Chiang Mai J. Sci. 2020; 47(6) : 1118-1129http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Cloning and Expression of Phytoene Desaturase Gene from Undaria pinnatifida SuringarTengteng Guan, Tingting Zhang, Yi Ma, Yinjie Li, Guicai Du* and Ronggui Li*College of Life Sciences, Qingdao University, Qingdao 266071, China.

*Author for correspondence; e-mail: duguicai@qdu.edu.cn, lrg@qdu.edu.cnReceived: 2 July 2019Revised: 1 June 2020

Accepted: 18 June 2020

ABSTRACT The phytoene desaturase is a key enzyme involved in the carotenoids biosynthesis pathway,

which catalyzes the conversion of ζ-carotene from phytoene. Undaria pinnatifida Suringar is an edible economic brown algae that produces high concentrations of fucoxanthin. In this study, the PDS gene (UpPDS) was isolated and characterized from U. pinnatifida, and the full-length cDNA sequence was 1707 bp in length and encoded 568 amino acid residues. Furthermore, the putative protein sequence showed a high homology to the PDS from other known plants. Phylogenetic analysis showed that the UpPDS was more related to that of brown algae compared to other species. By heterologous complementation and high-performance liquid chromatography analysis, desaturation activity of recombinant UpPDS was confirmed by production of ζ-carotene in engineering E. coli co-transformed with crtE, crtB and UpPDS. The L9(3

4) orthogonal test was used to optimize ζ-carotene fermentation conditions of the engineering bacteria. The results showed that temperature had the most significant effect on ζ-carotene production, the maximal yield of ζ-carotene could reach 5.0mg/L under the optimal conditions.

Keywords: phytoene desaturase, gene cloning, ζ-carotene, expression, Undaria pinnatifida Suringar

1. INTRODUCTIONFucoxanthin is a kind of light yellow to

brown carotenoids, and rich in brown algae such as U. pinnatifida [1]. Carotenoids constitutes a diverse group of natural pigments synthesized by all algae, higher plants, some fungi and bacteria [2]. In addition to the colorless carotenoids such as phytoene, most carotenoids are red, orange-red and yellow. So far, more than 750 species of naturally occurring carotenoids have identified [3]. Carotenoids are widely distributed in photosynthetic membranes in photosynthetic organisms, which play an important role in photosynthesis, not only as an important structural component of

the photosynthetic antenna and reaction center complex, but also as a protective agent for photooxidation [4]. Carotenoids are the main pigments in many flowers and fruits, which contribute to the bright color of many fruits and flowers [5]. Some carotenoids are precursors of phytohormone and abscisic acid (ABA) that regulate plant development [6], or precursors of vitamin A and retinoid complexes required for animal morphogenesis. Besides, carotenoids are essential ingredients in the human diet and can act as antioxidants with anticancer effects [7].

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The main biosynthetic pathways of carotenoids in plants have been elucidated. However, the biosynthetic mechanism of some special caroteinoids such as fucoxanthin in brown algae is still controversial. Carotenoids are synthesized from the precursor molecule, isoprene pyrophosphate (IPP), which is converted to geranylgeranyl pyrophosphate (GGPP) under the catalysis of IPP isomerase and geranyl geranyl pyrophosphate synthase. The first colorless carotenoid molecule, phytoene, is formed by condensation of two molecules of GGPP, which is catalyzed by phytoene synthase (PSY) [8]. Phytoene is desaturated by phytoene desaturase (PDS) to produce ζ-carotene [9], and then ζ-carotene desaturase (ZDS) modulates ζ-carotene to form lycopene. Subsequently, lycopene is cyclized twice catalyzed by two separate cyclases, lycopene ε-cyclase (LYCE) and lycopene β-cyclase (LYCB) to produce α-carotene and β-carotene, respectively [10, 11]. The α-carotene is further hydrolyzed to form lutein.

