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Introduction of #-3 desaturase obviously changed the fatty acid profile and sterol content of Schizochytrium sp. Lujing Re, Xiaoyan Zhuang, Sheglan Chen, Xiao-Jun Ji, and He Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04238 • Publication Date (Web): 23 Oct 2015 Downloaded from http://pubs.acs.org on October 26, 2015

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Journal of Agricultural and Food Chemistry

Introduction of ω-3 desaturase obviously changed the fatty acid profile and sterol content of Schizochytrium sp. Lu-jing Ren, Xiao-yan Zhuang, Sheng-lan Chen, Xiao-jun Ji, He Huang* State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China

*Corresponding author. Tel./fax: +86 25 58139942. E-mail: [email protected] (H. Huang)

ACS Paragon Plus Environment


1

Abstract:

2

ω-3 fatty acids play significant roles in brain development and cardiovascular

3

diseases prevention and have been widely used in food additives and pharmaceutical

4

industries. The aim of this study was to access the feasibility of ω-3 desaturase for

5

regulating fatty acid composition and sterol content in Schizochytrium sp. The

6

exogenous ω-3 desaturase gene driven by ubiqutin promoter was introduced by 18S

7

homologous sequence to the genome of Schizochytrium sp.. Genetically modified

8

strains had greater size and lower polar lipids than wild type strains. In addition, the

9

introduction of ω-3 desaturase improved ω-3/ω-6 ratio from 2.1 to 2.58 and converted

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3% DPA to DHA. Furthermore, squalene and sterol contents in lipid of genetically

11

modified strain reduced by 37.19% and 22.31%. The present study provided an

12

advantageous genetically engineered Schizochytrium sp. for DHA production and

13

effective metabolic engineering strategy for fatty acid producing microbes.

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Keywords: Schizochytrium sp., ω-3 desaturase gene, fatty acids, sterol, squalene


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Introduction

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Omega-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid

17

(DHA, 22:6 ω-3) and eicosapentaenoic acid (EPA), are beneficial to the healthy

18

growth of human body and have been widely used in food additives and

19

pharmaceutical industries. They paly significant roles in preventing cardiovascular

20

diseases, malignant tumors, alzheimer's disease, as well as promoting the intellectual

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development of infant 1. The recommended ω-6 /ω-3 PUFA ratio in diet is 1:1, but this

22

proportion in today’s diet is around 10–20:1 2. The human body cannot adjust the

23

proportion of ω-3 and ω-6 PUFAs, so improving the diet ω3 PUFAs have attracted

24

considerable interest.

25

Schizochytrium sp., a marine microalga, grow fast and could accumulate above

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50% lipids in cell dry weight with 40% of DHA in total fatty acids 3, which is

27

noteworthy and often considered as a satisfactory strain for DHA industrialization.

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Besides DHA, the lipid from Schizochytrium sp. also contains C14:0, C16:0, DPA and

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a small amount of ARA and EPA 4. DPA, belongs to omega-6 polyunsaturated fatty

30

acids, lack one double bond in the C-19 position of carbon backbone comparing with

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DHA. They were both synthesized by polyketide synthase pathway. Many previous

32

works 5 have concentrated on the regulation of fatty acid composition but is not very

33

effective. Interestingly, the proportion of DHA and DPA often increased and

34

decreased collectively as the environmental changes 6. It is very difficult to change the

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ratio of DHA/DPA by traditional fermentation condition optimization and regulation.

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ω-3 desaturase

7

can convert ω-6 PUFA into ω-3 PUFA effectively and this



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enzyme have been widely used to change the fatty acid composition in animals, plants

38

and microorganism. Lai et al

39

expressing the fat-1 gene, encoding a kind of ω-3 fatty acid desaturase. Liu et al.

40

introduced ω-3 desaturase gene from soybean into rice and found that α-linolenic

41

acid content in the seeds increased from 0.36 mg g-1 to 8.57 mg g-1. ω-3 desaturases

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isolated from different species have distinct substrate preferences. The ω-3 desaturase

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derived from Saprolegnia diclina and Pythium irregular were proved to catalyze the

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18-carbon, 20-carbon and 22-carbon fatty acids

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convert 8.4 percentage of 22:4 n-6 to 22:5 n-3 11. The ω-3 desaturase from C. elegans

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could also catalyze 22-carbon fatty acids and enable non-DHA producing Arabidopsis

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generate 2.31% of DHA in its fatty acids 12.

