Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis

ABSTRACT Microbial chemical production is a rapidly growing industry, with much of the growth fueled by advances in synthetic biology. New approaches have enabled rapid strain engineering

for the production of various compounds; however, translation to industry is often problematic because native phenotypes of model hosts prevent the design of new low-cost bioprocesses. Here,

we argue for a new approach that leverages the native stress-tolerant phenotypes of non-conventional microbes that directly address design challenges from the outset. Growth at high

temperature, high salt and solvent concentrations, and low pH can enable cost savings by reducing the energy required for product separation, bioreactor cooling, and maintaining sterile

conditions. These phenotypes have the added benefit of allowing for the use of low-cost sugar and water resources. Non-conventional hosts are needed because these phenotypes are polygenic

and thus far have proven difficult to recapitulate in the common hosts _Escherichia coli_ and _Saccharomyces cerevisiae_. Access through your institution Buy or subscribe This is a preview

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS SCREENING NON-CONVENTIONAL YEASTS FOR ACID TOLERANCE AND

ENGINEERING _PICHIA OCCIDENTALIS_ FOR PRODUCTION OF MUCONIC ACID Article Open access 31 August 2023 PHYSIOLOGICAL LIMITATIONS AND OPPORTUNITIES IN MICROBIAL METABOLIC ENGINEERING Article 02

August 2021 GENOME-SCALE TARGET IDENTIFICATION IN _ESCHERICHIA COLI_ FOR HIGH-TITER PRODUCTION OF FREE FATTY ACIDS Article Open access 17 August 2021 REFERENCES * Carlson, R. Estimating the

biotech sector’s contribution to the US economy. _Nat. Biotechnol._ 34, 247–255 (2016). THIS PERSPECTIVE ARTICLE PROVIDES A DETAILED ANALYSIS OF THE US INDUSTRIAL BIOTECHNOLOGY SECTOR.

Article  CAS  PubMed  Google Scholar  * Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. _Science_ 337, 816–821 (2012). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Caruthers, M. H. A brief review of DNA and RNA chemical synthesis. _Biochem. Soc. Trans._ 39, 575–580 (2011). Article  CAS  PubMed  Google Scholar  * Chao,

R., Mishra, S., Si, T. & Zhao, H. Engineering biological systems using automated biofoundries. _Metab. Eng._ 42, 98–108 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Hong, K. K. & Nielsen, J. Metabolic engineering of _Saccharomyces cerevisiae_: a key cell factory platform for future biorefineries. _Cell. Mol. Life Sci._ 69, 2671–2690 (2012). Article

  CAS  PubMed  Google Scholar  * Pontrelli, S. et al. _Escherichia coli_ as a host for metabolic engineering. _Metab. Eng._ 50, 16–46 (2018). Article  CAS  PubMed  Google Scholar  *

Shapouri, H. & Gallagher, P. USDA’s 2002 Ethanol Cost-of-Production Survey. _Agricultural Economics Report Number 841_ (US Department of Agriculture, Office of Energy Policy and New

Uses, 2005). * Blanch, H.W. & Clark, D.S. _Biochemical Engineering_, xii (M. Dekker, 1996). * Lam, F. H., Ghaderi, A., Fink, G. R. & Stephanopoulos, G. Biofuels. Engineering alcohol

tolerance in yeast. _Science_ 346, 71–75 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rich, J. O., Leathers, T. D., Bischoff, K. M., Anderson, A. M. & Nunnally, M. S.

Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation. _Bioresour. Technol._ 196, 347–354 (2015). Article  CAS  PubMed  Google Scholar  * Löbs, A. K.,

Engel, R., Schwartz, C., Flores, A. & Wheeldon, I. CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in _Kluyveromyces marxianus_.

_Biotechnol. Biofuels_ 10, 164 (2017). Article  PubMed  PubMed Central  CAS  Google Scholar  * Shui, W. et al. Understanding the mechanism of thermotolerance distinct from feat shock

response through proteomic analysis of industrial strains of _Saccharomyces cerevisiae_. _Mol. Cell. Proteom._ 14, 1885–1897 (2015). Article  CAS  Google Scholar  * Richter, K., Haslbeck, M.

& Buchner, J. The heat shock response: life on the verge of death. _Mol. Cell_ 40, 253–266 (2010). Article  CAS  PubMed  Google Scholar  * Phipps, B. M. et al. Structure of a molecular

chaperone from a thermophilic Archaebacterium. _Nature_ 361, 475–477 (1993). Article  CAS  Google Scholar  * Parsell, D. A. & Lindquist, S. The function of heat-shock proteins in stress

tolerance: degradation and reactivation of damaged proteins. _Annu. Rev. Genet._ 27, 437–496 (1993). Article  CAS  PubMed  Google Scholar  * Strassburg, K., Walther, D., Takahashi, H.,

Kanaya, S. & Kopka, J. Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress. _OMICS_ 14, 249–259 (2010). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. _Science_ 353, aac4354 (2016). Article  PubMed  CAS  Google Scholar  *

Verghese, J., Abrams, J., Wang, Y. & Morano, K. A. Biology of the heat shock response and protein chaperones: budding yeast (_Saccharomyces cerevisiae_) as a model system. _Microbiol.