The phytoene desaturase (PDS) is a key enzyme in the carotenoid metabolism pathway that regulates the conversion of phytoene to ζ-carotene [12, 13]. In a heterologous expression system, the accumulation of ζ-carotene was achieved by introducing the PDS gene into engineered E. coli [14]. Mutations in the chlorella PDS gene resulted in loss of phytoene desaturase activity and impaired ζ-carotene biosynthesis [14]. The over-expression of the PDS gene could promote the accumulation of carotenoids in flowers and fruits [15]. So far, the PDS gene has been isolated and identified from soybean [16], Arabidopsis [17], tomato [18] and other species. However, fewer studies on the phytoene desaturase gene in U. pinnatifida (UpPDS) have been reported.

U. pinnatifida Suringar, also known as sea mustard, is a widely planted economic brown algae belonging to the family Undaria. The leafy body of U. pinnatifida with high content of caroteinoids is not only edible, but also can be used as a traditional Chinese medicine [19]. Modern chemistry and pharmacology studies have

shown that U. pinnatifida has excellent antiviral [20] and antitumor activity [21], and good effects in lowering blood pressure [22], losing weight [23] and preventing cardiovascular and cerebrovascular diseases. In this study, the UpPDS gene was isolated and its coded phytoene desaturase was analyzed by bioinformatics. Besides, the function of UpPDS was confirmed by functional complementation in engineered E. coli co-expressing GGPP synthase and phytoene synthase. This study offers a basis for elucidation of pathway of carotenoids biosynthesis in U. pinnatifida Suringar and construction of engineering bacteria to produce ζ-carotene.

2. MATERIALS AND METHODS2.1 Materials

U. pinnatifida Suringar samples were collected from the coast of Qingdao, China from March 2018 to May 2018. The fresh samples were washed with sterile water and stored in a -80°C refrigerator for RNA extraction.

2.2 Total RNAs Extraction and cDNA SynthesisU. pinnatifida Suringar samples were quickly

milled into powder using a chilled mortar and pestle in liquid nitrogen. Total RNAs were extracted from U. pinnatifida Suringar tissue using RNAprep Pure Plant Kit (TIANGEN, China), the quality and concentration of total RNAs were tested by 1% denatured agarose gel electrophoresis and measurement of absorbance at 260 nm, respectively. The first strand cDNA synthesis kit was used to synthesize the first strand of complementary DNA (cDNA) from 2 μg of total RNAs using oligo (dT) primers according to the manufacturer’s instructions (TaKaRa, China).

2.3 Cloning of the Full-length cDNA of UpPDS According to the published sequence of PDS

from U. pinnatifida Suringar, a set of gene-specific primers PDSF1 (5′-ATC GGC AGT GGC AGC GTA G-3′) and PDSR1 (5′-CTA CGC TGC CAC TGC CGA T-3′) were designed for reverse transcription-polymerase chain reaction (RT-PCR).

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The full-length cDNA of PDS was amplified by PCR using the primers PDSF1 and PDSR1, and the first strand of cDNA as the template. The PCR was performed according to the following program: denaturation at 94°C for 5 min, 30 cycles of 94°C for 45s, 58°C for 45s and 72°C for 90s, and a final extension at 72°C for 10 min. The amplified product was separated by agarose gel electrophoresis, purified by M5 Gel Extraction Kit (Mei5, China), and ligated into the cloning vector pMD-18T to construct pMD-18T-UpPDS. The cDNA amplified by PCR in pMD-18T-UpPDS was verified by sequencing.

2.4 Bioinformatic Analysis The physical and chemical properties of the

protein encoded by UpPDS were carried out by ProtParam online software (http://web.expasy.org/protparam/). The NPS@:SOPMA software was used to analyze the secondary structure of proteins. The similarity analysis of amino acid sequences was carried out using BLASTp at web servers of the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast). SignalP4.1Server(http://www.cbs.dtu.dk/services/SignalP/), TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/) and ChloroP 1.1 Server (http://www.cbs.dtu.dk/services/ChloroP/) were used to predict the potential signal peptide, subcellular localization and the transit peptide, respectively. MEGA7 was used to construct the phylogenetic tree based on the neighbour-joining (NJ) method.

