Heterologous Expression of Leghemoglobin in <i>Saccharomyces cerevisiae</i>

XUE Changlu; ZHANG Pengfei; BAI Lijun; XIAO Gongnian; WEI Peilian

doi:10.13386/j.issn1002-0306.2022120029

Volume 44 Issue 20

Oct. 2023

Turn off MathJax

Article Contents

Abstract

References

Science and Technology of Food Industry > 2023 > 44(20): 101-107. > DOI: 10.13386/j.issn1002-0306.2022120029

XUE Changlu, ZHANG Pengfei, BAI Lijun, et al. Heterologous Expression of Leghemoglobin in Saccharomyces cerevisiae[J]. Science and Technology of Food Industry, 2023, 44(20): 101−107. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022120029.

Citation:

PDF (3012 KB)

Heterologous Expression of Leghemoglobin in Saccharomyces cerevisiae

XUE Changlu^{1, 2,},
ZHANG Pengfei^{1, 2},
BAI Lijun^{1, 2},
XIAO Gongnian^{1, 2},
WEI Peilian^{1, 2, ,}

1.
Zhejiang University of Science and Technology, Hangzhou 310023, China
2.
Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Product, Hangzhou 310023, China

More Information

Received Date: December 14, 2022
Available Online: August 01, 2023

Graphical Abstract

Abstract

Abstract

Leghemoglobin is a kind of plant-derived hemoglobin, which can be used as an important flavor catalyst and colorant in the processing of plant protein-based meat because of the property of increasing the fidelity of the product greatly. In order to realize the heterologous expression of leghemoglobin in food-grade microorganisms, the plasmid PESC-TRP carrying leghemoglobin LBC2 gene was transformed into S. cerevisiae CEN.PK2-1C. Kozak sequence was added to promote protein translation, and different promoter sequences were selected to improve the expression of leghemoglobin. The recombinant strain was fermented, and the expression level of LegH protein was analyzed by western blot. The results showed that the inducible promoter GAL1,10 had a significant advantage over the three constitutive promoters TEF1, ADH1 and GAP in LegH expression ability, which was 3.93 times higher than the lowest yield ADH1 promoter. Among the constitutive promoters, TEF1 promoter had the best ability, which was 2.91times that of the ADH1 promoter and 1.2 times that of the GAP promoter. Finally, the protein was concentrated and purified by Ni-column affinity chromatography, and the fermentation concentration of LegH was 2.79 mg/L. This study successfully achieved the heterologous expression of leghemoglobin in Saccharomyces cerevisiae, and after subsequent optimization, it would become another way of leghemoglobin heterologous expression.
- Saccharomyces cerevisiae,
- leghemoglobin,
- promoter optimization,
- heterologous expression,
- western blot

FullText(HTML)

References (30)