8

generated cloned pigs rich in ω-3 fatty acids by 9

10

. Using this enzyme, yeast could

48

In this study, we established a transformation system for Schizochytrium sp. The

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zeocin resistance gene (ble) driven with a CYC1II promoter-PEF1 terminator system,

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and ω-3 desaturase gene from Saprolegnia diclina driven with an ubiquitin

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promoter-terminator system were specifically incorporated into 18S ribosomal DNA

52

(rDNA) by homologous recombination. Then, cell growth, fatty acid composition and

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sterol content were compared between the ω-3 desaturase transformants and the wild

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type strains, aiming at accessing the feasibility of ω-3 desaturase for regulating the

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fatty acid composition and sterol content in Schizochytrium sp.


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Materials and methods

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Microorganism and medium

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Schizochytrium sp. HX-308 (CCTCC M209059), stored in China Center for Type 13

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Culture Collection (CCTCC)

, was used in the present study. This strain was

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preserved in 20% (v/v) glycerol at -80oC. The seed culture medium and the conditions

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were as same as our previous study 14. The culture preserved in the glycerine tube was

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inoculated into a 250-ml flask with 50 ml medium and cultivated for 24 h. After three

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generation cultivation, the seed culture was inoculated into a 5 L fermentor with the

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fed-batch fermentation. The initial cell dry weight at the beginning of the

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fermentation is about 1 g/L. The aeration rate and agitation speed in the preliminary

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experiment were 1 vvm and 350 rpm, the initial glucose concentration was 40 g/L and

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incubated at 30 °C. Glucose solution was fed into the bioreactor when the residual

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glucose concentration was below 20 g/L to keep the residual glucose concentration at

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40 g/L.

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Construction of expression vector

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As shown in Fig 1, the zeocin resistance gene expression cassette, which

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contains the TEF1 promoter, zeocin resistance gene (ble) and CYC1 terminator, was

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amplified from pGAPZaA and inserted into pBlueScript II SK by EcoRI and BamHI.

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This resulting plasmid was designated pBS-Zeo. The 18S rDNA fragment of

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Schizochytrium sp. HX-308 was amplified using universal primers and cloned into a

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T-vector (Takara, Dalian, China), named PMD-18S. The 18S upper and downstream



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segments were amplified using primers in Table 1. The segments for homologous

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recombination were amplified from PMD-18S-up and PMD-18S-down and cloned

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into pBS-Zeo, resulting in the targeting vector pBS-Zeo-18S. The ω-3 desaturase gene

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from

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promoter/terminator were respectively synthetized from the Genewiz, a company

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providing the service of DNA synthesis and sequencing, and ligated into pUC57

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Simple, generating pUC-ω-3, pUC-promoter, and pUC-terminator. After being

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sequenced, the amplified fragments were further modified by PCR using primers P1

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to P6 were list in Table 1, thus generating DNA fragments of ω-3 desaturase gene,

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ubiquitin promoter, and ubiquitin terminator, respectively. Subsequently, overlap

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extension PCR was applied once to splice the above three fragments in order, making

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the full-length product promoter-ω-3-terminator. The spliced fragment was then

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digested with BamHI, and the digested fragment was inserted into the multiple

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cloning site (MCS) of PBZ-Zeo-18S, resulting in the plasmid PBZ-18S+omega3 (Fig

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1).

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Transformation of Schizochytrium sp.

Saprolegnia

diclina

encoding

ω-3

desaturase

and

the

ubiquitin

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The Schizochytrium sp. cells in a logarithmic growth phase were harvested in

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ice-cold polypropylene pipes by centrifugation (5000rpm, 4°C, 10 min) and washed

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with ice-cold sterile water and 1M sorbitol twice respectively. Then suspend cell with

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1M sorbitol. The targeting plasmid PBZ-18S+omega3 were digested with the

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restriction enzyme BamHI for linearization before transformation. The competent

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cells were mixed with linearized plasmid DNA and transferred to a 0.2-cm cuvette for


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electroporation. The parameters of cell electroporation were 0.75 KV, 200 Ω, 50 µF.

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After electroporation, 1ml seed medium were added to incubate the cell at 200rpm,

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30 °C for 1 h, then the putative transformants were selected by plating them on solid

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media containing 1.5 ug/ml zeocin at 28 °C.