2.5 Prokaryotic Expression of UpPDSAccording to the UpPDS sequence, a pair

of primers mPDSF1 (5′- CGG GAT CCA TGA TGG GAG TGC GAG GCA AT -3′) and mPDR1 (5′- TGC TCG AGC TAC GCT GCC ACT GCC GAT GCC G -3′) were designed. The open reading frame (ORF) of UpPDS was amplified from pMD-18T-UpPDS using mPDSF1 and mPDSR1 primers. The PCR product digested with BamHI and XhoI was ligated into pET-28a

(+) digested with the same endonucleases to construct pET-28a-UpPDS. The recombinant plasmid pET-28a-UpPDS was transformed into E. coli BL21 (DE3) to construct engineering bacteria. One colony of engineering bacteria was inoculated into 2 mL LB medium supplied with 50 mg/mL kanamycin and cultured overnight in a shaker at 37°C. The culture was inoculated into 50 mL LB medium with the same antibiotic and cultured for 4 h under shaking conditions, and then isopropyl β-D-thiogalactoside (IPTG) was added to the culture to a final concentration of 0.5 mmol / L to induce protein expression at 16°C for 24 h. The culture of 1 mL was centrifuged at 10,000 rpm at room temperature, and the pellet was resuspended in 20 μL of distilled water, mixed with 20 μL of the loading buffer, and then boiled for 10 min. The recombinant protein was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). E. coli BL21 (DE3) transformed with pET-28a (+) was used as control.

2.6 Functional Assay of UpPDS in E. coliIn this study, the plasmid pAC-PHYTipi

was selected to construct an E. coli expression strain required for genetic complementation experiments. Plasmid pAC-PHYTipi contains crtE (encoding gene for GGPP synthase) and crtB (encoding gene for phytoene synthase) from Erwinia uredovora, which are responsible for the synthesis of phytoene from IPP. Under the action of UpPDS derived from pET-28a-UpPDS, the genetically complementary E. coli strain will be able to produce ζ-carotene. If ζ-carotene is successfully synthesized in the complementary E. coli strain, the biological function of UpPDS can be verified.

For the heterologous complementation assay, pET-28a-UpPDS and pAC-PHYTipi were co-transformed into E.coli BL21 (DE3), and then the co-transformed E. coli was plated onto a LB plate containing 50 μg/mL kanamycin and 50 μg/mL chloramphenicol. The plate was

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incubated in the dark at 28°C for 48 h, and bacterial growth and ζ-carotene production was observed. The phytoene dehydrogenase activity encoded by UpPDS was verified by color change of bacterial colonies from white to pale yellow. E.coli BL21 (DE3) co-transformed with pAC-PHYTipi and pET-28a(+) was used as negative control.

2.7 HPLC Analysis of ζ-caroteneOne colony of E.coli BL21 (DE3) co-transformed

with pET-28a-UpPDS and pAC-PHYTipi was inoculated into 50 mL LB medium supplied with 50 mg/mL kanamycin and 50 μg/mL chloramphenicol, and cultured overnight in a shaker at 37°C. The culture was transferred to the 2000 mL fresh liquid LB medium containing the same antibiotics, and shake-cultured at 37°C for 3 h. IPTG was then added to a final concentration of 1.0 mmol /L and the engineering bacteria was further cultured at 28°C for 24 h with agitation (160 rpm) in the dark. The bacterial cells were collected by centrifugation at 13400 rpm for 5 min. The precipitate obtained was suspended in 400 μL acetone and incubated at 28°C for 30 min, and the mixture was centrifuged at 12,000 rpm, 4°C for 2 min, and the supernatant was collected. The precipitate was re-suspended with 400 μL ethyl acetate, and centrifuged at 12,000 rpm for 2 min. The supernatant was collected, mixed with the acetone extract, and filtered through a 0.22 μm filter for HPLC analysis. All procedures were performed in the dark to protect ζ-carotene from photodegradation. The test sample was separated on a standard Poroshell 120 EC-C18

reverse phase column on a high performance liquid chromatography instrument (Shimadzu, LC-20A), using acetonitrile/ethyl acetate as a mobile phase at a flow rate of 1 mL / min. ζ-carotene in the samples was detected at 450 nm and identified by comparing its retention time to that of ζ-carotene standard.

