Abstract
An exponential increase in our understanding of genomes, proteomes, and metabolomes provides greater impetus to address critical biotechnological issues such as sustainable production of biofuels and bio-based chemicals and, in particular, the development of improved microbial biocatalysts for use in industrial biorefineries. Because these studies involve the evaluation of large numbers of genes and proteins, high-throughput integrated robotic molecular biology platforms that have the capacity to rapidly synthesize, clone, and express heterologous gene open reading frames (ORFs) in bacteria, cell-free extracts, and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving fungal strains for industrial production of biofuels and bio-based chemicals. We describe a system of four robotic platforms required for continuous operation of this process: (1) synthesis and screening of mutagenized gene ORFs by systematically replacing codons using an amino acid scanning mutagenesis algorithm, which evaluates all codons for functionality, in a multiplexed format to produce a library of optimized ORFs; (2) one-step construction of a synthetic yeast artificial chromosome (YAC) containing the optimized ORFs in a polyprotein cassette for expression of multiple genes such as those for enzymes in metabolic pathways or for valuable peptide or protein coproducts behind an optimized promoter with custom expression fusion tags selected for desired expression levels and protein locations inside or outside the industrial strain; (3) selection of a host strain that has been subjected to mutagenesis by irradiation and/or incubation at elevated temperatures anaerobically to produce a strain capable of robust growth in the particular biomass feedstock or waste stream or agricultural product required for profitable and environmentally friendly operations at a biorefinery and transformation of this host strain with these collections of synthetic YACs; and (4) high-throughput screening of the transformed strains for desired industrial traits. This system produces an improved industrial microorganism by manipulation of the host strain, assembly of an optimized synthetic chromosome, and stable transformation of the chromosome into the engineered improved host strains for use in production of biofuels via biorefinery operations.
Keywords
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Abdel-Banat BM, Hoshida H, Ano A, Nonklang S, Akada R (2010) High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol 85(4):861–867
Agrestia JJ, Antipov E, Abate AR, Ahn K, Rowat AC, Baret J-C, Marquez M, Klibanov AM, Griffiths AD, Weitz DA (2010) Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc Natl Acad Sci U S A 107(9):4004–4009
An FW, Tolliday N (2010) Cell-based assays for high-throughput screening. Mol Biotechnol 45:180–186
Angov E (2011) Codon usage: nature’s roadmap to expression and folding of proteins. Biotechnol J 6:650–659
Arnak R, Bruschi CV, Tosato V (2012) Yeast Artificial Chromosomes. In: Encyclopedia of Life Sciences (eLS). John Wiley & Sons Ltd, Chichester; Wiley online Library. http://www.els.net. doi:10.1002/9780470015902.a0000379.pub3
Bruschi CV, Gjuracic K (2002) Yeast Artificial Chromosomes. In Encyclopedia of Life Sciences. John Wiley & Sons, Wiley online Library. doi:10.1038/npg.els.0000379
Burke DT, Carle GF, Olson MV (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806–812
Chatzifragkou A, Makri A, Belka A, Bellou S, Mavrou M, Mastoridou M, Mystrioti P, Onjaro G, Aggelis G, Papanikolaou S (2011) Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36(2):1097–1108
Dellomonaco C, Fava F, Gonzalez R (2010) The path to next generation biofuels: successes and challenges in the era of synthetic biology. Microb Cell Fact 9:3
Didiot M-C, Serafini S, Pfeifer MJ, King FJ, Parker CN (2011) Multiplexed reporter gene assays: monitoring the cell viability and the compound kinetics on luciferase activity. J Biomol Screen 16:786–793
Dymond JS, Richardson SM, Coombes CE, Babatz T, Müller H, Annaluru N, Blake WJ, Schwerzmann JW, Junbiao D, Lindstrom DL, Boeke AC, Gottschling DE, Chandrasegaran S, Bader JS, Boeke JD (2011) Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477(7365):471–476
