烯酰还原酶基因的替换对裂殖壶菌合成二十碳五烯酸的影响

doi:10.11949/0438-1157.20201818

化工学报 ›› 2021, Vol. 72 ›› Issue (7): 3768-3779.DOI: 10.11949/0438-1157.20201818

烯酰还原酶基因的替换对裂殖壶菌合成二十碳五烯酸的影响

杨瑞雄¹(),郑鑫¹,陆涛¹,赵誉泽¹,杨庆华¹,卢英华^1,^2,³,何宁^1,²,凌雪萍^1,^2,³()

^1.厦门大学化学化工学院，福建厦门 361005
^2.福建省海洋生物资源开发利用协同创新中心，福建厦门 361005
^3.福建省化学生物学重点实验室（厦门大学），福建厦门 361005

收稿日期:2020-12-15 修回日期:2021-02-27 出版日期:2021-07-05 发布日期:2021-07-05
通讯作者: 凌雪萍
作者简介:杨瑞雄（1996—），女，硕士研究生，285157801@qq.com
基金资助:
国家自然科学基金重点项目(21736009);国家自然科学基金面上项目(31871779)

Effects of substitution of ER domains on the synthesis of eicosapentaenoic acid in Schizochytrium limacinum SR21

YANG Ruixiong¹(),ZHENG Xin¹,LU Tao¹,ZHAO Yuze¹,YANG Qinghua¹,LU Yinghua^1,^2,³,HE Ning^1,²,LING Xueping^1,^2,³()

^1.College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China
^2.Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen 361005, Fujian, China
^3.The Key Laboratory for Chemical Biology of Fujian Province (Xiamen University), Xiamen 361005, Fujian, China

Received:2020-12-15 Revised:2021-02-27 Online:2021-07-05 Published:2021-07-05
Contact: LING Xueping

摘要/Abstract

摘要：

二十碳五烯酸（eicosapentaenoic acid，EPA）因具有调节血脂、降低纤维蛋白、预防心血管疾病以及抗炎抗过敏的生理功能，被广泛应用于食品、医疗、化妆品以及饲料添加等领域。目前，EPA的需求日益增加，但是富含多不饱和脂肪酸（polyunsaturated fatty acids，PUFAs）的鱼油等自然资源逐渐匮乏，利用改造的微生物菌种发酵生产EPA的方式成为主要趋势。本研究利用同源重组技术将Shewanella sp. SCRC2738的烯酰还原酶基因（enoyl-reductase，sh-ER）分别敲入Schizochytrium limacinum SR21的ORFB-ER和ORFC-ER基因中，调控多不饱和脂肪酸合成的偏好性以提高EPA的产量。研究表明，sh-ER替换ORFB-ER基因的重组菌株（B-sh-ER）中EPA含量提高了85.7%，聚酮合酶（polyketide synthases，PKS）途径相关基因转录水平明显上调。而sh-ER替换ORFC-ER基因的重组菌株（C-sh-ER）中PUFAs合成基本不变。在5 L发酵罐中放大培养后，B-sh-ER重组菌株总油脂提高28.7%，达到73.2 g/L；EPA产量提高71.6%，达到732.4 mg/L。本研究为促进裂殖壶菌PUFAs高产提供了新的策略，同时也为调控裂殖壶菌合成EPA提供了新的视角。

关键词: 裂殖壶菌, 希瓦氏菌, 二十碳五烯酸, 烯酰还原酶, 分子生物学, 发酵, 代谢

Abstract:

