colonies with different CrtZ/CrtW combinations appeared as a range of colors from yellow via orange to red. According to Zelcbuch et al. [ 15], the characteristic color pattern for each colony was attributed to the distinct accumulation profile of carotenoid contents, and the colony with higher astaxanthin production presented darker red pigments. Eventually, nine strains with intense red color were visually picked up and cultured in shake flasks with YPD medium for 82 h. Then their carotenoid composition was analyzed by HPLC (Fig. S2). As a result, stains with different CrtZ/CrtW pairs exhibited diverse levels of carotenoid accumulation (Fig. 2(C)). And the constructed strain SyBE_Sc118060 harboring AspCrtZ and BDC263CrtW achieved the highest astaxanthin yield (3.1 mg/g DCW) with less intermediate metabolites accumulation (Fig. 2(C) and Fig. S2). Our results indicated that co-expression of crtZ and crtW can be beneficial for astaxanthin production, which is consistent with that reported by Ukibe et al. [ 3]. Compared to Chang et al. [ 5], astaxanthin production was further improved here by screening CrtZ and CrtW sources in parallel. Thus, strain SyBE_Sc118060 would be a promising candidate for further optimization.
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

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The conversion from β-carotene to astaxanthin catalyzed by CrtZ and CrtW involves a series of complicated and interlaced reactions. However, the most favorable reaction order for the co-catalysis by CrtZ and CrtW has not been thoroughly revealed yet [ 2, 27, 28]. Because astaxanthin production can be clarified by color intensity, screening different CrtZ/CrtW combinations might be an efficient strategy rather than setting a specific route in yeast. The combination as AspCrtZ-BDC263CrtW was the best one among thirty tested groups in terms of astaxanthin yield (Figs. 2(B) and 2(C)), whereas strains processing algal CrtW or CrtZ achieved lower astaxanthin yield (Fig. 2(C)). To better interpret these results, phylogenetic analysis was conducted by building phylogenetic trees according to the protein sequences of CrtZ and CrtW, respectively (Fig. 2(A)). In the meanwhile, another four strains with orange pigment and seven yellow clones were selected for astaxanthin measurement. Including data from previous nine redder colonies, all the results indicated that CrtW seems to be more crucial to astaxanthin production than CrtZ (Figs. 2(C‒E)). And the conversion efficiency from β-carotene to astaxanthin represented remarkable correlation with the evolutionary distances of CrtWs (Fig. 2(E)). Although the primary structures of the selected CrtWs and CrtZs are highly conserved (>30% protein identity), CrtWs exhibited larger differences on evolutionary distances than CrtZs (Fig. 2(A)). And bacterial CrtWs with shorter evolutionary distances from root (<0.4) were more primordial than algal CrtWs with longer evolutionary distances (>0.6, Fig. 2(A)). Nam et al. [ 29] have pointed out that ancestral enzymes appears more broad substrate specificity than evolved proteins. Sandmann [ 30] and Schaub et al. [ 31] both reported that carotenoid desaturase CrtI from lower species including archaea, bacteria and fungi can catalyze the conversion of phytoene to lycopene via four steps of desaturation, which was catalyzed by three separated enzymes in advanced organisms such as cyanobacteria and higher plants. Therefore, bacterial CrtWs might exhibit more substrate promiscuity than algal CrtWs, and thus afford a smoother multi-step reaction for higher astaxanthin production. In our study, the strains carrying bacterial CrtWs presented higher astaxanthin yield and BDC263CrtW with the shortest evolutionary distances as 0.2 showed a better performance (Fig. 2). Choi et al. [ 13] has proved the general substrate diversity for BDC263CrtW in E. coli. Thus, to some extent, protein evolution analysis matches our experiment results quite well, and is likely to be a powerful predicting and interpreting strategy to improve carotenoid heterologous biosynthesis.

