Effects of <i>Vitreoscilla</i> Hemoglobin Gene on Growth and Intracellular Oxidation State of <i>Saccharomyces cerevisiae</i>

doi:10.13523/j.cb.2106049

China Biotechnology

2021, Vol. 41

Issue (11): 32-39 DOI: 10.13523/j.cb.2106049

Effects of Vitreoscilla Hemoglobin Gene on Growth and Intracellular Oxidation State of Saccharomyces cerevisiae

XUE Zhi-yong,DAI Hong-sheng,ZHANG Xian-yuan,SUN Yan-ying,HUANG Zhi-wei(

)

Key Lab of Science & Technology of Eco-textile (Ministry of Education), College of Chemistry & Chemical Engineering and Biotechnology,Donghua University, Shanghai 201620, China

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Abstract

Heterologous expression of Vitreoscilla hemoglobin gene vgb in a variety of research and industrial fermentation bacteria can solve the problem of dissolved oxygen rate in high-density fermentation. Saccharomyces cerevisiae is a classic eukaryotic model and has an important application value in the fermentation industry. However, the impact of vgb in Saccharomyces cerevisiae on cell growth is unclear. In this study, a heterologous expression plasmid YEplac195-ADH1pr-vgb containing Vistreoscilla hemoglobin gene vgb was constructed with Adh1 as promoter, and transformed into Saccharomyces cerevisiae BY4741. Growth sensitivity test showed that the heterologous expression of vgb inhibited the growth of the strain in both fermentation and non fermentation carbon sources. Then, we found the accumulation of reactive oxygen species (ROS), the change of membrane permeability and lipid peroxidation in S. cerevisiae cells overexpressing vgb by DCFH-DA and PI staining and lipid peroxidation products assay. The results showed that the overexpression of vgb changed the oxidation state in cells, promoted the accumulation of reactive oxygen (ROS), and oxidative stress caused the growth inhibition of the strain.

Key words： Vitreoscilla hemoglobin Saccharomyces cerevisiae Growth inhibition Oxidative stress

Received: 28 June 2021 Published: 01 December 2021

ZTFLH:

Q819

Corresponding Authors: Zhi-wei HUANG E-mail: zhiweih@dhu.edu.cn

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	Zhi-yong XUE
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	Yan-ying SUN
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XUE Zhi-yong,DAI Hong-sheng,ZHANG Xian-yuan,SUN Yan-ying,HUANG Zhi-wei. Effects of Vitreoscilla Hemoglobin Gene on Growth and Intracellular Oxidation State of Saccharomyces cerevisiae. China Biotechnology, 2021, 41(11): 32-39.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2106049 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I11/32

Table 1 PCR primers used to construct plasmid YEplac195-ADH1pr-vgb

Fig.1 The construction of YEplac195-ADH1pr-vgb and the expression in Saccharomyces cerevisiae (a) Map of plasmid YEplac195-ADH1pr-vgb (b) The verification of ADH1pr-vgb amplification fragment E. coli PCR; M:Molecular weight; 1:Negative control; 2,3:Positive clone (c) Semi-quantitative RT-PCR of transformed strain 1,2:Positive clone; 3,4:Negative control

Fig.2 Overespression of vgb inhibits cell growth under aerobic respiratory conditions (a) Growth sensitivity test of Saccharomyces cerevisiae under aerioxidation conditions (b) Growth curve of Saccharomyces cerevisiae under aerobic breathing conditions

Fig.3 Overespression of vgb inhibits cell growth under oxygen-free respiratory conditions (a) Growth sensitivity test of Saccharomyces cerevisiae in 2% glycerol (b) Growth sensitivity test of Saccharomyces cerevisiae in 2% ethanol

Fig.4 Overexpression of vgb promotes ROS accumulation in Saccharomyces cerevisiae (a) Fluorescent pictures of ROS accumutation in Saccharomyces cerevisiae without or with 1 mmol/L H₂O₂ (b) Fluorescence intensity of H2DCF-DA in Saccharomyces cerevisiae without or with 1mmol/L H₂O₂ * :P<0.05 or ** :P<0.01 vs the corresponding wild type strain (Con group), respectively; ## :P<0.01 vs the corresponding treated strain

Fig.5 Overexpression of vgb damages the integrity of cell membrane and causes intracellular lipid peroxidation (a) PI dyeing fluorescence picture and dyeing cell percentage quantitative picture in Saccharomyces cerevisiae without or with 1 mmol/L H₂O₂ ** :P<0.01 vs the corresponding wild type strain (Con group); ## :P<0.01 vs the corresponding untreated strain (b) TBRAS content in Saccharomyces cerevisiae without or with 1 mmol/L H₂O₂ ** :P<0.01 vs the corresponding wild type strain (Con group), respectively; ## :P<0.01 vs the corresponding untreated strain


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