Eicosapentaenoic acid (EPA) is widely used in the fields of food, medical treatment, cosmetics and feed additives because of its physiological functions of regulating blood lipids, reducing fibrin, preventing cardiovascular diseases, anti-inflammatory and anti-allergic. At present, the supply of EPA cannot meet the market demand, due to the natural resources such as fish oil rich in polyunsaturated fatty acids (PUFAs) are gradually scarce. Therefore, more and more research focus on using the modified microbial strains to produce EPA. In this study, homologous recombination technology was used to knock the enoyl-reductase gene (sh-ER) of Shewanella sp.SCRC2738 into the ORFB-ER and ORFC-ER domains of Schizochytrium limacinum SR21, in order to regulate the preference for polyunsaturated fatty acid synthesis to increase the yield of EPA. The results showed that the EPA content of the B-sh-ER strain increased by 85.7% and the transcriptional level of polyketide synthases (PKS) pathway related genes were significantly up-regulated. The synthesis of PUFAs in C-sh-ER strains presented little change. In 5 L fed-batch fermentation, total lipids yield of the B-sh-ER strains increased by 28.7% reaching to 73.2 g/L; the EPA yield increased by 71.6% reaching to 732.4 mg/L. This study supplies a new strategy for promoting the high production of PUFAs and provides a novel perspective for regulating the synthesis of EPA in Schizochytrium sp.

Key words: Schizochytrium limacinum, Shewanella, EPA, enoyl-reductase, molecular biology, fermentation, metabolism

中图分类号:

Q 819

杨瑞雄, 郑鑫, 陆涛, 赵誉泽, 杨庆华, 卢英华, 何宁, 凌雪萍. 烯酰还原酶基因的替换对裂殖壶菌合成二十碳五烯酸的影响[J]. 化工学报, 2021, 72(7): 3768-3779.

YANG Ruixiong, ZHENG Xin, LU Tao, ZHAO Yuze, YANG Qinghua, LU Yinghua, HE Ning, LING Xueping. Effects of substitution of ER domains on the synthesis of eicosapentaenoic acid in Schizochytrium limacinum SR21[J]. CIESC Journal, 2021, 72(7): 3768-3779.

图/表 12

图1 希瓦氏菌、裂殖壶菌SR21、B-sh-ER菌株和C-sh-ER菌株中的PKS基因簇

Fig.1 PKS gene clusters of Shewanella sp. SCRC2738, Schizochytrium limacinum SR21, B-sh-ER strain and C-sh-ER strain

图2 B-sh-ER载体构建（a）;C-sh-ER载体构建（b）

Fig.2 Construction of B-sh-ER vector(a); Construction of C-sh-ER vector (b)

图3 含博来霉素抗性平板上生长的野生型菌株（a），B-sh-ER 菌株（b）和C-sh-ER菌株（c）

Fig.3 Wild strains(a), B-sh-ER strains (b) and C-sh-ER strains (c) grown on media containing zeocin

图4 B-sh-ER菌株中博来霉素表达框的基因组PCR (a); C-sh-ER菌株中博来霉素表达框的基因组PCR(b)

Fig.4 Genomic PCR of zeocin expression cassette in the B-sh-ER strains(a); Genomic PCR of zeocin expression cassette in the C-sh-ER strains (b)

图5 野生型菌株和B-sh-ER菌株中生物量（a），总油脂量（b）和油脂含量（c）；野生型菌株和C-sh-ER菌株生物量（d）、总油脂量（e）和油脂含量（f）

Fig.5 Biomass (a), total lipids yield (b) and lipids content (c) in the wild strain and B-sh-ER strains; Biomass (d), total lipids yield (e) and lipids content (f) in the wild strain and C-sh-ER strains

表1 野生型菌株、B-sh-ER菌株和C-sh-ER菌株脂肪酸组成

Table 1 Fatty acid composition of wild strains， C-sh-ER strains and B-sh-ER strains

Fatty acid	Composition/%
Fatty acid	Wild strain	B-sh-ER strain	C-sh-ER strain
C14:0	3.39 ± 0.05	3.59 ± 0.13	4.09 ± 0.30
C14:1	1.20 ± 0.02	1.81 ± 0.11^a	1.69 ± 0.14^a
C16:0	53.02 ± 0.60	53.88 ± 0.71	55.38 ± 0.10
C18:0	1.75 ± 0.01	2.61 ± 0.15^a	2.04 ± 0.16
ARA	0.16 ± 0.02	0.37 ± 0.01^a	0.53 ± 0.08^a
EPA	0.42 ± 0.01	0.78 ± 0.01^b	0.37 ± 0.02
DPA	6.51 ± 0.03	6.08 ± 0.10	4.45 ± 0.18^a
DHA	29.76 ± 0.25	27.63 ± 0.77	29.52 ± 0.62
EPA×100/DHA	1.61 ± 0.03	2.82 ± 0.03^b	1.25 ± 0.07^a
SFAs	58.15 ± 0.65	60.08 ± 0.69	61.51 ± 0.57^a
PUFAs	37.45 ± 0.27	34.71 ± 0.70^a	34.87 ± 0.32^a

