CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Li, Mengwan; Lang, Xuye; Moran Cabrera, Marcos; De Keyser, Sawyer; Sun, Xiyan; Da Silva, Nancy; Wheeldon, Ian

doi:10.1186/s13068-020-01852-3

Title: CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Journal Article · Wed Jan 06 00:00:00 EST 2021 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-020-01852-3· OSTI ID:1816782

Li, Mengwan ^[1]; Lang, Xuye ^[1]; Moran Cabrera, Marcos ^[1]; De Keyser, Sawyer ^[1]; Sun, Xiyan ^[1]; Da Silva, Nancy ^[2];

^[3]

Univ. of California, Riverside, CA (United States). Dept. of Chemical and Environmental Engineering
Univ. of California, Riverside, CA (United States). Dept. of Chemical and Biomolecular Engineering
Univ. of California, Riverside, CA (United States). Dept. of Chemical and Environmental Engineering; Univ. of California, Riverside, CA (United States). Center for Industrial Biotechnology

Background: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4^K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7^G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Univ. of California, Riverside, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Science Foundation (NSF)

Grant/Contract Number:: SC0019093; NSF 1803630; NSF 180367

OSTI ID:: 1816782

Journal Information:: Biotechnology for Biofuels, Vol. 14, Issue 1; ISSN 1754-6834

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

References (52)