2.8 Determination of Culture Conditions for ζ-Carotene Accumulation

E. coli BL21 (DE3) co-transformed with pET-28a-UpPDS and pAC-PHYTipi was cultured in 50 mL LB medium supplied with 50 mg/mL kanamycin and 50 μg/mL chloramphenicol, and ζ-carotene was induced to accumulate by addition of IPTG into the culture. To detect the effects of initial OD600 of the culture, IPTG concentrations, induction temperatures and induction time, the initial OD600 of the culture was selected at 0.3, 0.5, 0.8, 1.2and 1.9, the IPTG concentrations were adjusted to 0, 0.25 ,0.50 ,0.75 and 1.00 mmol/L, the induction temperatures were set at 13, 17, 22, 27, 32 and 37°C, and the induction time lasted 4, 9, 14, 19, 24 and 29 h, respectively. Three independent replicates were performed. The amount of ζ-carotene in the cultures was measured by HPLC as described above. Based on the results of single factor test, the L9(3

4) orthogonal test was designed based on initial OD600 value, IPTG concentration, induction temperature and induction time, each group was designed with 3 parallels and then averaged. Experiments were carried out according to Table 1 to determine the optimal culture conditions.

Table 1. The factors and levels of fermentation conditions in orthogonal design.

Levels and Factors Temperature (ºC) Time(h)

IPTG concentration(mmol/L) OD600

1 13 19 0.25 0.8

2 17 24 0.5 1.2

3 22 29 0.75 1.9

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2.9 Statistical AnalysisThe data of orthogonal test were subjected

to analysis of variance (ANOVA) for statistical significance (P < 0.05) using SPSS (Ver. 15, Chicago, USA), and analyzed by Orthogonal Experiment Design Assistant (V3.1).

3. RESULTS3.1 PDS Cloning and Bioinformatics Analysis

Total RNAs were extracted from U. pinnatifida Suringar, and a 1707 bp cDNA was successfully obtained by RT-PCR and designated as UpPDS. Sequence analysis indicated that the cDNA contained a complete open reading frame coding 568 amino acid residues. The protein molecular weight was predicted to be 62 kDa by Expasy software, and the isoelectric point (pI) was 5.79. The molecular formula of phytoene desaturase encoded by the UpPDS gene was C2803H4409N737O808S29, and the instability coefficient of phytoene desaturase was 37.65, indicating that the protein was a stable protein. The total average hydrophilicity (GRAVY) of UpPDS protein was -0.012, indicating it belonged to a class of stable hydrophilic proteins. Amino acid composition analysis showed that the phytoene desaturase encoded by UpPDS was rich in alanine and leucine, with 49.2% non-polar amino acids and 50.8% polar amino acids. Analysis by the SignalP4.0 and ChloroP programs showed that then-terminus 22 amino acid residues of UpPDS protein constituted the signal peptide which localized phytoene desaturase to chloroplast. In addition, Multiple sequence comparisons showed that the protein has three conserved domains: the dinucleotide binding motif, putative substrate carrier motif and carotenoid binding domain (Figure 1). BLASTp search indicated that the deduced amino acids of UpPDS showed a high homology with other plant PDS, with highest homology of 90% to that of Desmarestia viridis, which confirmed that UpPDS belonged to PDS family of brown algae (Figure 1). The phylogenetic tree of PDS proteins, including those from higher plants, phaeophyta, cyanobacteria, chlorobi

and chlorophyta, was constructed by MEGA7 software, which showed that UpPDS was closer in relationship to that of phaeophyta than other plants PDS (Figure 2). Analysis of the secondary structure and tertiary structure UpPDS showed that it was rich in α helix of 37.68%, followed by 38.73% random coil, 17.25% extended strand and 6.34% β turn (Figure 3).

3.2 Expression of Recombinant UpPDS ProteinIn order to characterize the phytoene

desaturase encoded by the UpPDS, the recombinant plasmid pET-28a-UpPDS was constructed, and this plasmid was introduced into E. coli BL21 (DE3). The recombinant phytoene desaturase was overexpressed in the transformants upon IPTG induction with a molecular weight of 62 kDa as judged by SDS-PAGE (Figure 4). The transformants also showed a relatively low-level expression due to expression leakage of UpPDS gene, whereas E. coli BL21 (DE3) transformed with pET-28a (+) only could not produce this protein (Figure 4).