Mol. Biol. Rev._ 76, 115–158 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gasch, A. P. et al. Genomic expression programs in the response of yeast cells to environmental

changes. _Mol. Biol. Cell_ 11, 4241–4257 (2000). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rodrigues, J. L. & Rodrigues, L. R. Potential applications of the _Escherichia

coli_ heat shock response in synthetic biology. _Trends Biotechnol._ 36, 186–198 (2018). Article  CAS  PubMed  Google Scholar  * Lewin, A., Wentzel, A. & Valla, S. Metagenomics of

microbial life in extreme temperature environments. _Curr. Opin. Biotechnol._ 24, 516–525 (2013). Article  CAS  PubMed  Google Scholar  * Jacquemet, A., Barbeau, J., Lemiègre, L. &

Benvegnu, T. Archaeal tetraether bipolar lipids: structures, functions and applications. _Biochimie_ 91, 711–717 (2009). Article  CAS  PubMed  Google Scholar  * Li, P. et al. The

transcription factors Hsf1 and Msn2 of thermotolerant _Kluyveromyces marxianus_ promote cell growth and ethanol fermentation of _Saccharomyces cerevisiae_ at high temperatures. _Biotechnol.

Biofuels_ 10, 289 (2017). Article  PubMed  PubMed Central  CAS  Google Scholar  * Seong, Y. J. et al. Physiological and metabolomic analysis of _Issatchenkia orientalis_ MTY1 with multiple

tolerance for cellulosic bioethanol production. _Biotechnol. J._ 12, 1700110 (2017). Article  CAS  Google Scholar  * Blaby, I. K. et al. Experimental evolution of a facultative thermophile

from a mesophilic ancestor. _Appl. Environ. Microbiol._ 78, 144–155 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rudolph, B., Gebendorfer, K. M., Buchner, J. & Winter,

J. Evolution of _Escherichia coli_ for growth at high temperatures. _J. Biol. Chem._ 285, 19029–19034 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Caspeta, L. et al.

Biofuels. Altered sterol composition renders yeast thermotolerant. _Science_ 346, 75–78 (2014). Article  CAS  PubMed  Google Scholar  * Shi, D. J., Wang, C. L. & Wang, K. M. Genome

shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of _Saccharomyces cerevisiae_. _J. Ind. Microbiol. Biotechnol._ 36, 139–147 (2009). Article  CAS  PubMed 

Google Scholar  * Bhandiwad, A. et al. Metabolic engineering of _Thermoanaerobacterium saccharolyticum_ for n-butanol production. _Metab. Eng._ 21, 17–25 (2014). Article  CAS  PubMed  Google

Scholar  * Lin, P. P. et al. Consolidated bioprocessing of cellulose to isobutanol using _Clostridium thermocellum_. _Metab. Eng._ 31, 44–52 (2015). Article  CAS  PubMed  Google Scholar  *

Cripps, R. E. et al. Metabolic engineering of _Geobacillus thermoglucosidasius_ for high yield ethanol production. _Metab. Eng._ 11, 398–408 (2009). Article  CAS  PubMed  Google Scholar  *

Yue, H. T. et al. A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant _Halomonas campaniensis_ LS21 grown in mixed substrates. _Biotechnol.

Biofuels_ 7, 108 (2014). THIS PAPER SHOWS THE POWER OF STRESS-TOLERANT MICROBES. ENGINEERED HALOPHILIC _H. CAMPANIENSIS_ WAS USED IN NON-ASEPTIC BIOREACTORS TO PRODUCE HIGH TITERS OF

POLYHYDROXYBUTYRATE FROM MIXED SUBSTRATE FEEDSTOCKS. Article  CAS  Google Scholar  * Voronovsky, A. Y., Rohulya, O. V., Abbas, C. A. & Sibirny, A. A. Development of strains of the

thermotolerant yeast _Hansenula polymorpha_ capable of alcoholic fermentation of starch and xylan. _Metab. Eng._ 11, 234–242 (2009). Article  CAS  PubMed  Google Scholar  * Löbs, A. K., Lin,

J. L., Cook, M. & Wheeldon, I. High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and

C12 carbon sources. _Biotechnol. J._ 11, 1274–1281 (2016). Article  PubMed  CAS  Google Scholar  * Xiao, H., Shao, Z., Jiang, Y., Dole, S. & Zhao, H. Exploiting _Issatchenkia orientalis_

SD108 for succinic acid production. _Microb. Cell Fact._ 13, 121 (2014). Article  PubMed  PubMed Central  CAS  Google Scholar  * Löbs, A. K., Schwartz, C., Thorwall, S. & Wheeldon, I.

