Enhancement of Torularhodin Production in ... - ACS Publications

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Biotechnology and Biological Transformations

Enhancement of torularhodin production in Rhodosporidium toruloides by Agrobacterium tumefaciens-mediated transformation and culture conditions optimization Ruiqi Bao, Ning Gao, Jing Lv, Chaofan Ji, Huipeng Liang, Shengjie Li, Chenxu Yu, Zhenyu Wang, and Xinping Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04667 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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Journal of Agricultural and Food Chemistry

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Enhancement of torularhodin production in Rhodosporidium toruloides by

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Agrobacterium tumefaciens-mediated transformation and culture conditions

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optimization

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Ruiqi Baoa, Ning Gaob, Jing Lva, Chaofan Jia, Huipeng Lianga, Shengjie Lia, Chenxu Yuc,

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Zhenyu Wang a,*, Xinping Lina,*

6

a

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Engineering Research Center of Seafood, 1 Qingogng Yuan, Dalian 116034, PR China.

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b

9

Sciences, 457 Zhongshan Road, Dalian 116023, PR China

School of Food Science and Technology, Dalian Polytechnic University, National

Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of

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c

11

USA

12

*Corresponding

13

[email protected], [email protected]

Department of Agricultural and Biosystems Engineering, Iowa State University, IA 50011,

author

(Tel.:

+86041186318675,

fax:

1

ACS Paragon Plus Environment

+86041186318655,

E-mail:


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ABSTRACT: Nine transformants of Rhodosporidium toruloides with significant changes in

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carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation

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(ATMT), including a white, three red and four yellow mutants. A red mutant A1-15-BRQ that

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showed a high torularhodin production was selected for culture condition optimization.

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Results indicated that torularhodin yield was boosted with glucose as carbon source, at a C/N

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ratio of 22, at a loading volume of 75 mL, and at 28°C. The torularhodin yield of 21.3 mg/L

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consisting of 94.4% of total carotenoids was obtained by Box-Behnken designed (BBD)

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experiments. The torularhodin yield was 17.0-fold higher than that of wild type with time

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shorten from 9 days to 3 days. This study reports an effective strategy for improving

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torularhodin production, and provides a candidate R. toruloides strain for highly selective

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production of torularhodin.

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Rhodosporidium

toruloides,

torularhodin,

Agrobacterium

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KEYWORDS:

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tumefaciens-mediated transformation, response surface method, selective production

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INTRODUCTION

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Carotenoids are terpenoids with many conjugated double bonds, and are widely used in

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food, cosmetics and pharmaceutical industries.1, 2 Red yeast Rhodosporidium toruloides is one

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of the well-known producers of carotenoids.3 Carotenoids produced by R. toruloides mainly

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consist of β-carotene, torulene, torularhodin and γ-carotene (Figure 1).4 It has been shown that

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the antioxidant capacity of carotenoid species is positively correlated with their number of

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conjugated double bonds.5 Hence, torularhodin (Figure 1 a), which has more double bonds, is

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a stronger antioxidant than β-carotene (Figure 1 d). Torularhodin is capable of maintaining

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stability of cell membranes under stress, regulating immune system, reducing risk of diseases

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such as certain cancer and prostate disease, and improving antimicrobial ability of Titanium

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materials.6-9 Although β-carotene has been widely studied and utilized in recent years,

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application of torularhodin is still rare due to its high production cost. Therefore,

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finding/cultivating microbial species with a high yield of torularhodin is of crucial importance

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in reducing the process cost and promoting industrial production of torularhodin.

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(Figure 1)

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Mutagenesis is a common technique to induce microbial strains to produce a high yield of

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carotenoid. Zhang et al. reported the selection of a dark-red mutant strain XR-2 of red yeast

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with high carotenoids and lipids production, by combining physical mutagenesis (atmospheric

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and room temperature plasma) with chemical mutagenesis (nitrosoguanidine).10 Gharibzahedi

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et al. utilized UV radiation and EMS treatment to obtain a high canthaxanthin content Dietzia

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natronolimnaea mutant.11 Agrobacterium tumefaciens-mediated transformation (ATMT) is

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one of the most frequently used mutagenesis strategies developed in recent years.12,

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13


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Compared with other mutagenesis methods, ATMT yields have much higher mutation rate due

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to direct gene modification. ATMT had been applied successfully in R. toruloides in previous

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studies. Liu et al. reported the first successful transformation of R. toruloides14 and Lin et al.