3.3 Functional Verification of UpPDS ProteinColor complementation assays have been

conveniently and efficiently used to identify genes in carotenoids biosynthesis pathway [24]. To examine the catalytic activity of phytoene desaturase encoded by UpPDS, the plasmid pET-28a-UpPDS was co-transformed with the plasmid pAC-PHYTipi into E. coli BL21 (DE3). The co-transformed E. coli BL21 (DE3) could form pale yellow colonies (Figure 5A), while colonies of E. coli BL21 (DE3) co-transformed with pAC-PHYTipi and pET-28a (+) were white (Fig.5A), which indicated that carotenoids were probably synthesized in E. coli BL21 (DE3) harboring pAC-PHYTipi and pET-28a-UpPDS.

To further confirm the activity of the UpPDS protein in engineering bacteria, HPLC analysis was performed to determine the accumulation of ζ-carotene in the transformants. Results showed that E. coli co-transformed with pET-28a-UpPDS and

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20

Figure 1.

Figure 2.

Figure 1. Comparison of predicted amino acid sequences of UpPDS with other plants. The black boxes represent signal peptides and conserved motifs in the PDS proteins. UpPDS, Undaria pinnatifida PDS (AUE44544.1); DvPDS, Desmarestia viridis PDS (AUE44543.1); SdPDS, Scytosiphon dotyi PDS (AUE44550.1); SmPDS, Sargassum muticum PDS(AUE44553.1); PhPDS, Petunia x hybrida PDS (AKM12416.1); LcPDS, Lycium chinense PDS (AHN92038.1); ZmPDS, Zea mays PDS (AAA99519.1).

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Figure 2. Phylogenetic tree constructed from the amino acid sequences of PDS encoded proteins of U. pinnatifida and other species. A phylogenetic tree was generated using MEGA7 with neighbor joining. Within the brackets are the accession numbers of the reference proteins of other species, and the branch length indicating the evolution distance is measured by its scale of 0.2.

Figure 3. The secondary structure of PDS from U. pinnatifida. Helix, Sheet, Turn, Coil were indicated with blue, red, green, purple, respectively.

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Figure 5. Identification of PDS enzyme activity in color complementation experiments. A1. pAC-PHYTipi and pET-28a-UpPDS A2. pAC-PHYTipi and pET-28a(+). B. HPLC analysis of carotenoid pigments in E. coli co-transformation system.B1. ζ-Carotene standard. B2. pAC-PHYTipi and pET-28a-UpPDS together. B3. pAC-PHYTipi and pET-28a(+) together. B4. pAC-PHYTipi only. B5. pET-28a-UpPDS only. B6. fermentation products that have not been transformed into any plasmid.

Figure 4. SDS-PAGE of UpPDS recombinant protein. M, protein marker; lane1, cell extract from induced bacteria containing pET-28a(+) empty vector; lanes2 and 3, cell extracts from uninduced or induced bacteria harbouring pET-28a-UpPDS.

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pAC-PHYTipi produced one kind of caroteinoids which had the same retention time as that of ζ-carotene standard (Figure 5B). Correspondingly, no pigment was detected in E. coli transformed with pET-28a-UpPDS only, or co-transformed with pET-28a (+) and pAC-PHYTipi (Figure 5B). This result verified the function of UpPDS protein, which could catalyze the conversion of phytoene to ζ-carotene.

3.4 Effects of Culture Conditions on ζ-carotene Accumulation

The engineering bacteria, E. coli BL21 (DE3) co-expressing crtB, crtE and UpPDS, could accumulate ζ-carotene. In this study, effects of the initial OD600 of the culture, IPTG concentration, induction time and inducing temperature on ζ-carotene synthesis were investigated. Single factor analysis indicated that the culture accumulated highest amount of ζ-carotene when the initial OD600 was 1.2, IPTG concentration was 0.5 mmol/L, induction time lasted for 24 h, and inducing temperature was 17ºC (Figure 6). According to the orthogonal experimental results, the optimal fermentation conditions were summarized as follows: initial OD600 value was 1.9, IPTG concentration was 0.5 mmol /L, induction time 19 h, and induction temperature was 17°C. Under this optimal condition, the production of ζ-carotene reached 250 μg for 50 mL culture, and we calculated that the yield of ζ-carotene could reach 5.0 mg/L (Table 2). According to the R value in the Table 2, the order of the four factors influencing ζ-carotene accumulation of engineering bacteria was temperature > TPTG concentration > time > OD600. The results of variance analysis showed that the effect of temperature is significant (P < 0.05), which indicates that the temperature has the greatest effect on the accumulation of ζ-carotene in engineering bacteria.