References

[1]	JIN Y, HE X Y, ANDOH-KUMI K, et al. Evaluating potential risks of food allergy and toxicity of soy leghemoglobin expressed in Pichia pastoris[J]. Molecular Nutrition & Food Research,2018,62(1):13.
[2]	APPLEBY C A. Leghemoglobin and rhizobium respiration[J]. Annual Review of Plant Physiology,1984,35(1):443−478. doi: 10.1146/annurev.pp.35.060184.002303
[3]	HARGROVE M S, BARRY J K, BRUCKER E A, et al. Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants[J]. Journal of Molecular Biology,1997,266(5):1032−1042. doi: 10.1006/jmbi.1996.0833
[4]	SIMSA R, YUEN J, STOUT A, et al. Extracellular heme proteins influence bovine myosatellite cell proliferation and the color of cell-based meat[J]. Foods,2019,8(10):521. doi: 10.3390/foods8100521
[5]	FRASER R Z, SHITUT M, AGRAWAL P, et al. Safety evaluation of soy leghemoglobin protein preparation derived from Pichia pastoris, intended for use as a flavor catalyst in plant-based meat[J]. International Journal of Toxicology,2018,37(3):241−262. doi: 10.1177/1091581818766318
[6]	周景文, 张国强, 赵鑫锐, 等. 未来食品的发展:植物蛋白肉与细胞培养肉[J]. 食品与生物技术学报,2020,39(10):1−8. [ZHOU Z W, ZHANG G Q, ZHAO X R, et al. Future of food: plant-based and cell-cultured meat[J]. Journal of Food Science and Technology,2020,39(10):1−8. doi: 10.3969/j.issn.1673-1689.2020.10.001 ZHOU Z W, ZHANG G Q, ZHAO X Y, et al. Future of food: plant-based and cell-cultured meat[J]. Journal of Food Science and Technology, 2020, 39(10): 1-8. doi: 10.3969/j.issn.1673-1689.2020.10.001
[7]	陈林杰, 薛常鲁, 苏悦, 等. 豆血红蛋白在毕赤酵母中的表达条件优化[J]. 微生物学通报,2022,49(6):2050−2061. [CHEN L J, XUE C L, SU Y, et al. Optimization of leghemoglobin expression conditions in Pichia pastoris[J]. Microbiology China,2022,49(6):2050−2061. doi: 10.13344/j.microbiol.china.210944 CHEN L J, XUE C L, SU Y, et al. Optimization of leghemoglobin expression conditions in Pichia pastoris[J]. Microbiology China, 2022, 49(6): 2050-2061. doi: 10.13344/j.microbiol.china.210944
[8]	SHAO Y R, XUE C L, LIU W J, et al. High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis[J]. Bioresource Technology,2022,363:127884. doi: 10.1016/j.biortech.2022.127884
[9]	ARREDONDO-PETER R, MORAN J F, SARATH G, et al. Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli (purification and characterization of the recombinant protein)[J]. Plant Physiology,1997,114(2):493−500. doi: 10.1104/pp.114.2.493
[10]	LIU L F, MARTINEZ J L, LIU Z H, et al. Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae[J]. Metabolic Engineering,2014,21:9−16. doi: 10.1016/j.ymben.2013.10.010
[11]	ZHANG B H, ZHAO X R, WANG Z W, et al. Efficient secretory expression and purification of food-grade porcine myoglobin in Komagataella phaffii[J]. Journal of Agricultural and Food Chemistry,2021,69(35):10235−10245. doi: 10.1021/acs.jafc.1c04124
[12]	ZHAO X R, ZHOU J W, DU G C, et al. Recent advances in the microbial synthesis of hemoglobin[J]. Trends in Biotechnology,2021,39(3):286−297. doi: 10.1016/j.tibtech.2020.08.004
[13]	SUMAN S P, JOSEPH P. Myoglobin chemistry and meat color[J]. Annual Review of Food Science and Technology,2013,4(1):79−99. doi: 10.1146/annurev-food-030212-182623
[14]	FRASER R, BROWN P O, KARR J, et al. Impossible Foods Inc. Methods and compositions for affecting the flavor and aroma profile of consumables: US, 9700067B2[P]. 2017−07−11.
[15]	SHANKAR S, HOYT M A. Impossible Food Inc. Expression construction and methods of genetically engineering methylotrophic yeast: US, WO 2016/183163A1[P]. 2018−01−19.
[16]	苏悦. 微生物高效表达异源豆血红蛋白的研究[D]. 杭州: 浙江大学, 2020 SU Y. Study on efficient expression of heterologous leghemoglobin in microorganisms[D]. Hangzhou: Zhejiang University, 2020.
[17]	CURRELL D L, LEVIN J. The oxidative effect of bacterial lipopolysaccharide on native and cross-linked human hemoglobin as a function of the structure of the lipopolysaccharide[J]. Febs Journal,2010,269(18):4635−4640.
[18]	MARTINEZ J L, LIU L F, PETRANOVIC D, et al. Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae[J]. Biotechnology and Bioengineering,2015,112(1):181−188. doi: 10.1002/bit.25347
[19]	MOULD R M, HOFMANN O M, BRITTAIN T. Production of human embryonic haemoglobin (Gower II) in a yeast expression system[J]. Biochemical Journal,1994,298(3):619−622. doi: 10.1042/bj2980619
[20]	BUISSON N, LABBE-BOIS R. Flavohemoglobin expression and function in Saccharomyces cerevisiae: No relationship with respiration and complex response to oxidative stress[J]. Journal of Biological Chemistry,1998,273(16):9527. doi: 10.1074/jbc.273.16.9527
[21]	OUTTEN C E, CULOTTA V C. Alternative start sites in the Saccharomyces cerevisiae GLR1 gene are responsible for mitochondrial and cytosolic isoforms of glutathione reductase[J]. The Journal of Biological Chemistry,2004,279(9):7785−7791. doi: 10.1074/jbc.M312421200
[22]	STUART J A, HARPER J A, BRINDLE K M, et al. Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria[J]. The Journal of Biological Chemistry,2001,276(21):18633−9. doi: 10.1074/jbc.M011566200
[23]	GROTE A, HILLER K, SCHEER M, et al. JCat: A novel tool to adapt codon usage of a target gene to its potential expression host[J]. Nucleic Acids Research, 2005, 33(Web Server issue): W526.
[24]	安伯格. 酵母遗传学方法实验指南[M]. 第二版. 北京: 科学出版社, 2009: 98 ANBERG D C. Methods in yeast genetics[M]. Second edition. Beijing: Science Press, 2019: 98.
[25]	JIN Q, PAN F, HU C F, et al. Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers[J]. Metabolic Engineering,2022,70:102−114. doi: 10.1016/j.ymben.2022.01.009
[26]	MNAIMNEH S, DAVIERWALA A P, HAYNES J, et al. Exploration of essential gene functions via titratable promoter alleles[J]. Cell,2004,118(1):31−44. doi: 10.1016/j.cell.2004.06.013
[27]	SUN J, SHAO Z Y, ZHAO H, et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae[J]. Biotechnology & Bioengineering,2012,109(8):2082−2092.
[28]	邱玲, 钱俊佳, 康振, 等. 乙肝表面抗原在酿酒酵母中的异源表达[J]. 食品与生物技术学报,2015,34(9):906−913. [QIU L, QIAN J J, KANG C, et al. Heterologous expression of HBsAg in Saccharomyces Cerevisiae[J]. Journal of Food Science and Biotechnology,2015,34(9):906−913. doi: 10.3969/j.issn.1673-1689.2015.09.002 QIU L, QIAN J J, KANG C, et al. Heterologous expression of HBsAg in Saccharomyces Cerevisiae[J]. Journal of Food Science and Biotechnology, 2015, 34(9): 8. doi: 10.3969/j.issn.1673-1689.2015.09.002
[29]	夏炳乐, 李敏莉, 刘清亮, 等. 烟草过氧化物酶Ⅰ的紫外-可见吸收光谱研究[J]. 光谱学与光谱分析,2004,24(7):830−833. [XIA C L, LI M L, LIU Q L, et al. Uv-vis absorption spectral characteristics of tobacco peroxidase Ⅰ from nicotiana tabacum[J]. Spectroscopy and Spectral Analysis,2004,24(7):830−833. doi: 10.3321/j.issn:1000-0593.2004.07.017 XIA C L, LI M L, LIU Q L, et al. Uv-vis absorption spectral characteristics of tobacco peroxidase Ⅰ from nicotiana tabacum[J]. Spectroscopy and Spectral Analysis, 2004, 24(7): 4. doi: 10.3321/j.issn:1000-0593.2004.07.017
[30]	DOMINGUES E, BRILLET T, VASSEUR C, et al. Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin[J]. Plasmid,2009,61(1):71−77. doi: 10.1016/j.plasmid.2008.09.006

