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Construction of a light-controlled expression system and its application in
Yarrowia lipolytica - Synthetic Biology Journal Vol. 2, Issue 5, Pages: 778-791(2021)
- 作者机构：
1.华东理工大学微分析与生物系统工程实验室，生物反应器工程国家重点实验室，上海 200237
2.浙江工业大学工程生物学与健康研究中心，长三角绿色制药协同创新中心，药学院，浙江 杭州 310014
- 作者简介：
- 基金信息：
DOI：10.12211/2096-8280.2021-018
CLC： Q819Received：05 February 2021，
Revised：2021-08-13，
Published：31 October 2021
- 稿件说明：
移动端阅览
张萍， 魏文平， 周英， 叶邦策. 解脂耶氏酵母中光控表达系统的构建及其应用研究［J］. 合成生物学， 2021， 2（5）： 778-791
ZHANG Ping， WEI Wenping， ZHOU Ying， YE Bangce. Construction of a light-controlled expression system and its application in Yarrowia lipolytica［J］. Synthetic Biology Journal， 2021， 2（5）： 778-791
张萍， 魏文平， 周英， 叶邦策. 解脂耶氏酵母中光控表达系统的构建及其应用研究［J］. 合成生物学， 2021， 2（5）： 778-791 DOI： 10.12211/2096-8280.2021-018.
ZHANG Ping， WEI Wenping， ZHOU Ying， YE Bangce. Construction of a light-controlled expression system and its application in Yarrowia lipolytica［J］. Synthetic Biology Journal， 2021， 2（5）： 778-791 DOI： 10.12211/2096-8280.2021-018.
摘要
光作为诱导因子具有响应速度快、无损、可逆和独特的时空特异性等优势，已广泛应用于生物底盘细胞的工程化改造中。本研究构建重组解脂耶氏酵母（
Yarrowia lipolytica
）的光控表达系统，成功实现了对香豆酸和柚皮素合成。该光控系统基于绿光响应调控因子CarH以及转录激活因子VPR-HSF1，构建了光响应复合体CarH-VPRH，而该复合体在绿光照射条件下无法聚合，从而不能调控目标基因的转录与表达。结果表明，基于光控因子CarH-VPRH、诱导型启动子2CarOTEF以及报告基因mCherry构建的光诱导传感器能够显著响应光
照，并在黑暗条件下激活mCherry转录，72 h和120 h时产生的荧光信号分别是光照下的43倍和143倍。在此基础上，将该元件应用于对香豆酸及柚皮素的生物合成及合成途径的动态调控中，实现黑暗条件下的对香豆酸产量是绿光诱导下的2.0倍，达到99.1 mg/L；柚皮素产量是绿光诱导下的2.6倍达到117.1 mg/L；在细胞生长方面，绿光照射组的生长量高于黑暗组，特别是在绿光照射24 h，然后切换成黑暗条件，细胞的生长量有较为明显的优势，显示了绿光诱导系统在调控
Y. lipolytica
中产物合成与细胞生长平衡的潜力。因此该绿光响应基因调控系统可有效应用于
Y. lipolytica
细胞中目标基因的转录调控，具有廉价易得、无毒害、灵活添加及易去除等优势，因此具有大规模合成和生产目的产物的潜力。
Abstract
Light has the advantages of fast response
non-destructive
reversible
and unique temporal and spatial specificity as an inducing factor
which make it widely used in the metabolic engineering of biological chassis cells. In this study
a light-controlled expression system for recombinant
Yarrowia lipolytica
was developed
which successfully improved the synthesis efficiency and yield of
p
-coumaric acid and naringenin. Based on the green light response regulator CarH and the transcription activator VPR-HSF1
the light response complex CarH-VPRH
the core component of the light control system
was constructed
which cannot polymerize under green light irradiation conditions
and thus cannot regulate the transcription and expression of target genes. The results showed that the light-induced sensor based on CarH-VPRH
2CarOTEF and mCherry could respond to green light significantly to inhibit the expression of target genes
while mCherry transcription was activated under dark conditions. The fluorescent signals of mCherry at 72 h and 120 h were 43 times and 143 times of that detected under green light irradiation conditions. Furthermore
the sensor was applied to induce the synthesis of
p
-coumaric acid and naringenin in
Y. lipolytica
successfully
and the yields of
p
-coumarin acid and naringenin under dark culture conditions were 2.0 times and 2.6 times of that produced under the induction of green light
reaching 99.1 mg/L
and 117.1 mg/L
respectively. In terms of cell growth
the
growth of the green light irradiation group was better than that of the dark group
and especially when the dark condition was switched on after 24 h of green light irradiation
the cell growth was substantially improved
indicating the potential of the green light-induced system to regulate product synthesis and cell growth balance in
Y. lipolytica
. Therefore
it can be concluded that the green light-responsive gene regulation system can be applied to the transcription regulation of target genes in
Y. lipolytica
as an effective regulatory element with the advantages of low cost
nontoxicity
flexible insertion and removal. Therefore
it has a potential for the large-scale synthesis and production of target products.
2
关键词
Keywords
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