Construction of a light-controlled expression system and its application in Yarrowia lipolytica

doi:10.12211/2096-8280.2021-018

Abstract

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.

Key words: CarH, green light induction, Yarrowia lipolytica, biosensor, naringenin

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。

关键词: CarH, 绿光诱导, 解脂耶氏酵母, 生物传感器, 柚皮素

CLC Number:

Q819

Ping ZHANG, Wenping WEI, Ying ZHOU, Bangce YE. 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.

Figures/Tables 8

Tab. 1 Plasmids and strains used in this study

Type	Characteristics	Source
Strains
Yarrowia lipolytica Po1f	URA3^-, Leu2^-	This laboratory
Yl-CVH	Po1f with plasmid p13-CV-2CarO-Tm	This study
Yl-CA	Po1f with plasmid p13-CV-2CarO-TAL	This study
Yl-NA	Yl-CA with plasmid p12-4CC	This study
Plasmids
pINA1312	The plasmid contains a strong constitutive promoter hp4d, ura3 gene expression cassette and kanamycin resistance gene	This laboratory
pINA1269	The plasmid contains a strong constitutive promoter hp4d, leu2 gene expression cassette and ampicillin resistance gene	This laboratory
pSET152-Pj23119-2CarO-GFP	The plasmid is a commonly used plasmid pSET152 of Streptomyces which contains CarO site	This laboratory
pUC57-CarH	The plasmid contains codon optimized CarH gene	This laboratory
pUC57-VPRH	The plasmid contains codon optimized VPR and HSF1 genes	This laboratory
p13PxoTAL	The plasmid pINA1312 contains codon optimized tal gene and Ptef promoter	This laboratory
p13-Tm	The plasmid pINA1312 contains the mcherry expression module controlled by the TEF promoter	This laboratory
p13-CV	The plasmid pINA1312 contains the CarH-VPRH expression module controlled by the hp4d promoter	This study
p13-CV-2CarO-Tm	The plasmid is a fluorescent protein expression module 2CarO-TEF-mcherry controlled by an inducible promoter inserted into the plasmid p13-CV	This study
p13-CV-2CarO-TAL	The plasmid is a TAL expression module controlled by an inducible promoter inserted into the plasmid p13-CV	This study
p12-4CL	The plasmid pINA1269 contains codon optimized 4cl gene with FBAin promoter	This laboratory
pCas-AXPCHSCHI	The plasmid contains codon optimized chs gene with Pexp1 promoter and codon optimized chi gene with FBAin promoter, ampicillin resistance gene	This laboratory
p12-4CC	The plasmid pINA1269 contains the 4cl gene with FBAin promoter, chs gene with Pexp1 promoter and chi gene with FBAin promoter	This study

Tab. 2 Primers used in this study

Primers	Sequence (5′- 3′)
1312CarH-F	acacatacaaccacacacatccacgtgatgacctcctccggcgtctacaccat
CarH-VPRH-R	tcgtccttgtagtcggatccagaggatccgatagcctccttctcaggacctcgaggca
CarH-VPRH-F	tgagaaggaggctatcggatcctctggatccgactacaaggacgacgacgacaagcct
KpnIHSF1-R	tggggacaggccatggaggtaccttaggacacagtagggtccttagccttaggaggct
SalI-Pj23119F	aagcactttgctagatagagtcgacttgacagctagctcagtcctaggtataatgcta
CarO-TEF-R	acggtggtcttttatacccttggctcgtcgtccttgtagtccatcatatgtctagct
CarO-TEF-F	agctagacatatgatggactacaaggacgacgagccaagggtataaaagaccaccgt
StuI-XPR2-277R	agctctgtacaccgagaaacaggcctacaatatctggtcaaatttcagtttcgttaca
TEF-TAL-F	agcatttccttctgagtataagaatcattcaaagctcctcgacctacttcgcagtcgc
1269-SpeI-XPR2-F	aacgcgcgaggcagcagatccactagtgggaggttaagagaattatcaccggcaaact
FBAin-CYCt-R	gggacgctcgaaggctttaatttgccagtgtacgcagtactatagaggaacaattgcc
FBAin-CYCt-F	tgttcctctatagtactgcgtacactggcaaattaaagccttcgagcgtcccaaaacc
1269-SpeI-XPR2-R	agatccgcggccgcataggccactagtgggaggttaagagaattatcaccggcaaact