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Analytical methods

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Glucose was measured enzymatically using a bioanalyzer (SBA-40C, Institute of

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Biology, Shandong Academy of Sciences, Jinan, China). Ten millilitres fermentation

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broth was extracted by pipette and injected into a dried centrifuge tube. Then the

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broth was centrifuged, and the supernatant was discarded. Then all the cells were

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transferred to the filter paper which was dried and weighted, and the filter paper was

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put into an oven at 60°C to dry until the drying did not reduce the weight. The

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methods of lipid extraction and fatty acid methyl esters (FAMEs) preparation were the

111

same as those used in our previous study 15.

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Analysis of lipid fractions was performed according to Fan et al.

16

with some

113

modifications. The total lipid (2.5 g) was fractionated to neutral lipids (NLs) and polar

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lipids (PLs) by elution on a silica column, initially with petroleum ether/diethyl ether

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(9:1) and then with methanol. After evaporation of the eluate, the amount of each lipid

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fraction was determined gravimetrically. Unsaponifiable matters were isolated from

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lipid by saponification and analyzed by GC-MS

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three times to ensure the precision of data.

17

. Each experiment was conducted



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Results and Discussion

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Expression of ω-3 desaturase gene in Schizochytrium sp.

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Our previous studies have verified the feasibility of ble gene transformation in

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Schizochytrium sp. and proved that using 18SrDNA as the recombination site for

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Schizochytrium has no obvious effect on cell growth and fatty acid composition. Then,

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we expressed ω-3 desaturase gene using an omega-3 expression cassette driven by

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ubiquitin promoter/terminator (Fig 2A). The omega-3 expression cassette, obtained by

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the overlap extension PCR (Fig 2B), was inserted to the plasmid PBS-Zeo-18S to

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generate the PBS-Zeo-omega 3 (Fig 2C). 2.6 kbp PCR products corresponding to the

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size of omega-3 expression cassette were amplified from the genome DNA of the

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transformates (Fig 3D), indicating that the exogenous gene has been incorporated into

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the genome of Schizochytrium sp. As shown in Fig 3E, omega-3 transformate cells

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were slightly greater than the original strain.

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18SrDNA sequence is a suitable recombination site for genetic engineering.

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Therefore, we chose the 18S sequence as the homologous recombination sites in this

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study. Generally, the 18SrDNA sequence exists in multiple copies and locates at

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transcriptional active regions in organisms. Thus, this homologous sequence provided

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more integration chances and higher expression levels of exogenous gene.

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Effect on cell growth and lipid accumulation

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To further investigate the discrepancy between the wild-type strain and the

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genetically modified strain, these two strains were further cultivated in a 5 L

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fermentor respectively. The time courses of substrate consumption, cell growth and


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lipid accumulation were shown in Fig. 3. During the fermentation, wild-type strains

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consumed 209 g of glucose, while the genetically modified strain consumed 178 g of

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glucose. Thence, cell dry weight and lipid yield of GM strain were all slightly lower

144

than that of wild type strain. But the conversion rate of glucose to cell dry weight and

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lipid were approximately the same. These phenomena indicated that the genetically

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modified strain has lower glucose consumption rate but the same substrate conversion

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rate. As shown in Fig 3A, DO of genetically modified strain maintained at around 5%

148

while the value of wild type strain were always zero after 12h, indicating that

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genetically modified strain consumed less oxygen than the original strains. Therefore,

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the introduction of ω-3 desaturase genes reduced the glucose consumption rate and

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cell oxygen demand. Yan et al

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synthetase gene into Schizochytrium sp. TIO1101, and they found cell growth rate and

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glucose consumption rate of the modified strain was also lower than the wild type

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strain, which also indicated that the introduced exogenous gene might increase the

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burden of the cell.

18

introduced the Escherichia coli acetyl-CoA

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The introduction of ω-3 desaturase gene also caused significant changes in lipid

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fractions of Schizochytrium sp. HX-308. As shown in Table 2, the proportion of NLs

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increased from 77.70% of wild-type strain to 89.77% of genetically modified strain.