Supplements (0)

Cited By

Cited by

Periodical cited type(7)

1.	刘亚兵，罗学尹，戴宇樵，王敏，蒲璐璐，潘科，刘忠英，李琴. 灵芝菌处理对夏秋黑茶梗品质的影响. 沈阳农业大学学报. 2023(03): 289-295 .
2.	孟圆，夏婷，程艳，耿贝贝，权冰艳，宋睿喆，于金浩，王敏，白晓丽. 碱法提取普洱茶渣膳食纤维的工艺优化. 食品研究与开发. 2023(18): 158-164 .
3.	高丽娟，郜春霞，吴佳琪，吴修祯，李凯. 响应面法优化爬山虎不溶性膳食纤维反提取工艺. 河南农业. 2023(36): 56-59 .
4.	许婧. 茶叶保健食品加工技术及发展趋势分析. 现代食品. 2022(04): 70-73 .
5.	王彤辉，相堂永，徐姗，顾依，任舒静，江勇，杨帆，陈志鹏. 萌芽黑青稞喷干粉的制备工艺优化. 食品研究与开发. 2022(10): 156-165 .
6.	皮小弟，罗瑞婷，李叶青，邹志群，吴思毅，黄志远. 豆渣水溶性膳食纤维的复合酶法提取及其应用于可食性膜研究. 保鲜与加工. 2022(10): 56-62 .
7.	牛潇潇，王杰，王宁，梁亮，韩育梅，杨杨. 超微粉碎对马铃薯渣理化性质和微观结构的影响. 中国粮油学报. 2022(12): 84-91 .