Evaluation of UPR Response in Yeast by Using <i>UPRE-lac</i> Z as a Reporter Gene

doi:10.13523/j.cb.2006036

China Biotechnology

2020, Vol. 40

Issue (10): 1-9 DOI: 10.13523/j.cb.2006036

Evaluation of UPR Response in Yeast by Using UPRE-lac Z as a Reporter Gene

ZHANG Xiao-mao¹,GUO Jing-han¹,HONG Jie-fang²,LU Hai-yan¹,DING Juan-juan¹,ZOU Shao-lan^1,^***(

),FAN Huan^3,^***(

)

1 School of Chemical Engineering and Technology, Tianjin University, Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin 300072, China
2 Tianjin R&D Center for Petrochemical Technology, Tianjin University, Tianjin　300072, China
3 Tianjin Animal Science and Veterinary Research Institute, Tianjin　300381, China

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Abstract

Objective: Unfolded Protein Response (UPR) is one of the most important protein quality control mechanisms, the information about UPR with help to optimize heterologous protein synthesis by secretory pathway and cell self protection from inhibitory substances such as acid and ethanol.Methods: The indicating strains, W303- 1A (leu 2::UPRE-lac Z) and An-a (leu 2::UPRE-lac Z), abbreviated as WZ and AZ, respectively, were first constructed by CRISPR/Cas9 technology and using UPRE-lac Z as a reporter.Results: The growth curve test showed that there was no significant difference between two strains and their host ones. The β-galactosidase activity was assayed at 4 h of incubation time after adding four reagents into media as followings, 1 μg/ml tunicamycin, 8%(v/v) ethanol, 0.3%(v/v) acetic acid, 5% (v/v) ethanol + 0.1%(v/v)acetic acid, respectively. The values of strain AZ were 8.2, 26.4, 1.1 and 7.9 times of that of blank control, respectively, while the results of strain WZ were 12.6, 2.4, 1.0 and 1.0 times, respectively. Further β-glucosidase was expressed by YEplac195 vector into strain AZ and WZ cell. The assay results after 24 h cultivation in 2% cellobiose media showed that the β-glucosidase activities were 0.35 and 6.12 U/ml, respectively, and the responding LacZ values were 3.1 and 5.4 times of that of blank control, respectively.Conclusions: There exists significant difference on the UPR response spectrum between two strains, which also implies their divergent direction and strategy of genetic modification. This study lays the foundation of analytical methods for investigating the key limiting factors in cell inhibitor tolerance and heterologous protein expression in order to make better use of yeast ER and UPR pathway engineering.

Key words： Yeast UPRE-lacZ UPR response Inhibitor tolerance Secretory expression

Received: 22 June 2020 Published: 10 November 2020

ZTFLH:

Q291

Corresponding Authors: Shao-lan ZOU,Huan FAN E-mail: slzhou@tju.edu.cn;fanhuan1971@163.com

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	Xiao-mao ZHANG
	Jing-han GUO
	Jie-fang HONG
	Hai-yan LU
	Juan-juan DING
	Shao-lan ZOU
	Huan FAN

Cite this article:

ZHANG Xiao-mao,GUO Jing-han,HONG Jie-fang,LU Hai-yan,DING Juan-juan,ZOU Shao-lan,FAN Huan. Evaluation of UPR Response in Yeast by Using UPRE-lac Z as a Reporter Gene. China Biotechnology, 2020, 40(10): 1-9.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2006036 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I10/1

Table 1 All the strains and plasmids used in this study

Table 2 All the primers used in this study

Fig.1 Agarose gel electrophoresis of PCR products and donor DNA fragments (a)PCR for pRS42H-gLEU2 construction, 6 528bp (b) Lane 1, PCR1, 364bp; lane 2, PCR2, 302bp (c) Lane 1, PCR3, 3 095bp; lane 2, PCR13, 3 439bp; lane 3, PCR123, 3 721bp; M, 1kb DNA Ladder (d)Structural map for donor DNA segment containing UPRE-lacZ cassette

Fig.2 Chromogenic plate (left: W303-1A host; right: An-a host)

Fig.3 Agarose gel electrophoresis of PCR products (left: W303-1A host; right: An-a host)

Fig.4 The growth curve of four strains

Fig.5 OD₆₀₀ value and β-galactosidase activity at 4 h of growth time in different media under limited oxygen condition

Fig.6 Enzymatic activities of the strains WZ and AZ at 24 h growth time in YPC media


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