双功能脱毒用酿酒酵母的构建

doi:10.13304/j.nykjdb.2023.0130

摘要/Abstract

摘要：

饲料中真菌毒素污染普遍，已经成为目前阻碍畜禽健康养殖的一大重要因素。真菌毒素种类繁多，其中黄曲霉毒素，尤其是黄曲霉毒素B₁（aflatoxin B₁，AFB₁）影响范围广、危害大，开发新方法用于饲料真菌毒素的脱毒是目前的研究热点。本研究分离并比较了4株不同的酿酒酵母菌株对AFB₁的吸附能力，发现较其他方式（加热和甘露聚糖酶处理）和菌株，经葡聚糖酶处理后的酿酒酵母CEN.PK菌株对AFB₁吸附率最高，可达53.3%±8.2%。随后，再以该菌株为宿主菌组成型表达来自于约氏黄杆菌的肌醇氧化酶基因FjMiox，经改造后工程菌可将1 g·L^-1的肌醇全细胞转化为0.273 g·L^-1的葡萄糖醛酸。在动物肝脏内，葡萄糖醛酸可经酶催化和真菌毒素结合增强其水溶性使其易于排除，因此可合成葡萄糖醛酸的工程化酿酒酵母结合了肠道内吸附脱毒和肝脏内结合解毒2种功能，为解决黄曲霉毒素在饲料中的污染提供了新的可能解决方案。

关键词: 黄曲霉毒素, 酿酒酵母, 肌醇氧化酶, 葡萄糖醛酸, 毒素脱毒

Abstract:

The contamination of mycotoxins is common in feed and becomes one major hurdle preventing healthy breeding of animals. There are many types of mycotoxins， among which aflatoxins，and particularly aflatoxin B₁（AFB₁） have the highest impact and harm. Therefore， developing new methods for detoxification of mycotoxins in feeds is currently a hot research topic. In this study， Saccharomyces cerevisiae strains were first isolated and four were compared for their absorption rates of AFB₁. It was then discovered that， in comparison with other methods including heating and mannanase treatment and strains， glucanase treatment of CEN.PK had the highest absorption of AFB₁， which could reach as high as 53.3%±8.2%. Then， CEN.PK was used as the host cell to constitutively express a myo-inositol oxidase gene from Flavobacterium johnsoniae （FjMiox）. The engineered yeast could convert 1 g·L^-1 of myo-inositol into 0.273 g·L^-1 of glucuronic acid. In the liver of animals， the glucuronic acid could be conjugated with mycotoxins by enzymatic catalysis to increase their water solubility， thereby facilitating their excretion. Therefore， the engineered Saccharomyces cerevisiae able to synthesize glucuronic acid combines the functions of binding the mycotoxin in the gut and conjugating to the mycotoxin in the liver， which could provide a new solution to combat the contamination of aflatoxins in feeds.

Key words: aflatoxins, Saccharomyces cerevisiae, myo-inositol oxygenase, glucuronic acid, mycotoxin detoxification

中图分类号:

TS261.1⁺1

王懿珺, 黄火清, 苏小运. 双功能脱毒用酿酒酵母的构建[J]. 中国农业科技导报, 2024, 26(2): 226-233.

Yijun WANG, Huoqing HUANG, Xiaoyun SU. Construction of Bifunctional Saccharomyces cerevisiae with Ability to Detoxify Mycotoxins[J]. Journal of Agricultural Science and Technology, 2024, 26(2): 226-233.

图/表 6

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引物Primer	序列 Sequence （5’- 3’）	用途 Usage
ITS-1	TCCGTAGGTGAACCTGCGG	扩增ITS片段 Amplifying ITS
ITS-2	TCCTCCGCTTATTGATATGC	扩增ITS片段 Amplifying ITS
F1	ATAAGCTTGATATCGAATTCTCATTATCAATACTGCCATT	扩增TDH3启动子片段 Amplifying TDH3 promoter fragment
R1	GTATCAATATGTTTTTTCATTTTGTTTGTTTATGTGTGTT	扩增TDH3启动子片段 Amplifying TDH3 promoter fragment
F2	AACACACATAAACAAACAAAATGAAAAAACATATTGATAC	扩增FjMiox基因 Amplifying FjMiox gene
R2	TAAAGACATAAGAGATCCGCTTAAAATTTCAATTTTTCAG	扩增FjMiox基因 Amplifying FjMiox gene
F3	CTGAAAAATTGAAATTTTAAGCGGATCTCTTATGTCTTTA	扩增ADH2终止子片段 Amplifying ADH2 terminator fragment
R3	CACCGCGGTGGCGGCCGCATGAGAAATATCGAGGG	扩增ADH2终止子片段 Amplifying ADH2 terminator fragment

引物Primer	序列 Sequence （5’- 3’）	用途 Usage
ITS-1	TCCGTAGGTGAACCTGCGG	扩增ITS片段 Amplifying ITS
ITS-2	TCCTCCGCTTATTGATATGC	扩增ITS片段 Amplifying ITS
F1	ATAAGCTTGATATCGAATTCTCATTATCAATACTGCCATT	扩增TDH3启动子片段 Amplifying TDH3 promoter fragment
R1	GTATCAATATGTTTTTTCATTTTGTTTGTTTATGTGTGTT	扩增TDH3启动子片段 Amplifying TDH3 promoter fragment
F2	AACACACATAAACAAACAAAATGAAAAAACATATTGATAC	扩增FjMiox基因 Amplifying FjMiox gene
R2	TAAAGACATAAGAGATCCGCTTAAAATTTCAATTTTTCAG	扩增FjMiox基因 Amplifying FjMiox gene
F3	CTGAAAAATTGAAATTTTAAGCGGATCTCTTATGTCTTTA	扩增ADH2终止子片段 Amplifying ADH2 terminator fragment
R3	CACCGCGGTGGCGGCCGCATGAGAAATATCGAGGG	扩增ADH2终止子片段 Amplifying ADH2 terminator fragment

组别 Group	处理方式 Ways of treatment	菌体量 Cell quantity/（CFU·mL^-1）	处理温度 Treatment temperature/℃	处理时间 Treatment time/min
T1	—	4×10⁷	—	—
T2	葡聚糖酶 Glucanase	4×10⁷	25	120
T3	甘露聚糖酶 Mannanase	4×10⁷	25	120
T4	加热 Heating	4×10⁷	100	5

组别 Group	处理方式 Ways of treatment	菌体量 Cell quantity/（CFU·mL^-1）	处理温度 Treatment temperature/℃	处理时间 Treatment time/min
T1	—	4×10⁷	—	—
T2	葡聚糖酶 Glucanase	4×10⁷	25	120
T3	甘露聚糖酶 Mannanase	4×10⁷	25	120
T4	加热 Heating	4×10⁷	100	5

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