4. DISCUSSIONCarotenoids are the most widely distributed

pigments in nature [25].It not only plays an important role in plant photosynthesis, but also is

widely used in food, cosmetics and health products. Fucoxanthin is a major kind of carotenoids produced in brown algae and shows a multifunctions. At present, several genes encoding key enzymes in the carotenoid synthesis pathway have been cloned from diatoms and green algae, and few reports on the cloning and identification of these genes from brown algae have been reported [26]. As the main rate-limiting enzyme in the carotenoid biosynthesis pathway, phytoene desaturase has received more research interest. In this study, we successfully cloned the full-length ORF of UpPDS from U. pinnatifida Suringar, which encoded a protein of 568 amino acids with an estimated molecular weight of 62 kDa.

The high homology between the sequence of the UpPDS protein and PDS of other plants showed that the UpPDS had maintained four conservative structure and function domains during evolution. The first conserved domain is the signal peptide, which is located at the N-terminus of PDS and responsible for transportation of PDS to plastids. Dinucleotide binding motif (GXGX2GX3AX2LX3GX6EX5GG), which contains a β-sheet-helix-β-sheet configuration, is a characteristic sequence binding nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or flavin adenosine dinucleotide (FAD). All known carotenoid desaturases and cis-trans-isomerases contain dinucleotide-binding features[27]. Besides, putative substrate carrier motif found commonly in phytoene synthase and lycopene β-cyclase [28] and carotenoid binding domain [29] have also been identified in UpPDS protein.

A great deal of researches have been done through the genetic engineering to increase carotenoids in plants, and promising progresses have been made [30]. Many engineering bacteria for producing various carotenoids such as lycopene, β-carotene and zeaxanthin, have also been constructed and their culturing conditions were also well investigated [31]. However, there is few study on production of ζ-carotene in engineering

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Figure 6. Univariate analysis of the production of ζ-carotene. A. Induced temperature on the production of ζ-carotene. B. Effect of induced time on production of ζ-carotene. C. IPTG induced concentration on the production of ζ-carotene. D. The amount of ζ-carotene produced by the induced initial OD600 value. E. ζ-carotene standard curve.

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E. coli. In this study, we constructed engineering bacteria accumulating ζ-carotene, and studied effects of the culture conditions on ζ-carotene biosynthesis in order to provide a new resource to prepare ζ-carotene. From the test results, we found that induction temperature had significant influence on ζ-carotene accumulation, and the optimal induction temperature was only 17ºC, which was a very low temperature compared with the optimal culturing temperature of E. coli. This was very probably due to the hard refolding of recombinant phytoene desaturase (data not shown), and lowering induction temperature might facilitate the refolding of phytoene desaturase. ACKNOWLEGEMENT

This work was supported by the Project for Demonstration Cities for Innovative Development in Marine Economy (201606), Applied Basic Research Special Fund For Young Talents of Original

Innovation Project of Qingdao (18-2-2-54-jch) and Postdoctoral Applied Research Project of Qingdao (Grant Number 2015142).

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Table 2. ζ-carotene production (μg) and analysis in in an orthogonal experiment.

Factor Temperature (ºC)

Time(h)

IPTG concentration(mmol/L) OD600

ζ-carotene production(μg)

Level

L1 13 19 0.25 0.8 53.5±4.40g

L2 13 24 0.5 1.2 60.5±1.55g

L3 13 29 0.75 1.9 59±0.75g

L4 17 19 0.5 1.9 250±17.73a

L5 17 24 0.75 0.8 210±6.25b

L6 17 29 0.25 1.2 190±11.11c

L7 22 19 0.75 1.2 140±4.69e

L8 22 24 0.25 1.9 120±6.27f

L9 22 29 0.5 0.8 150±2.79d

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K3 136.6 133.0 136.3 143.0

R 159.0 17.66 32.33 12.83

Data were means ± SD of three replicates. Values in the column followed by the same letter did not differ significantly at P < 0.05.

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