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

doi:10.11949/0438-1157.20201818

Abstract

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

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

CLC Number:

Q 819

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.

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.

Figures/Tables 12

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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

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

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

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

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