图6 B-sh-ER菌株相关基因转录水平分析(a) 裂殖壶菌烯酰还原酶基因ORFB-ER;(b) 希瓦氏菌烯酰还原酶基因sh-ER;(c) 裂殖壶菌烯酰还原酶基因ORFC-ER;(d)酰基转移酶(AT)基因

Fig.6 The transcription level of related gene in B-sh-ER strain(a) Schizochytrium limacinum SR21 enoyl-reductase gene ORFB-ER; (b) Shewanella sp. SCRC2738 enoyl-reductase gene sh-ER; (c) Schizochytrium limacinum SR21 enoyl-reductase gene ORFC-ER; (d) acyl transferase (AT) gene

图7 野生型菌株(a)和B-sh-ER菌株(b)在5 L发酵罐中分批补料发酵生长曲线

Fig.7 Growth curves of fed-batch fermentation in 5 L fermentator by wild strain (a) and B-sh-ER strain (b)

图8 野生菌株和B-sh-ER菌株的补料分批发酵参数(a) 生物量;(b) 总油脂量;(c) EPA产量;(d) DHA产量;(e) EPA×100/DHA

Fig.8 Fed-batch fermentation parameters of wild strain and B-sh-ER strain

表2 补料分批发酵野生型菌株和B-sh-ER菌株的脂肪酸组成

Table 2 Fatty acid composition of the wild strain and B-sh-ER strain in fed-batch fermentation

Lipid profile	Wild strain	B-sh-ER strain
total lipid/(g/L)	56.90 ± 1.36	73.24 ± 1.09^b
PUFAs/%	42.34 ± 0.76	32.02 ± 1.21^a
DHA/%	33.22 ± 0.38	25.10 ± 0.68^a
DPA/%	8.37 ± 0.46	5.92 ± 0.49^a
EPA/%	0.75 ± 0.02	1.00 ± 0.02^a
C16:0/%	49.54 ± 0.89	57.40 ± 0.72^a

表3 相关菌株EPA产量及占比

Table 3 EPA yield and proportion of related strains

Strains	DCW/(g/L)	EPA yield/(mg/L)	EPA proportion/%
Schizochytrium limacinum SR21	159.7	732.4	1.0
Thraustochytrid Aurantiochytrium^[10]	144.4	2700.0	3.1
Schizochytrium sp. MYA1381^[29]	182	1650.0	1.45
Nitzschia laevis^[9]	22.1	695.2	3.3
Nitzschia laevis^[9]	—	1112.0	2.4
Phaeodactylum tricornutum^[40]	16.2	356.4	2.2
Mortierella alpine ATCC 32222^[41]	20.1	588.5	—
Mortierella alpina ST1358^[42]	10	1800.0	26.4

表A1

Table A1 Primers for RT-qPCR

引物名称	引物序列
ORFB-ER-F	GTTGAAGCCTCCGCCTTTATG
ORFB-ER-R	CCTGCTCTTGGGTGATCTCGC
ORFC-ER-F	CAGCCTGCTCCTTGGTAATCT
ORFC-ER-R	CATCAAGAACCGCATCATCG
AT-F	TGCTACACGGTGCTGCTCTCTGATG
AT-R	ACGTACATTAGCGCTAGGCTGGGC
sh-ER-F	TCGCTCAAACCGCTGACATCGTA
sh-ER-R	CGGCGTAGTAGGCGTACTTCACG

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烯酰还原酶基因的替换对裂殖壶菌合成二十碳五烯酸的影响

Effects of substitution of ER domains on the synthesis of eicosapentaenoic acid in Schizochytrium limacinum SR21

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