Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (7): 83-91    DOI: 10.13523/j.cb.20160712
    
Design and Construction of highβ-carotene Producing Saccharomyces cerevisiae
WANG Rui-zhao, PAN Cai-hui, WANG Ying, XIAO Wen-hai, YUAN Ying-jin
1 Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University;Key Laboratory of Systems Bioengineering(Ministry of Education, Tianjin 300072, China;
2 SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and technology, Tianjin University, Tianjin 300072, China
Download: HTML   PDF(1695KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Saccharomyces cerevisia is generally regarded as safe and widely used in industrial fields. Fine-tuning the expression level of key genes involved inβ-carotene biosynthetic pathway is the key issue for β-carotene production in S. cerevisia. β-carotene producing strain library was constructed by integrating heterologous genes (crtE, crtI, crtYB) from Xanthophyllomyces dendrorhous through Delta site integration.28 strains in darker yellow were picked out and the titer was testified in 96 well plate coupled with shake flask. There were significant difference inβ-carotene titers, ranging from 5.70mg/L to 61.88mg/L. Then the strain with highest titer(SyBE_Sc118012)was selected for further engineering. By means of integrate one copy of tHMG1 and fusion gene BTS1-ERG20 at endogenous ypl062w gene site, the production ofβ-carotene was improved by 1.65 fold, up to 162.1mg/L, which is the highest reported titer at shake flask level. The research highlighted the importance of Delta site integration together with precursor enhancement strategy in heterologous biosynthesis in S. cerevisia.



Key wordsβ-carotene      site      Precursor supply      Saccharomyces cerevisiae      Synthetic biology      &delta      Gene copy number     
Received: 04 January 2016      Published: 16 March 2016
ZTFLH:  Q753  
Cite this article:

WANG Rui-zhao, PAN Cai-hui, WANG Ying, XIAO Wen-hai, YUAN Ying-jin. Design and Construction of highβ-carotene Producing Saccharomyces cerevisiae. China Biotechnology, 2016, 36(7): 83-91.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20160712     OR     https://manu60.magtech.com.cn/biotech/Y2016/V36/I7/83