Fine-tuning the expression ratio of CrtZ to CrtW

As illustrated in Fig. 2(C), canthaxanthin was accumulated as the dominant intermediate in strain SyBE_Sc118060, suggesting hydroxylation of keto-substrate by CrtZ is a rate-limiting step. Scaife et al. [ 28] and Zhou et al. [ 2] have reported that the ratio of CrtZ to CrtW is another critical factor for enhancing the astaxanthin biosynthesis. Thus, the relative expression level of CrtZ to CrtW was adjusted by up-regulating the promoter activity of gene crtZ. After replacing the original CrtZ promoter FBA1p with a stronger one TEF1p (generating strain SyBE_Sc118076) [ 32], both the initial strain (strain SyBE_Sc118060) and the modified one were cultured in shake flasks in YPD medium with 2%, 4%, 5% and 10% (w/v) glucose for 82 h. As shown in Fig. 3(A), there was no significant difference between strain SyBE_Sc118060 (FBA1p-crtZ) and strain SyBE_Sc118076 (TEF1p-crtZ) at the same glucose concentration. Furthermore, strain SyBE_Sc118076 in medium with 2% or 4% glucose achieved higher astaxanthin yield than that with 5% or 10% glucose (Fig. 3(B)), suggesting that a glucose concentration greater than 4% was benifit to higher biomass but not astaxanthin yield. Thus, the transcription level of CrtZ and CrtW in strains SyBE_Sc118060 and SyBE_Sc118076 cultured in 2% and 4% glucose media were analyzed by real-time PCR. Samples were harvested after 10 h (early exponential phase), 14 h (middle exponential phase) and 30 h (late exponential phase) during the cultivation. For 2% glucose, although the modification on CrtZ promoter significantly reduced the transcription level of both crtZ and crtW (Figs. 3(C) and 3(D)) during the whole time course, but the ratio of crtZ to crtW were increased at mid-, late-exponential phases (Fig. 3(E)), resulting in no improvement on astaxanthin yield but reduction on intermediate accumulation (Fig. 3(B) and Fig. S3). Meanwhile, for 4% glucose, astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW) after the alteration on crtZ promoter, which might mainly due to the 33.5-fold increase on crtZ transcription level (Fig. 3(C)) and 39.1-fold enhancement on the ratio of crtZ to crtW (Fig. 3(E)) at early exponential phase. Eventually, strain SyBE_Sc118076 was choose as the candidate for further bioreactor experiments due to its potential on astaxanthin production.
At present, it is hard to explain this constitutive promoters pattern in our study as it has been reported that the promoters are unaffected by glucose concentration [ 33]. Anyway, the results as shown in Fig. 3 demonstrated that the increase on the expression ratio of CrtZ to CrtW would be benefit to the conversion efficiency from β-carotene to astaxanthin, which is consistent with the previous research [ 18]. Because genes crtZ and crtW were carried by a multiple copy plasmid pRS425k, it is difficult to accurately adjust and calculate the optimal ratio between CrtZ and CrtW under current circumstance. Moreover, plasmid stability during the cultivation is another important issue to address in future study.
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

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Optimization of astaxanthin production in bioreactor

To achieve high cell density fermentation, strain SyBE_Sc118076 was cultivated in a 5-L bioreactor under glucose restriction strategy (Figs. 4(A) and 4(B)). Although astaxanthin yield was higher under 4% glucose, considering supplementing nitrogen source earlier and reducing bioproduct (ethanol etc.) formation, the glucose concentration in batch medium was reset as 2%. A 50% glucose solution was fed periodically into the medium after the initial glucose was depleted in batch medium. The glucose concentration was maintained at lower than 1 g/L by controlling the feeding rate (Fig. 4(B)). Meanwhile, 1% yeast extract was added into the bioreactor every 24 h to fulfill the necessary of nitrogen source. Eventually, a titer of 81.0 mg/L astaxanthin was obtained after 168 h cultivation, which was the highest reported astaxanthin titer in S. cerevisiae.
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

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However, the astaxanthin titer did not meet the requirement for industrialization, and more efforts including response surface methodology would be taken in further study for developing optimal fermentation process. Meanwhile, because Zhou et al. [ 2] once described the role of Fe2+ as a cofactor for both CrtZ and CrtW in enhancing astaxanthin accumulation, cultivation of the astaxanthin producing strain in the medium with appropriate Fe2+ concentration would be applied in future. Moreover, considering the oxidative stress tolerance ability exhibited by astaxanthin accumulation strains, adaptive laboratory evolution performed by periodic hydrogen peroxide shocking schemes [34] could also be attempted in further study.

Conclusions

Heterologous β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) were employed in an existing β-carotene high producing S. cerevisiae strain for astaxanthin biosynthesis. CrtZs and CrtWs from diverse sources were combinatorially screening, and CrtW from B. vesicularis DC263 and CrtZ from Alcaligenes sp. strain PC-1 were verified to be the best combination for astaxanthin accumulation. Moreover, by enhancing the promoter activity of crtZ to increase the ratio of CrtZ to CrtW, astaxanthin yield were increased by 30.4% in a medium with 4% (w/v) glucose. Finally, the highest reported astaxanthin titer (81.0 mg/L) was achieved in a 5-L bioreactors. This study sets a good reference for metabolic web enhancement by screening key enzyme sources and manipulating expression to overproduce the desired molecules in microbes.

Acknowledgements

This work was supported by the International S&T Cooperation Program of China (2015DFA00960), the National Natural Science Foundation of China (Grant Nos. 31600052 and 21676192) and Innovative Talents and Platform Program of Tianjin (16PTSYJC00050).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11705-017-1628-0 and is accessible for authorized users.
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