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Corresponding, the proportions of PLs decreased from 22.30% to 10.23%. As we

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know, neutral lipids were the main storage lipids and polar lipids were the main

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components of membrane structure

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strain might be related to the change of cell morphology. The cell size of genetically

19

. The decrease of PLs in genetically modified



163

modified strain was greater than that of the wild-type strain (Fig 2). For a certain

164

weight of cells, larger ones requires less number of cells and need fewer membrane

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structures to breeding, which means need less polar lipids.

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Effect on ω-3/ω-6 ratio

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In the genetically modified strain, the ratio of ω-3/ω-6 fatty acids and DHA/DPA

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were all higher than that of wild-type strain at different stages of the fermentation (Fig

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5). In particular, the ratio of ω-3/ω-6 at 24h in GM strain reached the maximum of

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2.58, which was 25.85% higher than that of wild type strain, indicating that ω-3

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desaturase had the highest expression at the beginning of the fermentation. At this

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time, nitrogen did not exhaust and cell grew fast. The balanced nutrition conditions

173

might give good environment for the expression of exogenous genes. After 24h, the

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ratio of ω-3/ω-6 decreased from 2.58 to 2.38 and then held constant until the end of

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the fermentation, suggesting that the ω-3 desaturase expressed stable in lipid

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accumulation stage. As we known, the reaction from DPA to DHA catalyzed by ω-3

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desaturase need oxygen as an important substrate

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dissolved oxygen during the fermentation was also an important reason for the

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decrease of ω-3/ω-6 ratio. But the ω-3/ω-6 ration and DHA/DPA ratio of wide type

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strain kept constant during fermentation, this might be related to the PKS pathway,

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which does not need oxygen, so the values could keep constant during the

182

fermentation no matter the dissolve oxygen decrease or not. At the end of

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fermentation, DPA and DHA percentage in total fatty acids in genetically modified

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strain reached 18.67% and 49.23%. Compared with the value of 21.65% and 46.17%

20

, so we think the decrease of


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of wild-type strain, 3% DPA was converted to DHA by the introduced ω-3 desaturase.

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In fact, there are many other studies trying to modify the fatty acid compositions 17

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by optimizing the culture conditions. We previously

investigated the differential

188

effects of nutrient limitations on biochemical constituents and docosahexaenoic acid

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production of Schizochytrium sp. and found that the ratios of DHA/DPA at different

190

nutrient conditions were basically constant at 2.15, no big difference. Kavita P. Patil 21

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studied the effect of different media supplements on DHA yields in Schizochytrium

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limacinum SR21 and found that DHA yield and DPA yield mutually increased or

193

decreased. In addition, the DHA/DPA ratio could increase from 5.58 to 6.47 when

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changing ammonium acetate to sodium acetate in the medium, but cell dry weight

195

decreased a half, which is very uneconomical. Song et al 22 found that DHA and DPA

196

percentage in total fatty acids could increase from 35.30% and 7.02% to 61.4 % and

197

12.32% respectively when adding pentanoic acid in the medium. The DHA/DPA

198

ratios in the two cultures were all around 5.0, which also indicated that DPA and DHA

199

percentages often increased and decreased collectively.

200

In addition, the ratio change in this study was also not too high, but it indeed

201

decreased the DPA percentage and increased the DHA percentage simultaneously,

202

which proved the introduction of ω-3 desaturase. The low conversion efficiency might

203

be ascribed to the pathway shift and enzyme catalysis efficiency. As we known, DPA

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and DHA were synthesized by polyketide synthase pathway

205

but the mechanism of the double bond formation and chain elongation were still

206

unclear, especially how cell selectively synthesized DHA and DPA, the two molecules


23

in Schizochytrium sp,


207

with so similar structure but only differ with one double bond in the carbon chains. In

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this study, the introduction of ω-3 desaturase gene integrated into the genome of

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Schizochytrium sp. catalyzed parts of the intermediates of polyketide synthase

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pathway, especially ω-6 fatty acids, to ω-3 fatty acids. Comparing with the conversion

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efficiency

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DPA was converted to DHA. This might be ascribed to the reason that polyketide

213

synthase pathway was independent of the elongation and saturation pathways, ω-6

214

fatty acids, such as DPA and ARA, obtained from polyketide synthase pathway might

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need more time and space to complete the pathway transformation.