[1] Vickers C E,Bongers M,Liu Q,et al.Metabolic engineering of volatile isoprenoids in plants and microbes.Plant,Cell&Environment,2014,37(8):1753-1775.
[2] Siddiqui M S,Thodey K,Trenchard I,et al.Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools.Fens Yeast Res,2012,12(2):144-170.
[3] Da S N,Srikrishnan S.Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.Fems Yeast Res,2012,12(2):197-214.
[4] Ro D,Ouellet M,Paradise E,et al.Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor,artemisinic acid.Bmc Biotechnol,2008,8(21):83.
[5] Sun J,Shao Z,Zhao H,et al.Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae.Biotechnol Bioeng,2012,109(8):2082-2092.
[6] Fink G,Farabaugh P,Roeder G,et al.Transposable elements (Ty) in yeast.Cold Spring Harb Symp Quant Biol,1981,45(Pt 2):575-580.
[7] Dujon B.The yeast genome project:what did we learn.Trends Genet,1996,12(7):263-270.
[8] Parekh R N,Shaw M R,Wittrup K D.An integrating vector for tunable,high copy,stable integration into the dispersed Ty delta sites of Saccharomyces cerevisiae.Biotechnol Prog,1996,12(1):16-21.
[9] Lee F W,Da S N.Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae.Appl Microbiol Biotechnol,1997,48(3):339-345.
[10] Lee F W,Da S N.Sequential delta-integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae.Biotechnol Prog,1997,13(4):368-373.
[11] Yamada R,Taniguchi N,Tanaka T,et al.Cocktail delta-integration:a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains.Microb Cell Fact,2010,9:32.
[12] Kato H,Matsuda F,Yamada R,et al.Cocktail δ-integration of xylose assimilation genes for efficient ethanol production from xylose in Saccharomyces cerevisiae.J Biosci Bioeng,2013,116(3):333-336.
[13] Bai Flagfeldt D,Siewers V,Huang L,et al.Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae.Yeast,2009,26(10):545-551.
[14] 郭睿,丁明珠,元英进.产青蒿二烯的人工酵母细胞的构建及发酵优化.化工学报,2015,66(1):378-385.Guo R,Ding M,Yuan Y.Construction of artificial yeast cell for producing amorphadiene,and optimization of fermentation.CIESC Journal,2015,66(1):378-385.
[15] Shi S,Liang Y,Zhang M,et al.A highly efficient single-step,markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.Metab Eng,2016,33:19-27.
[16] Jin Z,Wong A,Foo J L,et al.Engineering Saccharomyces cerevisiae to produce odd chain-length fatty alcohols.Biotechnol Bioeng,2016,113(4):842-851.
[17] Namitha K K,Negi P S.Chemistry and biotechnology of carotenoids.Crit Rev Food Sci,2010,50(8):728-760.
[18] Mayne S T.Beta-carotene,carotenoids,and disease prevention in humans.Faseb J,1996,10(7):690-701.
[19] Ye V M,Bhatia S K.Pathway engineering strategies for production of beneficial carotenoids in microbial hosts.Biotechnol Lett,2012,34(8):1405-1414.
[20] Yang J,Guo L.Biosynthesis of beta-carotene in engineered E.coli using the MEP and MVA pathways.Microb Cell Fact,2014,13(1):1-11.
[21] Wang C,Kim J,Kim S.Synthetic biology and metabolic engineering for marine carotenoids:new opportunities and future prospects.Mar Drugs,2014,12(9):4810-4832.
[22] Verwaal R,Wang J,Meijnen J P,et al.High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.Appl Environ Microb,2007,73(13):4342-4350.
[23] Xie W,Liu M,Lv X,et al.Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae.Biotechnol Bioeng,2014,111(1):125-133.
[24] Verwaal R,Jiang Y,Wang J,et al.Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response.Yeast,2010,27(12):983-998.
[25] Yan G,Liang H,Duan C,et al.Enhanced production of β-carotene by recombinant industrial wine yeast using grape juice as substrate.Curr Microbiol,2012,64(2):152-158.
[26] Özaydin B,Burd H,Lee T S,et al.Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production.Metab Eng,2013,15:174-183.
[27] Xie W,Ye L,Lv X,et al.Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae.Metab Eng,2015,28:8-18.
[28] Reyes L H,Gomez J M,Kao K C.Improving carotenoids production in yeast via adaptive laboratory evolution.Metab Eng,2014,21:26-33.
[29] Brachmann C B,Davies A,Cost G J,et al.Designer deletion strains derived from Saccharomyces cerevisiae S288C:a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.Yeast,1998,14(2):115-132.
[30] Shao Z,Zhao H,Zhao H.DNA assembler,an in vivo genetic method for rapid construction of biochemical pathways.Nucleic Acids Res,2008,37(2):e16.
[31] 元英进,贾斌,林秋卉,等.多片段DNA的酵母快速组装方法:中国,CN 201310389392 A.2015-03-18.Yuan Y,Jia B,Lin Q,et al.Fast Aseembly in Yeast Via Multiple DNA Fragments:China,CN 201310389392 A.2015-03-18.
[32] Gietz R D,Schiestl R H.High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.Nat Protoc,2007,2(1):31-34.
[33] 惠伯棣.类胡萝卜素化学及生物化学.北京:中国轻工业出版社,2005:68-199.Hui B L.Carotenoid Chemistry and Biochemistry.Beijing:China Light Industry Press,2005:68-199.
[34] Partow S,Siewers V,Bjørn S,et al.Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae.Yeast,2010,27(11):955-964.
[35] Giaever G,Chu A M,Ni L,et al.Functional profiling of the Saccharomyces cerevisiae genome.Nature,2002,418(6896):387-391.
[36] Nikawa J,Kawabata M.PCR-and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.Nucleic Acids Res,1998,26(3):860-861.