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applied ATMT to increase carotenoid production in R. toruloides by establishing R. toruloides

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NP11 mutant libraries and obtaining three mutants whose carotenoid yields were higher than

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in wild type NP1115. However, ATMT method hasn’t been utilize to alter carotenoid

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production profile in R. toruloides.

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Biosynthesis of carotenoid in red yeast can be influenced by numerous factors. Bhosale et

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al. reported effects of carbon to nitrogen (C/N) ratio and media components (carbon sources,

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nitrogen sources and salts) on carotenoid production by Rhodotorula glutinis.16 Zhang et al..

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discovered that illumination levels and temperature can significantly affect carotenoid

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biosynthesis in R. glutinis.17 Parreira showed that both carbon concentration and oxygen

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availability were factors that affect carotenoid production by R. toruloides NCYC 921.18 To

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optimize culture conditions for carotenoid production, response surface method (RSM) is

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often used to find the best parameters. With RSM, Saenge et al.19 optimized initial chemical

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oxygen demand in palm oil mill effluent, C/N ratio, and Tween 20 concentration for

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concomitant production of lipids and carotenoids; Singh et al.20 optimized glucose

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concentration, NaCl concentration, culture time for lipids and carotenoids production in

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R.toruloides under osmotic stress. However, most optimization studies focused on the total

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carotenoids production in red yeast, while few reports concentrated on optimization of

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torularhodin production.

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In this study, ATMT was tested as a way to selectively boost torularhodin production in R.

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toruloides. It was used as a tool to insert gene randomly into R. toruloides NP11. Two mutant

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libraries were constructed. According to their phenotypes, several mutants with different

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colors were screened, and mutants with high torularhodin production were selected. In order

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to further improve torularhodin production and shorten fermentation time, culture conditions

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for the selected mutants were optimized by single factor experiments (carbon source, C/N

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ratio, loading volume, and temperature) and Box-Behnken designs (BBD), and RSM was

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utilized to identify the optimal conditions.

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MATERIALS AND METHODS

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Strains and Culture Media.

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R. toruloides NP11 GDMCC 2.224 and Agrobacterium tumefaciens strains AGL1-HYG1

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and AGL1-HYG2 were prepared according to Lin et al.15 Transformants were maintained on

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YEPDH agar, which is YEPD agar with 50 mg/L hygromycin and 300 mg/L cephalosporin.

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Transformants were cultivated in SD medium.15 In the later optimization experiments, mutant

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A1-15-BRQ was cultivated in basal medium. Basal medium was prepared according to lipid

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production medium in Wu’s study21 with some modifications (20 g/L glucose, 2 g/L

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(NH4)2SO4, 5 g/L KH2PO4, 0.15 g/L MgSO4 and 10 mL/L trace mineral solution, pH 6.0). The

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composition of trace element solution21 was as follow: 4 g/L CaCl2·2H2O, 0.52 g/L citric acid

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monohydrate, 0.55 g/L FeSO4·7H2O, 0.076 g/L MnSO4·H2O, 0.10 g/L ZnSO4·7H2O,100 μL

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of 18 M H2SO4 (pH 6.0).

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Fermentation Conditions.

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SD-fermentation.

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NP11 and all mutants were grown in SD medium at 28°C with agitation of 200 rpm for

5



95

36 h as pre-culture. Then 5 mL pre-culture was extracted and added to 45 mL fresh SD

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medium in a 250 mL conical flask. Subsequently fermentation was carried out at 28°C with

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agitation of 200 rpm for 216 h.

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Basal liquid medium fermentation.