Functional characterization of rose phenylacetaldehyde reductase (PAR), an enzyme involved in the biosynthesis of the scent compound 2-phenylethanol Chen, Xiao-Min; Kobayashi, Hiromi; Sakai, Miwa Journal of Plant Physiology, Vol. 168, Issue 2 https://doi.org/10.1016/j.jplph.2010.06.011	journal	January 2011
High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources Löbs, Ann-Kathrin; Lin, Jyun-Liang; Cook, Megan Biotechnology Journal, Vol. 11, Issue 10 https://doi.org/10.1002/biot.201600060	journal	September 2016
Sequential gene integration for the engineering of Kluyveromyces marxianus Pecota, Douglas C.; Rajgarhia, Vineet; Da Silva, Nancy A. Journal of Biotechnology, Vol. 127, Issue 3 https://doi.org/10.1016/j.jbiotec.2006.07.031	journal	January 2007
Putting bugs to the blush: Metabolic engineering for phenylpropanoid-derived products in microorganisms van Summeren-Wesenhagen, Philana V.; Marienhagen, Jan Bioengineered, Vol. 4, Issue 6 https://doi.org/10.4161/bioe.23885	journal	November 2013
Production of 2-Phenylethyl Alcohol by Kluyveromyces marxianus Fabre, C. E.; Blanc, P. J.; Goma, G. Biotechnology Progress, Vol. 14, Issue 2 https://doi.org/10.1021/bp9701022	journal	April 1998
Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain Nambu-Nishida, Yumiko; Nishida, Keiji; Hasunuma, Tomohisa Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-08356-5	journal	August 2017
Evolution of feedback-inhibited / barrel isoenzymes by gene duplication and a single mutation Hartmann, M.; Schneider, T. R.; Pfeil, A. Proceedings of the National Academy of Sciences, Vol. 100, Issue 3 https://doi.org/10.1073/pnas.0337566100	journal	January 2003
Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses Lertwattanasakul, Noppon; Kosaka, Tomoyuki; Hosoyama, Akira Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0227-x	journal	March 2015
Genome-wide landscape of position effects on heterogeneous gene expression in Saccharomyces cerevisiae Wu, Xiao-Le; Li, Bing-Zhi; Zhang, Wen-Zheng Biotechnology for Biofuels, Vol. 10, Issue 1 https://doi.org/10.1186/s13068-017-0872-3	journal	July 2017
Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain Signori, Lorenzo; Passolunghi, Simone; Ruohonen, Laura Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/1475-2859-13-51	journal	January 2014
Metabolic Engineering of Escherichia coli for Production of 2-Phenylethanol and 2-Phenylethyl Acetate from Glucose Guo, Daoyi; Zhang, Lihua; Kong, Sijia Journal of Agricultural and Food Chemistry, Vol. 66, Issue 23 https://doi.org/10.1021/acs.jafc.8b01594	journal	May 2018
Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus McTaggart, Tami L.; Bever, Danielle; Bassett, Shane Biotechnology and Bioengineering, Vol. 116, Issue 7 https://doi.org/10.1002/bit.26976	journal	April 2019
Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica Schwartz, Cory; Shabbir-Hussain, Murtaza; Frogue, Keith ACS Synthetic Biology, Vol. 6, Issue 3 https://doi.org/10.1021/acssynbio.6b00285	journal	November 2016
High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Abdel-Banat, Babiker M. A.; Hoshida, Hisashi; Ano, Akihiko Applied Microbiology and Biotechnology, Vol. 85, Issue 4 https://doi.org/10.1007/s00253-009-2248-5	journal	October 2009
Metabolic Engineering of Saccharomyces cerevisiae for Rosmarinic Acid Production Babaei, Mahsa; Borja Zamfir, Gheorghe M.; Chen, Xiao ACS Synthetic Biology, Vol. 9, Issue 8 https://doi.org/10.1021/acssynbio.0c00048	journal	June 2020
Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae Hassing, Else-Jasmijn; de Groot, Philip A.; Marquenie, Vita R. Metabolic Engineering, Vol. 56 https://doi.org/10.1016/j.ymben.2019.09.011	journal	December 2019
High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042 Nonklang, Sanom; Abdel-Banat, Babiker M. A.; Cha-aim, Kamonchai Applied and Environmental Microbiology, Vol. 74, Issue 24 https://doi.org/10.1128/AEM.01854-08	journal	December 2008
CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis Cao, Mingfeng; Gao, Meirong; Ploessl, Deon Biotechnology Journal, Vol. 13, Issue 9 https://doi.org/10.1002/biot.201700598	journal	July 2018
Biotechnological production of 2-phenylethanol M., Etschmann; W., Bluemke; D., Sell Applied Microbiology and Biotechnology, Vol. 59, Issue 1 https://doi.org/10.1007/s00253-002-0992-x	journal	June 2002
Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus Löbs, Ann-Kathrin; Schwartz, Cory; Thorwall, Sarah ACS Synthetic Biology, Vol. 7, Issue 11 https://doi.org/10.1021/acssynbio.8b00331	journal	October 2018
Super life - how and why ‘cell selection’ leads to the fastest-growing eukaryote: Control of highest eukaryotic growth rate Groeneveld, Philip; Stouthamer, Adriaan H.; Westerhoff, Hans V. FEBS Journal, Vol. 276, Issue 1 https://doi.org/10.1111/j.1742-4658.2008.06778.x	journal	December 2008
De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris Kong, Sijia; Pan, Hong; Liu, Xiaoyun Enzyme and Microbial Technology, Vol. 133 https://doi.org/10.1016/j.enzmictec.2019.109459	journal	February 2020
Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus Lang, Xuye; Besada-Lombana, Pamela B.; Li, Mengwan Metabolic Engineering Communications, Vol. 11 https://doi.org/10.1016/j.mec.2020.e00145	journal	December 2020
Ethyl acetate production by the elusive alcohol acetyltransferase from yeast Kruis, Aleksander J.; Levisson, Mark; Mars, Astrid E. Metabolic Engineering, Vol. 41 https://doi.org/10.1016/j.ymben.2017.03.004	journal	May 2017
Bioproduction of the aroma compound 2-Phenylethanol in a solid-liquid two-phase partitioning bioreactor system by Kluyveromyces marxianus Gao, Fang; Daugulis, Andrew J. Biotechnology and Bioengineering, Vol. 104, Issue 2 https://doi.org/10.1002/bit.22387	journal	October 2009
Biosynthesis of 2-phenylethanol from glucose with genetically engineered Kluyveromyces marxianus Kim, Tae-Yeon; Lee, Sang-Woo; Oh, Min-Kyu Enzyme and Microbial Technology, Vol. 61-62 https://doi.org/10.1016/j.enzmictec.2014.04.011	journal	July 2014