Fischer CR, Klein-Marcuschamer D, Stephanopoulos G (2008) Selection and optimization of microbial hosts for biofuels production. Metab Eng 10(6):295–304
Gibson DG (2012) Oligonucleotide assembly in yeast to produce synthetic DNA fragments. Methods Mol Biol 852:11–21. doi:10.1007/978-1-61779-564-0_2
Gibson DG (2014) Programming biological operating systems: genome design, assembly and activation. Nat Methods 11(5):521–526
Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, Venter JC (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329(5987):52–56
Ha S-J, Galazka JM, Kim SR, Choi J-H, Yang X, Seo J-H, Glass NL, Cate JHD, Jin Y-S (2011) Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proc Natl Acad Sci U S A 108(2):504–509
Hranueli D, Starcevic A, Zucko J, Rojas JD, Diminic J, Baranasic D, Gacesa R, Padilla G, Long PF, Cullum J (2013) Synthetic biology: a novel approach for the construction of industrial microorganisms. Food Technol Biotechnol 51(1):3–11
Hughes SR, Riedmuller SB, Mertens JA, Li X-L, Bischoff KM, Cotta MA, Farrelly PJ (2005) Development of a liquid handler component for a plasmid-based functional proteomic robotic workcell. J Assoc Lab Autom 10:287–300
Hughes SR, Riedmuller SB, Mertens JA, Li X-L, Bischoff KM, Qureshi N, Cotta MA, Farrelly PJ (2006) High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell. Proteome Sci 4:10
Hughes SR, Dowd PF, Hector RE, Riedmuller SB, Bartolett S, Mertens JA, Qureshi N, Liu S, Bischoff KM, Li X, Jackson JS Jr, Sterner D, Panavas T, Cotta MA, Farrelly PJ, Butt T (2007) Cost-effective high-throughput fully automated construction of a multiplex library of mutagenized open reading frames for an insecticidal peptide using a plasmid-based functional proteomic robotic workcell with improved vacuum system. J Assoc Lab Autom 12(4):202–212
Hughes SR, Dowd PF, Hector RE, Panavas T, Sterner DE, Qureshi N, Bischoff KM, Bang SS, Mertens JA, Johnson ET, Li XL, Jackson JS, Caughey RJ, Riedmuller SB, Bartolett S, Liu S, Rich JO, Farrelly PJ, Butt TR, Labaer J, Cotta MA (2008) Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain. J Pept Sci 14(9):1039–1050
Hughes SR, Hector RE, Rich JO, Qureshi N, Bischoff KM, Dien BS, Saha BC, Liu S, Cox EJ, Jackson JS Jr, Sterner DE, Butt TR, Labaer J, Cotta MA (2009) Automated yeast mating protocol using open reading frames from Saccharomyces cerevisiae genome to improve yeast strains for cellulosic ethanol production. J Assoc Lab Autom 14:190–199
Hughes SR, Gibbons WR, Bang SS, Pinkelman R, Bischoff KM, Slininger PJ, Qureshi N, Kurtzman CP, Liu S, Saha BC, Jackson JS, Cotta MA, Rich JO, Javers JE (2012) Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars. J Ind Microbiol Biotechnol 39(1):163–173. doi:10.1007/s10295-011-1012-x. Epub 2011 Jul 12
Hughes SR, Bang SS, Cox EJ, Schoepke A, Ochwat K, Pinkelman R, Nelson D, Qureshi N, Gibbons WR, Kurtzman CP, Bischoff KM, Liu S, Cote GL, Rich JO, Jones MA, Cedeño D, Doran-Peterson J, Riaño-Herrera NM, Rodríguez-Valencia N, López-Núñez JC (2013) Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars. J Lab Autom 18(4):276–290. doi:10.1177/2211068213480037. Epub 2013 Mar 29
Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS (2007) High-throughput screening assays for the identification of chemical probes. Nat Chem Biol 3(8):466–479
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153(1):163
James AP, Kilbey BJ (1977) The timing of UV mutagenesis in yeast: a pedigree analysis of induced recessive mutation. Genetics 87:237–248
Kim B, Du J, Eriksen DT, Zhao H (2013a) Combinatorial design of a highly efficient xylose-utilizing pathway in Saccharomyces cerevisiae for the production of cellulosic biofuels. Appl Environ Microbiol 79(3):931–941
Kim SR, Skerker JM, Kang W, Lesmana A, Wei N, Arkin AP, Jin Y-S (2013b) Rational and evolutionary engineering approaches uncover a small set of genetic changes efficient for rapid xylose fermentation in Saccharomyces cerevisiae. PLoS One 8(2):e57048. doi:10.1371/journal.pone.0057048
Kuhn RM, Ludwig RA (1994) Complete sequence of the yeast artificial chromosome cloning vector pYAC4. Gene 141:125–127
Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu J, Johnson SD, Lin-Cereghino GP (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38(1):44–48
Linshiz G, Yehezkel TB, Kaplan S, Gronau I, Ravid S, Adar R, Shapiro E (2008) Recursive construction of perfect DNA molecules from imperfect oligonucleotides. Mol Syst Biol 4:191
Martis EA, Radhakrishnan R, Badve RR (2011) High-throughput screening: the hits and leads of drug discovery: an overview. J Appl Pharm Sci 01(01):02–10
Merryman C, Gibson DG (2012) Methods and applications for assembling large DNA constructs. Metab Eng 14:196–204
Nielsen J, Larsson C, van Maris A, Pronk J (2013) Metabolic engineering of yeast for production of fuels. Curr Opin Biotechnol 24:398–404
Nonklang S, Abdel-Banat BMA, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, Akada R (2008) High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042. Appl Environ Microbiol 74(24):7514–7521
Pang ZW, Liang JJ, Qin XJ, Wang JR, Feng JX, Huang RB (2010) Multiple induced mutagenesis for improvement of ethanol production by Kluyveromyces. Biotechnol Lett 32:1847–1851
Patent Application, Docket No. 0150.06 10—Stephen R. Hughes, Serial No. 13/246,096 “Amino Acid Scanning Mutagenesis Process for Automated Production of a Library of Synthetic Mutagenized Gene Sequences” filed 9-27-2011 LOG #275214
Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD (2012) Microbial engineering for the production of advanced biofuels. Nature 488:320–328
Perlack RD, Stokes BJ (Study Leads) (2011) U.S. billion-ton update: biomass supply for a bioenergy and bioproducts industry. United States Department of Energy ORNL/TM-2011/224. Oak Ridge National Laboratory, Oak Ridge, TN. 227 p. DOI10.2172/1023318. http://www1.eere.energy.gov/bioenergy/pdfs/billion_ton_update.pdf (accessed May 24, 2014).
Rodrussamee N, Lertwattanasakul N, Hirata K, Suprayogi LS, Kosaka T, Yamada M (2011) Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus. Appl Microbiol Biotechnol 90:1573–1586
Sanchez CP, Preuss M, Lanzer M (2002) Construction and screening of YAC libraries. In: Donlan DL (ed) Malaria methods and protocols, methods in molecular medicineTM, vol 72. Humana Press, New York, pp 291–304
Shabi U, Kaplan S, Linshiz G, Yehezkel TB, Buaron H, Mazor Y, Shapiro E (2010) Processing DNA molecules as text. Syst Synth Biol 4:227–236
Thorne N, Auld DS, Inglese J (2010) Apparent activity in high-throughput screening: origins of compound-dependent assay interference. Curr Opin Chem Biol 14(3):315–324. doi:10.1016/j.cbpa.2010.03.020. Epub 2010 Apr 22
Tsvetanova B, Peng L, Liang X, Li K, Yang JP, Ho T, Shirley J, Xu L, Potter J, Kudlicki W, Peterson T, Katzen F (2011) Genetic assembly tools for synthetic biology. Methods Enzymol 498:327–348. doi:10.1016/B978-0-12-385120-8.00014-0
Wohlbach DJ, Kuo A, Sato TK, Potts KM, Salamov AA, LaButti KM, Sun H, Alicia Clum A, Pangilinan JL, Lindquist EA, Lucas S, Lapidus A, Jin M, Gunawan C, Balan V, Dale BE, Jeffries TW, Zinkel R, Barry KW, Grigoriev IV, Gasch AP (2011) Comparative genomics of xylose-fermenting fungi for enhanced biofuel production. Proc Natl Acad Sci U S A 108(32):13212–13217
Young E, Lee S-M, Alper H (2010) Optimizing pentose utilization in yeast: the need for novel tools and approaches. Biotechnol Biofuels 3:24
Zanella F, Lorens JB, Link W (2010) High content screening: seeing is believing. Trends Microbiol 28(5):237–245
Zhang F, Rodriguez S, Keasling JD (2011) Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 22(6):775–783
Zhu JY, Zhuang XS (2012) Conceptual net energy output for biofuel production from lignocellulosic biomass through biorefining. Prog Energ Combust 38:583–598. doi:10.1016/j.pecs.2012.03.00
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Hughes, S.R., Riedmuller, S.B. (2015). Integrated Automation for Continuous High-Throughput Synthetic Chromosome Assembly and Transformation to Identify Improved Yeast Strains for Industrial Production of Biofuels and Bio-based Chemicals. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 2. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10503-1_16
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