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In single factor experiments, A1-15-BRQ was cultivated in YEPD medium at 28°C with

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200 rpm agitation for 24 h. 10% (v/v) seed culture was added to basal liquid medium in a 250

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mL conical flask. Subsequently, fermentation was carried out at 28°C with agitation of 200

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rpm for 72 h.

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To investigate the effects of culture conditions on A1-15-BRQ, firstly 20 g/L of glucose

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(Damao Chemical Reagent Factory, Tianjin, China), fructose (Sangon Biotech, Shanghai,

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China) and glycerol (Sangon Biotech, Shanghai, China) were added to different batches of

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basal liquid medium to select for the best carbon source. Secondly, the selected carbon source

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(i.e., glucose) was then used at different C/N ratios in subsequent experiments, namely, 9, 20,

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45, 63 g/L of glucose were added to obtain C/N ratios at 10, 22, 50 and 70, respectively.

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Thirdly, the loading volume was altered for A1-15-BRQ (25, 50, 75, 125 mL basal medium

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with C/N ratio of 22, respectively), and finally the temperature effect was investigated by

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cultivating A1-15-BRQ at 20, 25, 28, 35°C in 50 mL basal medium, respectively.

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Mutant Strains Construction by ATMT and Verification.

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The T-DNA binary vector PZPK was constructed according to Lin’s report.13 Mutant

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strains was constructed by Lin’s method.13 A. tumefaciens strains transformed with plasmid

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with PZPK-pPGK-HYG-Tnos plasmid13or PZPK-pGPD-HYG-Tnos plasmid13 were named as

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AGL1-HYG1 and AGL1-HYG2. Two mutant libraries were constructed with different

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colored transformants that were transformed with AGL1-HYG1 (i.e., PGK library) and

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AGL1-HYG2 (i.e., GPD library), respectively. Two reddish and three yellowish transformants

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were selected from the PGK library. A red, a white, and two yellowish transformants were

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selected from the GPD library. PCR was carried out to verify whether T-DNA was

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successfully inserted into NP11.

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Determination of Biomass and Residual Sugar.

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Samples of biomass and residual sugar were harvested at the end-point of fermentation.

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The cell pellets were collected from culture medium by centrifugation at 8000 rpm for 10 min.

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The precipitates were dried to a constant weight at 105°C, then measured gravimetrically.

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Biomass was expressed as dry cell weight (DCW, g/L). The supernatant was collected as well

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for determination of residual sugar (RS) concentration (g/L). RS concentration was

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determined by DNS method.22

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Extraction of Carotenoids.

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Carotenoids were extracted according to a previously published method with some

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modifications.23 Cells from 2 mL of production culture was harvested. The cell pellets were

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washed twice with deionized water, centrifuged, suspended in 1 mL 3 M HCl, kept for 20 min,

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boiled at 100°C for 4 min and cooled in ice bath for 5 min. The cell pellets were then

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collected again by centrifugation at 9600 × g for 10 min. Subsequently, the carotenoids of

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these cell pellets were extracted by 900 μL acetone under vigorous vortexing for 20 min. After

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centrifugation to remove cell debris, the organic phase was collected. The above extraction

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was repeated on the cell debris until the pellets became colorless. All the organic phase was

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combined for further analysis of carotenoids.

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Quantitation of Carotenoids.

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The extracted carotenoids were quantitated by high performance liquid chromatography

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(HPLC) equipped with a SunFireTM C18 column (5 μm: 4.6 mm  150 mm, Waters, Milford,

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USA) according to Lin et al.15 The mobile phase consisted of 89 to 100% acetone with

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ultrapure water, and flow rate was 1.0 mL/min. The total run time was 25 min. Carotenoids

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absorption was measured at 450 nm. Torularhodin, torulene, γ-carotene (CaroteNature,

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Lupsingen, Switzerland) and β-carotene (Aladdin, China) standards were used to identify and

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quantify the four carotenoids. The retention time of the carotenoids was shown at Figure

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S1(b). Total carotenoid content was obtained by summing up the four identified carotenoid

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species.