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Effect on squalene and sterol content

24

of ARA to EPA in Mortierella alpina 1S-4 by ω-3-desaturase, only 3%

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Squalene and sterol were two main kinds of unsaponifiable matters in DHA-rich

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oil of Schizochytrium sp. Squalene content of genetically modified strain decreased

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from 22.09 mg/g at 48 h to 17.37 mg/g at 96 h during lipid accumulation stage, then

220

dropped to 27.86 mg/g. Wild type strains had similar change trends. In particular,

221

squalene contents of genetically modified strain were all lower than that of wild-type

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strain during the whole fermentation process. The final squalene content of

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genetically modified strain was 37.19% lower than 27.86 mg/g of wild type strains,

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indicating that the introduction of ω-3 desaturase obviously decreased the squalene

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content (Table 3). As we know, squalene has antioxidant activity and may thus

226

provide a cellular defense against active oxygen that damage DNA, protein amino

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acids or polyunsaturated fatty acids

228

value of unsaponifiable matters, which was required to be below 4% in lipid

25

. But too much squalene would increase the


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according to the GB 26400-2011. The introduction of ω-3 desaturase can improve the

230

quality of DHA-rich oil to a certain extent.

231

Sterols in Schizochytrium sp. were also identified and analyzed by GC–MS and

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five main sterols, including cholesterol, stigmasterol, ergosterol, lanosterol and

233

cycloartenol, were detected and quantified throughout the fed-batch process (Table 3).

234

The sterol contents of genetically modified strain were all lower than the value of wild

235

type strain except ergosterol. The final lanosterol and cycloartenol content were only

236

0.492 mg/g and 0.152 mg/g respectively, which were 23.44% and 54.55% lower than

237

that of wild-type strain. Strangely, the ergosterol content of genetically modified strain

238

was three times of the value of wild type strains. The final value of ergosterol content

239

in modified strain reached 0.171 mg/g, while the value in wild type strain was only

240

0.052 mg/g. In wide type strain, the ergosterol content decreased during the

241

fermentation but the value in modified strain did not decrease. As we known, the

242

ergosterol belongs to the fungi sterol and is the common sterol in cell membrane. This

243

might indicate that the modified strain might need more ergosterol to maintain its

244

greater cell size.

245

Sterols and squalene were synthesized by the mevalonate pathway, in which

246

hydroxy-methylglutaryl CoA reductase (HMG CoA reductase) and fatty acyl-CoA

247

cholesterol acyltransferase (ACAT) were the main rate-limiting enzymes. Previous

248

studies

249

and increase the activity of ACAT. Therefore, the introduction of exogenous ω-3

250

desaturase gene improved the DHA content of Schizochytrium sp., and the increased

26

have found ω-3 PUFAs could reduce the activity of HMG CoA reductase



251

ω-3 polyunsaturated fatty acids might restrain the mevalonate pathway and thence

252

reduced the content of sterols and squalene 27.

253

In this study, the exogenous ω-3 desaturase gene was successfully incorporated

254

and expressed in Schizochytrium sp., and then the effect of ω-3 desaturase gene on

255

cell growth, lipid accumulation, and fatty acid synthesis were systematically

256

examined. The introduction of ω-3 desaturase not only improved the ratio of ω-3/ω-6

257

but also decreased the contents of unsaponifiable matters such as squalene and sterols.

258

This study proved the potential of ω-3 desaturase to improve the ratio of ω-3/ω-6 fatty

259

acids of oleaginous microorganism. In addition, this study provided an advantageous

260

genetically engineered Schizochytrium sp. for industrial DHA production and

261

effective metabolic engineering strategy for fatty acid producing microbes.

262

Author Information

263

Corresponding Authors

264

*He Huang Phone: +86 25 58139942. E-mail: [email protected] (H. Huang)

265

Funding

266

This work was financially supported by the National Science Foundation for

267

Distinguished Young Scholars of China (No. 21225626), the National Natural Science

268

Foundation of China (No. 21306085), the National High Technology Research and

269

Development Program of China (No. 2012AA021704 and No.2014AA021701) and

270

the Specialized Research Fund for the Doctoral Program of Higher Education (No.

271

20133221120008).

