PGASO: A synthetic biology tool for engineering a cellulolytic yeast

Biotechnology for Biofuels

Jui-Jen Chang³  Cheng-Yu Ho⁴  Feng-Ju Ho²  Tsung-Yu Tsai²  Huei-Mien Ke⁶  Christine H-T Wang²  Hsin-Liang Chen²  Ming-Che Shih¹  Chieh-Chen Huang⁴  Wen-Hsiung Li⁵ 

[1] Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan

[2] Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan

[3] Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan

[4] Biotechnology Center, National Chung Hsing University, Taichung, 115, Taiwan

[5] Department of Ecology and Evolution, University of Chicago, Chicago, IL, 60637, USA

[6] Microbial Genomics, National Chung Hsing University, Taichung, 402, Taiwan

关键词: Bio-ethanol;    Cellulolytic enzymes;    Yeast;    Synthetic biology;    Consolidated bioprocess;   

Others  :  798256
DOI  :  10.1186/1754-6834-5-53

 received in 2012-04-06, accepted in 2012-06-28,  发布年份 2012

【 摘 要 】

Background

To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome.

Results

A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO), that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei), a beta-glucosidase (from a cow rumen fungus), a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol.

Conclusions

This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

【 授权许可】

   
2012 Chang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Lynd LR, van Zyl WH, McBride JE, Laser M: Consolidated bioprocessing of cellulosic biomass: an update. CurrOpinBiotechnol 2005, 16:577-583.
• [2]Ilmén M, den Haan R, Brevnova E, McBride J, Wiswall E, Froehlich A, Koivula A, Voutilainen SP, Siika-Aho M, la Grange DC, Thorngren N, Ahlgren S, Mellon M, Deleault K, Rajgarhia V, van Zyl WH, Penttilä M: High level secretion of cellobiohydrolases bySaccharomyces cerevisiae. Biotechnol Biofuels. 2011, 4:30. BioMed Central Full Text
• [3]Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A: Direct ethanol production from cellulosic materials using a diploid strain ofSaccharomyces cerevisiaewith optimized cellulase expression. Biotechnol Biofuels 2011, 4:8. BioMed Central Full Text
• [4]Nonklang S, Abdel-Banat BM, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, Akada R: High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeastKluyveromycesmarxianusDMKU3-1042. Appl Environ Microbiol 2008, 74:7514-7521.
• [5]Fonseca GG, Heinzle E, Wittmann C, Gombert AK: The yeastKluyveromycesmarxianusand its biotechnological potential. ApplMicrobiolBiotechnol 2008, 79:339-354.
• [6]Sánchez M, Iglesias FJ, Santamaría C, Domínguez A: Transformation ofKluyveromyceslactisby Electroporation. Appl Environ Microbiol 1993, 59:2087-2092.
• [7]van Ooyen AJ, Dekker P, Huang M, Olsthoorn MM, Jacobs DI, Colussi PA, Taron CH: Heterologous protein production in the yeastKluyveromyceslactis. FEMS Yeast Res 2006, 6:381-392.
• [8]Liu Q, Li MZ, Leibham D, Cortez D, Elledge SJ: The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. CurrBiol 1998, 8:1300-1309.
• [9]Shuldiner AR, Scott LA, Roth J: PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products. Nucleic Acids Res 1920, 1990:18.
• [10]Aslanidis C, de Jong PJ: Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 1990, 18:6069-6074.
• [11]Hartley JL, Temple GF, Brasch MA: DNA cloning using in vitro site-specific recombination. Genome Res 2000, 10:1788-1795.
• [12]Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, van den Heuvel S, Vidal M: GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 2000, 328:575-592.
• [13]Pachuk CJ, Samuel M, Zurawski JA, Snyder L, Phillips P, Satishchandran C: Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments. Gene 2000, 243:19-25.
• [14]Tsuge K, Matsui K, Itaya M: One step assembly of multiple DNA fragments with a designed order and orientation inBacillus subtilisplasmid. Nucleic Acids Res 2003, 31:e133.
• [15]Shao Z, Zhao H: DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 2009, 37:e16.
• [16]Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R: USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res 2007, 35:1992-2002.
• [17]Li MZ, Elledge SJ: MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat Genet 2005, 37:311-319.
• [18]Li MZ, Elledge SJ: Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 2007, 4:251-256.
• [19]Marsischky G, LaBaer J: Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res 2004, 14:2020-2028.
• [20]Kegel A, Martinez P, Carter SD, Astrom SU: Genome wide distribution of illegitimate recombination events inKluyveromyceslactis. Nucleic Acids Res 2006, 34:1633-1645.
• [21]Quan J, Tian J: Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 2009, 4:e6441.
• [22]Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, Montague MG, Venter JC, Smith HO, Hutchison CA: One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. ProcNatlAcadSci USA 2008, 105:20404-20409.
• [23]Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA: Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6:343-345.
• [24]Beggs JD: Transformation of yeast by a replicating hybrid plasmid. Nature 1978, 275:104-109.
• [25]Gietz RD, Woods RA: Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 2002, 350:87-96.
• [26]Das S, Hollenberg CP: A high-frequency transformation system for the yeast Kluyveromyceslactis. Curr Genet 1982, 6:123-128.
• [27]Abdel-Banat BM, Nonklang S, Hoshida H, Akada R: Random and targeted gene integrations through the control of non-homologous end joining in the yeastKluyveromycesmarxianus. Yeast 2010, 27:29-39.
• [28]Chen HL, Lu MY, Chen YC, Chang JJ, Wang HT, Wang TY, Ruan SK, Wang TY, Hung KY, Cho HY, Ke HM, Lin WT, Shih MC, Li WH: A highly efficient β-glucosidase from the calf lumen fungusNeocallimastixpatriciarumW5. Biotechnol Biofuels 2012,  : - . in press
• [29]Rasmussen MA, Hespell RB, White BA, Bothast RJ: Inhibitory Effects of Methylcellulose on Cellulose Degradation by Ruminococcusflavefaciens. Appl Environ Microbiol 1988, 54:890-897.
• [30]Hong J, Wang Y, Kumagai H, Tamaki H: Construction of thermotolerant yeast expressing thermostablecellulase genes. J Biotechnol 2007, 130:114-123.
• [31]Yanase S, Hasunuma T, Yamada R, Tanaka T, Ogino C, Fukuda H, Kondo A: Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeastKluyveromycesmarxianusdisplaying cellulolytic enzymes. ApplMicrobiolBiotechnol 2010, 88:381-388.
• [32]Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F: Colorimetric method for determination of sugars and related substances. Anal Chem 1956, 28:350-356.