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Experimental Design for optimization of culture conditions with RSM.

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On the basis of the single factor experiment results, C/N ratio, loading volume, and

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temperature were selected as variables for response surface model inputs, which were labeled

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as X1, X2 and X3, respectively. The ranges of the three variables were shown in Table 1. A

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BBD model was designed, and 15 experiment runs were carried out containing three

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replications at the central points. The experiments were designed by using the Design Expert

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(Version 8.0.6, State Ease Inc., Minneapolis, USA). Data from the BBD were analyzed by

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multiple regressions to fit the following quadratic polynomial model (Eq. 1):

157 158

where Y is the predicted response variable, β0, βi, βii and βij are coefficients for intercept, linear,

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quadratic and interactive terms, respectively. Xi and Xj are independent variables (i

160

j). The

suitability of the model was evaluated by coefficient of determination (R2) and analysis of 8


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variance (ANOVA). Experiments were performed in duplicate and mean values were used.

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(Table 1)

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Statistical Analysis.

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The SPSS software (Version 22, SPSS Inc., Chicago, USA) and Design Expert were used

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for statistical analysis. F-test was used to analyze the significance of variations. When P
F

857.41

95.27

57.06

0.0002

intercept

19.71

0.70

X1

2.32

0.58

26.50

26.50

15.87

0.0105

X2

-1.68

0.49

19.68

19.68

11.79

0.0186

X3

13.82

1.24

206.45

206.45

123.65

0.0001

X1×X2

-0.71

0.48

3.60

3.60

2.16

0.2019

X1×X3

-1.03

0.97

1.88

1.88

1.12

0.3376

X2×X3

-1.89

0.73

11.24

11.24

6.73

0.0485

X12

-7.65

0.67

216.15

216.15

129.46

< 0.0001

X22

-4.24

0.38

210.08

210.08

125.83

< 0.0001

X32

-25.21

1.51

463.41

463.41

277.56

< 0.0001

residual

8.35

1.67

lack of Fit

7.81

2.60

9.60

0.0958

pure error

0.54

0.27

cor total

865.76 R2=0.9904

Adj R2=0.9730

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SNR=20.807

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Table

6.

Comparison

of

torularhodin

production

between

A1-15-BRQ

and

carotenoid-production strains from other studies. species R. toruloides A1-15-BRQ R. toruloides NP11 R. toruloides A1-15-BRQ S. johnsonni DBVPG 7467 R. glutinis DBVPG 6081 R. diobovatum DBVPG 6202 R. toruloides DBVPG 6739 R. toruloides CBS 5490 R. glutinis JMT 21978 R. toruloides CBS 5490

production medium glucose (29.6 g/L), (NH4)2SO4 (2 g/L), KH2PO4 (5 g/L), MgSO4 (0.15 g/L), trace mineral solution (1% v/v) glucose (20 g/L), yeast nitrogen base (6.7 g/L) glucose (20 g/L), yeast nitrogen base (6.7 g/L)

glucose (40.0 g/L), KH2PO4 (8 g/L), MgSO4.7H2O (0.5 g/L), yeast extract (3 g/L)

glycerol (60 g/L), peptone (20 g/L), yeast extract (10 g/L) glucose (50 g/L), yeast extract (5 g/L) glycerol (60 g/L), yeast extract (10 g/L), peptone(20 g/L), grape seed oil (20 % v/v)

time (d)

torularhodin production

ratio (%)

3

21.3 mg/L (5.1 mg/g)

94

9

1.3 mg/L

46

9

25.1 mg/L

97

5

41.2 μg/g

58

4

5

52.5 μg/g

47

4

5

37.8 μg/g

55

4

5

19.9 μg/g

19

4

12

19.7 mg/L

69

5

8

11.7 mg/L

65

38

12

0.6 mg/g

67

24

29


ref. This study This study This study


Figure 1.

Figure 2.

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Figure 3.

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Figure 4.

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Table of contents graphic

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Enhancement of Torularhodin Production in ... - ACS Publications

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