272


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Figure Captions Fig. 1 Structure of the targeting vector recombinant plasmid PBZ-18S+omega-3 Fig 2 Argarose gel of different fragements. (A) ω-3 desaturase gene, ubiqutin promoter and termenter; (B) the products of the overlap extension PCR; (C) PBZ-18S-omega-3 gene vector; (D) PCR product amplified from the genome DNA of the transformates; (E) Cell morphology of wild type strain and genetically modified strain in electron microscope. Fig.3 (A) Time course of cell dry weight and DO of two strains (B) Time course of glucose consumption and lipid accumulation of two strains. (W is wild-type strain, G is genetically modified strain). Fig.4 (A) Comparison of ω-3/ω-6 fatty acid ratio between W and G. (B) Comparison of DHA/DPA ratio between the W and G. (W is wild-type strain, G is genetically modified strain)



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Table 1 Primers used in this study Gene

Primer

Primer sequence(5’-3’)

name Ubiquitin

P-S

TCGGATCCCGTTAGAACGCGTAATAC

promoter

P-A

TTCGTCTTATCCTCAGTCATGTTGGCTAGTGTTGCTTAGGTCGCT

ω-3 desaturase

ω-3-S

CCTAAGCAACACTAGCCAACATGACTGAGGATAAGACGAAGGT

gene

ω-3-A

ATACTACAGATAGCTTAGTTTTAGTCCGACTTGGCCTTGG

Ubiquitin

T-S

CCAAGGCCAAGTCGGACTAAAACTAAGCTATCTGTAGTATGTGC

terminator

T-A

TCGGATCCACCGCGTAATACGACTCACTATAGGGAGACTGCAGTT

18SupS

5’- GGGTACCCGTAGTCATATGCTTGTCTC -3’

18SupA

5’- CCTCGAGGATTTCACCTCTAGCGAC -3’

18SdownS

5’- CGGATCCGATGCCGACTAGAGATT-3’

18SdownA

5’- GAGCTCTCCGCAGGTTCACCTACGGA-3’

18Sup

18Sdown


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Table 2 Comparison of kinetic parameters and lipid fractions of wild type and modified strains

Wild-type strain

Genetically modified strain

Yx/s

0.360±0.002

0.390±0.002

Yp/s

0.230±0.002

0.275±0.002

YDHA/s

0.116±0.001

0.141±0.001

NLs (%)

77.700±0.541

89.770±0.690

PLs (%)

22.301±0.130

10.230±0.342



Page 22 of 27

Table 3 . Differences of unsaponifiable matters during fed-batch cultivation of Schizochytrium sp.

Time

(W is wild-type strain, G is genetically modified strain)

Squalene

Ergosterol

Cholesterol

Stigmasterol

Lanosterol

Cycloartenol

（mg/g）

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

W

33.21±0.841

0.170±0.002

0.091±0.002

0.231±0.012

0.541±0.025

0.291±0.014

G

22.09±0.293

0.120±0.004

0.052±0.003

0.192±0.013

0.373±0.021

0.052±0.024

W

24.19±0.682

0.090±0.004

0.071±0.002

0.141±0.011

0.382±0.014

0.154±0.017

G

17.37±0.514

0.141±0.004

0.053±0.002

0.091±0.009

0.344±0.015

0.071±0.022

W

38.22±0.782

0.052±0.006

0.081±0.003

0.111±0.008

0.641±0.017

0.330±0.022

G

27.86±0.864

0.171±0.003

0.052±0.004

0.082±0.012

0.492±0.020

0.152±0.017

Strain

48h

72h

108h


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Fig. 1 Structure of the targeting vector recombinant plasmid PBZ-18S+omega-3



Ubi pro

A

Omega-3 desaturase Ubi ter

B

2000

Omega-3 desaturase cassette

C

PBZ-18S-omega3

D

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desaturase M( bp) expression cassette

10000 7000

1000

5000

750

3000

500

2000

5000 3000 2000 1000

2600

E

Modified strain

Wild type

Fig 2 Argarose gel of different fragements. (A) ω-3 desaturase gene, ubiqutin promoter and termenter; (B) the products of the overlap extension PCR; (C) PBZ-18S-omega-3 gene vector; (D) PCR product amplified from the genome DNA of the transformates; (E) Cell morphology of wild type strain and genetically modified strain in electron microscope.


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Fig.3 (A) Time course of cell dry weight and DO of two strains (B) Time course of glucose consumption and lipid accumulation of two strains. (W is wild-type strain, G is genetically modified strain).



Fig.4 (A) Comparison of ω-3/ω-6 fatty acid ratio between W and G. (B) Comparison of DHA/DPA ratio between the W and G. (W is wild-type strain, G is genetically modified strain)


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