Iterative algorithm-guided design of massive strain libraries, applied to itaconic acid production in yeast Young, Eric M.; Zhao, Zheng; Gielesen, Bianca E. M. Metabolic Engineering, Vol. 48 https://doi.org/10.1016/j.ymben.2018.05.002	journal	July 2018
Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis Thorwall, Sarah; Schwartz, Cory; Chartron, Justin W. Nature Chemical Biology, Vol. 16, Issue 2 https://doi.org/10.1038/s41589-019-0452-x	journal	January 2020
Tyrosine and Tryptophan Act through the Same Binding Site at the Dimer Interface of Yeast Chorismate Mutase Schnappauf, Georg; Krappmann, Sven; Braus, Gerhard H. Journal of Biological Chemistry, Vol. 273, Issue 27 https://doi.org/10.1074/jbc.273.27.17012	journal	July 1998
Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii Dalvie, Neil C.; Leal, Justin; Whittaker, Charles A. ACS Synthetic Biology, Vol. 9, Issue 1 https://doi.org/10.1021/acssynbio.9b00372	journal	December 2019
A genetic toolbox for metabolic engineering of Issatchenkia orientalis Cao, Mingfeng; Fatma, Zia; Song, Xiaofei Metabolic Engineering, Vol. 59 https://doi.org/10.1016/j.ymben.2020.01.005	journal	May 2020
Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose Shen, Li; Nishimura, Yuya; Matsuda, Fumio Journal of Bioscience and Bioengineering, Vol. 122, Issue 1 https://doi.org/10.1016/j.jbiosc.2015.12.022	journal	July 2016
Multilevel engineering of the upstream module of aromatic amino acid biosynthesis in Saccharomyces cerevisiae for high production of polymer and drug precursors Suástegui, Miguel; Yu Ng, Chiam; Chowdhury, Anupam Metabolic Engineering, Vol. 42 https://doi.org/10.1016/j.ymben.2017.06.008	journal	July 2017
Biotechnological production of flavours and fragrances Krings, U.; Berger, R. G. Applied Microbiology and Biotechnology, Vol. 49, Issue 1 https://doi.org/10.1007/s002530051129	journal	January 1998
Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica Schwartz, Cory; Cheng, Jan-Fang; Evans, Robert Metabolic Engineering, Vol. 55 https://doi.org/10.1016/j.ymben.2019.06.007	journal	September 2019
Dual N- and C-Terminal Helices Are Required for Endoplasmic Reticulum and Lipid Droplet Association of Alcohol Acetyltransferases in Saccharomyces cerevisiae Lin, Jyun-Liang; Wheeldon, Ian PLoS ONE, Vol. 9, Issue 8 https://doi.org/10.1371/journal.pone.0104141	journal	August 2014
Kluyveromyces marxianus: A yeast emerging from its sister's shadow Lane, Melanie M.; Morrissey, John P. Fungal Biology Reviews, Vol. 24, Issue 1-2 https://doi.org/10.1016/j.fbr.2010.01.001	journal	February 2010
Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus Lee, Ki-Sung; Kim, Jun-Seob; Heo, Paul Applied Microbiology and Biotechnology, Vol. 97, Issue 5 https://doi.org/10.1007/s00253-012-4306-7	journal	August 2012
CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus Löbs, Ann-Kathrin; Engel, Ronja; Schwartz, Cory Biotechnology for Biofuels, Vol. 10, Issue 1 https://doi.org/10.1186/s13068-017-0854-5	journal	June 2017
Understanding the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation based on integration of RNA-Seq and metabolite data Fu, Xiaofen; Li, Pengsong; Zhang, Lei Applied Microbiology and Biotechnology, Vol. 103, Issue 6 https://doi.org/10.1007/s00253-019-09637-x	journal	January 2019
Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: Quantification of metabolic impact Luttik, M. A. H.; Vuralhan, Z.; Suir, E. Metabolic Engineering, Vol. 10, Issue 3-4 https://doi.org/10.1016/j.ymben.2008.02.002	journal	May 2008
Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome Heo, Paul; Yang, Tae-Jun; Chung, Soon-Chun Journal of Biotechnology, Vol. 167, Issue 3 https://doi.org/10.1016/j.jbiotec.2013.06.020	journal	September 2013
Phylogeny of the genus Kluyveromyces inferred from the mitochondrial cytochrome-c oxidase II gene Belloch, C.; Querol, A.; Garcia, M. D. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, Vol. 50, Issue 1 https://doi.org/10.1099/00207713-50-1-405	journal	January 2000
Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis Rodriguez, Angelica; Kildegaard, Kanchana R.; Li, Mingji Metabolic Engineering, Vol. 31 https://doi.org/10.1016/j.ymben.2015.08.003	journal	September 2015
Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae Krappmann, S.; Lipscomb, W. N.; Braus, G. H. Proceedings of the National Academy of Sciences, Vol. 97, Issue 25 https://doi.org/10.1073/pnas.240469697	journal	November 2000
Metabolic engineering delivers next-generation biofuels Keasling, Jay D.; Chou, Howard Nature Biotechnology, Vol. 26, Issue 3 https://doi.org/10.1038/nbt0308-298	journal	March 2008
Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products Cao, Mingfeng; Gao, Meirong; Suástegui, Miguel Metabolic Engineering, Vol. 58 https://doi.org/10.1016/j.ymben.2019.08.008	journal	March 2020
Innovating a Nonconventional Yeast Platform for Producing Shikimate as the Building Block of High-Value Aromatics Gao, Meirong; Cao, Mingfeng; Suástegui, Miguel ACS Synthetic Biology, Vol. 6, Issue 1 https://doi.org/10.1021/acssynbio.6b00132	journal	September 2016
Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose Schwartz, Cory; Curtis, Nicholas; Löbs, Ann-Kathrin Biotechnology Journal, Vol. 13, Issue 9 https://doi.org/10.1002/biot.201700584	journal	May 2018
Engineering Yarrowia lipolytica as a Chassis for De Novo Synthesis of Five Aromatic-Derived Natural Products and Chemicals Gu, Yang; Ma, Jingbo; Zhu, Yonglian ACS Synthetic Biology, Vol. 9, Issue 8 https://doi.org/10.1021/acssynbio.0c00185	journal	July 2020
Biological Parts for Kluyveromyces marxianus Synthetic Biology Rajkumar, Arun S.; Varela, Javier A.; Juergens, Hannes Frontiers in Bioengineering and Biotechnology, Vol. 7 https://doi.org/10.3389/fbioe.2019.00097	journal	May 2019
Genome and metabolic engineering in non-conventional yeasts: Current advances and applications Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian Synthetic and Systems Biotechnology, Vol. 2, Issue 3 https://doi.org/10.1016/j.synbio.2017.08.002	journal	September 2017