[1] MA Ning,WANG Han-jie. Advances of Optogenetics in the Regulation of Bacterial Production[J]. China Biotechnology, 2021, 41(9): 101-109.
[2] HUANG Huan-bang,WU Yang,YANG You-hui,WANG Zhao-guan,QI Hao. Progress in Incorporation of Non-canonical Amino Acid Based on Archaeal Tyrosyl-tRNA Synthetase[J]. China Biotechnology, 2021, 41(9): 110-125.
[3] GUO Fang,ZHANG Liang,FENG Xu-dong,LI Chun. Plant-derived UDP-glycosyltransferase and Its Molecular Modification[J]. China Biotechnology, 2021, 41(9): 78-91.
[4] GUO Man-man,TIAN Kai-ren,QIAO Jian-jun,LI Yan-ni. Application of Phage Recombinase Systems in Synthetic Biology[J]. China Biotechnology, 2021, 41(8): 90-102.
[5] DONG Shu-xin,QIN Lei,LI Chun,LI Jun. Transcription Factor Engineering Harnesses Metabolic Networks to Meet Efficient Production in Cell Factories[J]. China Biotechnology, 2021, 41(4): 55-63.
[6] ZHENG Yi,GUO Shi-ying,SUI Feng-xiang,YANG Qi-yu,WEI Ya-xuan,LI Xiao-yan. Applications of Quorum Sensing Systems in Synthetic Biology[J]. China Biotechnology, 2021, 41(11): 100-109.
[7] CHA Ya-ping, ZHU Mu-zi, LI Shuang. Research Progress on In Vivo Continuous Directed Evolution[J]. China Biotechnology, 2021, 41(1): 42-51.
[8] GUO Er-peng, ZHANG Jian-zhi, SI Tong. Recent Advances in the High-throughput Engineering of Lanthipeptides[J]. China Biotechnology, 2021, 41(1): 30-41.
[9] CHANG Lu, HUANG Jiao-fang, DONG Hao, ZHOU Bin-hui, ZHU Xiao-juan, ZHUANG Ying-ping. A Review on Bioremediation and Detection of Heavy Metal Pollution by Synthetic Biological Engineered Microorganisms and Biofilms[J]. China Biotechnology, 2021, 41(1): 62-71.
[10] RAO Hai-mi,LIANG Dong-mei,LI Wei-guo,QIAO Jian-jun,CAI YIN Qing-ge-le. Advances in Synthetic Biology of Fungal Aromatic Polyketides[J]. China Biotechnology, 2020, 40(9): 52-61.
[11] ZHANG Yu-ting,LI Wei-guo,LIANG Dong-mei,QIAO Jian-jun,CAI YIN Qing-ge-le. Research Progress in Synthetic Biology of P450s in Terpenoid Synthesis[J]. China Biotechnology, 2020, 40(8): 84-96.
[12] PENG Xiang-lei,WANG Ye,WANG Li-nan,SU Yan-bin,FU Yuan-hui,ZHENG Yan-peng,HE Jin-sheng. Single-Primer PCR for Site-Directed Mutagenesis[J]. China Biotechnology, 2020, 40(8): 19-23.
[13] WANG Zhen,LI Xia,YUAN Ying-jin. Advances in Production of Caffeic Acid and Its Ester Derivatives in Heterologous Microbes[J]. China Biotechnology, 2020, 40(7): 91-99.
[14] ZHAO Xiao-yan,CHEN Yun-da,ZHANG Ya-qian,WU Xiao-yu,WANG Fei,CHEN Jin-yin. Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreS II from Myxococcus sp.V11[J]. China Biotechnology, 2020, 40(3): 79-87.
[15] SU Yong-jun,HU Die,HU Bo-chun,LI Chuang,WEN Zheng,ZHANG Chen,WU Min-chen. Improving the Enantioselectivity of an Epoxide Hydrolase towards p-Methylphenyl Glycidyl Ether by Site-directed Mutagenesis[J]. China Biotechnology, 2020, 40(3): 88-95.