Fig. 1 Design of the green light response sensor((a)Tool plasmid design of green light response sensor; green light response transcription factor CarH-VPRH is driven by constitutive promoter hp4d, expression of reporter gene mcherry is driven by inducible promoter 2CarO-TEF; (b)"Off" mode under green light; (c) "On" mode of expression in dark condition)

Fig. 2 Performance of the green light response sensor(a)Fluorescence protein detection spectrogram of sensing strain. Black curve is dark group, and green curve is green light irradiation group, excitation light: 570 nm, emission light: 590~700 nm; (b)Green light response signal output difference of strains; Tested strain was Yl-CVH; Culture medium YPD, 72 h; (c)Effect of cobamide vitamin B12 concentration on light-controlled induction system,48 h

Fig. 3 Dynamic response of the green light response sensor to light conditions(Green area in figure represents green light irradiation condition, and gray area represents dark condition. Culture conditions: YPD liquid medium, 30 ℃, 220 r/min, and supplemented with 15 μmol/L cobamide vitamin B12 at 0 h, 24 h, 48 h, 72 h and 96 h; Fluorescence value measurement conditions: excitation light: 580 nm, emission light: 608 nm; Sampling time interval: 12 h)

Fig. 4 Cell imagines regulated by green light irradiation(Imaging strains were suspended and preserved in PBS buffer solution with pH 7.4. Experimental strains were cultured in YPD medium, and added 15 μmol/L cobamide vitamin B12 at 0 h. The strain samples were obtained at 72 h)

Fig. 5 p-Coumaric acid synthesis regulated by green light irradiation(a) p-Coumaric acid synthesis pathway controlled by green light. PPP—pentose phosphate pathway; PEP—phosphoenolpyruvate; E4P— erythrose 4-phosphate; DAHP—3-deoxy-D-arabino-heptulosonic acid-7-phosphate; PPA—prephenate; HPP—4-hydroxy-phenylpyruvate; TAL—tyrosine ammonia lyase(b) HPLC chromatogram of strain Yl-CA. The red curve represents the 50 mg/L p-coumaric acid standard, the black curve represents the strain under dark conditions, and the green curve represents the strain under green light(c) Changes of p-coumaric acid concentration of strain Yl-CA under different light conditions; Medium YPD, and all added 15 μmol/L cobamide vitamin B12 at 0 h

Fig. 6 Naringenin synthesis regulated by green light irradiation(a) Naringenin synthesis pathway controlled by green light. PPP—pentose phosphate pathway; PEP—phosphoenolpyruvate; E4P—erythrose 4-phosphate; DAHP—3-deoxy-D-arabino-heptulosonic acid-7-phosphate; TAL—tyrosine ammonia lyase; 4CL—4-coumaroyl-CoA ligase; CHS—chalcone synthase; CHI—chalcone isomerase(b) HPLC chromatogram of strain Yl-NA. The red curve represents the 50 mg/L p-coumaric acid standard and 50 mg/L naringenin standard, the black curve represents the strain under dark conditions, and the green curve represents the strain under green light(c) Growth curve of strain Yl-NA(d) p-Coumaric acid fermentation curve of strain Yl-NA(e) Naringenin fermentation curve of strain Yl-NA; YPD medium, all added 15 μmol/L cobamide vitamin B12 at 0 h, and the two groups whose light conditions were switched from light to dark at 24 h and 48 h were added 15 μv cobamide vitamin B12 again at 24 h and 48 h, respectively

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