Linked Research (3)

Similar Records

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two–stage fine chemical production with Kluyveromyces marxianus

Journal Article · Fri May 07 00:00:00 EDT 2021 · Biotechnology Progress · OSTI ID:1816782

Hillman, Ethan T.; Li, Mengwan; Hooker, Casey A.; +3 more

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

Journal Article · Sun Mar 24 00:00:00 EDT 2019 · Biotechnology and Bioengineering · OSTI ID:1816782

McTaggart, Tami L.; Bever, Danielle; Bassett, Shane; +1 more

Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli

Journal Article · Fri Jan 04 00:00:00 EST 2019 · ACS Synthetic Biology · OSTI ID:1816782

Tian, Tian; Kang, Jing Wei; Kang, Aram; +1 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Metabolic engineering
Flavors and fragrances
Thermotolerance
Expression regulation

Additional file 1 of CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus Li, Mengwan; Lang, Xuye; Cabrera, Marcos Moran https://doi.org/10.6084/m9.figshare.13535705 The current record is supplemented by this text	text	January 2021
CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus Li, Mengwan; Lang, Xuye; Cabrera, Marcos Moran https://doi.org/10.6084/m9.figshare.c.5259821 The current record is supplemented by this collection	collection	January 2021
Additional file 1 of CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus Li, Mengwan; Lang, Xuye; Cabrera, Marcos Moran https://doi.org/10.6084/m9.figshare.13535705.v1 The current record is supplemented by this text	text	January 2021

Title: CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Citation Formats

References (52)

Linked Research (3)

Similar Records

Related Subjects