Highly multiplexed CRISPRi repression of respiratory functions enhances mitochondrial localized ethyl acetate biosynthesis in _Kluyveromyces marxianus_. _ACS Synth. Biol._ 7, 2647–2655

(2018). THIS PAPER DEMONSTRATES THE POWER OF CRISPR TECHNOLOGIES FOR RAPIDLY ENGINEERING NON-CONVENTIONAL HOSTS. HERE, MULTIPLEXED CRISPR INTERFERENCE (CRISPRI) WAS USED TO SCREEN VARIOUS

TRANSCRIPTIONAL PROGRAMS TO OPTIMIZE A NATIVE BIOSYNTHETIC PATHWAY IN THE THERMOTOLERANT YEAST _K. MARXIANUS_. Article  PubMed  CAS  Google Scholar  * Abdel-Banat, B. M., Hoshida, H., Ano,

A., Nonklang, S. & Akada, R. 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, 861–867 (2010). Article  CAS  PubMed  Google Scholar  * Cernak, P. et al. Engineering _Kluyveromyces marxianus_ as a robust synthetic biology

platform host. _MBio_ 9, e01410–e01418 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cheon, Y. et al. A biosynthetic pathway for hexanoic acid production in _Kluyveromyces

marxianus_. _J. Biotechnol._ 182-183, 30–36 (2014). Article  CAS  PubMed  Google Scholar  * Dmytruk, K., Kurylenko, O., Ruchala, J., Ishchuk, O. & Sibirny, A. in _Yeast Diversity in

Human Welfare_ (eds Satyanarayana, T. & Kunze, G.) 257–282 (Springer, 2017). * Lenton, S., Walsh, D. L., Rhys, N. H., Soper, A. K. & Dougan, L. Structural evidence for

solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations. _Phys. Chem. Chem. Phys._ 18, 18054–18062 (2016). Article  CAS  PubMed 

Google Scholar  * Oren, A. Microbial life at high salt concentrations: phylogenetic and metabolic diversity. _Saline Syst._ 4, 2 (2008). Article  PubMed  PubMed Central  CAS  Google Scholar

  * Padan, E., Venturi, M., Gerchman, Y. & Dover, N. Na+/H+ antiporters. _Biochim. Biophys. Acta_ 1505, 144–157 (2001). Article  CAS  PubMed  Google Scholar  * Purvis, J. E., Yomano, L.

P. & Ingram, L. O. Enhanced trehalose production improves growth of _Escherichia coli_ under osmotic stress. _Appl. Environ. Microbiol._ 71, 3761–3769 (2005). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Yang, L. et al. A primary sodium pump gene of the moderate halophile _Halobacillus dabanensis_ exhibits secondary antiporter properties. _Biochem. Biophys.

Res. Commun._ 346, 612–617 (2006). Article  CAS  PubMed  Google Scholar  * Ekberg, J. et al. Adaptive evolution of the lager brewing yeast _Saccharomyces pastorianus_ for improved growth

under hyperosmotic conditions and its influence on fermentation performance. _FEMS Yeast Res._ 13, 335–349 (2013). Article  CAS  PubMed  Google Scholar  * Schweikhard, E. S., Kuhlmann, S.

I., Kunte, H. J., Grammann, K. & Ziegler, C. M. Structure and function of the universal stress protein TeaD and its role in regulating the ectoine transporter TeaABC of _Halomonas

elongata_ DSM 2581(T). _Biochemistry_ 49, 2194–2204 (2010). Article  CAS  PubMed  Google Scholar  * Kunte, H., Lentzen, G. & Galinski, E. Industrial production of the cell protectant

ectoine: protection mechanisms, processes, and products. _Curr. Biotechnol._ 3, 10–25 (2014). Article  CAS  Google Scholar  * Chen, X., Yu, L., Qiao, G. & Chen, G. Q. Reprogramming

_Halomonas_ for industrial production of chemicals. _J. Ind. Microbiol. Biotechnol._ 45, 545–554 (2018). Article  CAS  PubMed  Google Scholar  * Qin, Q. et al. CRISPR/Cas9 editing genome of

extremophile _Halomonas_ spp. _Metab. Eng._ 47, 219–229 (2018). Article  CAS  PubMed  Google Scholar  * Chen, R. et al. Optimization of the extraction and purification of the compatible

solute ectoine from _Halomonas_ elongate in the laboratory experiment of a commercial production project. _World J. Microbiol. Biotechnol._ 33, 116 (2017). Article  PubMed  CAS  Google

Scholar  * Zaky, A. S., Greetham, D., Tucker, G. A. & Du, C. The establishment of a marine focused biorefinery for bioethanol production using seawater and a novel marine yeast strain.

_Sci. Rep._ 8, 12127 (2018). Article  PubMed  PubMed Central  CAS  Google Scholar  * Yuan, W. J., Zhao, X. Q., Ge, X. M. & Bai, F. W. Ethanol fermentation with _Kluyveromyces marxianus_

from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater. _J. Appl. Microbiol._ 105, 2076–2083 (2008). Article  CAS  PubMed  Google Scholar  * Ramos,

J. L., Duque, E., Godoy, P. & Segura, A. Efflux pumps involved in toluene tolerance in _Pseudomonas putida_ DOT-T1E. _J. Bacteriol._ 180, 3323–3329 (1998). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Rühl, J., Schmid, A. & Blank, L. M. Selected _Pseudomonas putida_ strains able to grow in the presence of high butanol concentrations. _Appl. Environ.

Microbiol._ 75, 4653–4656 (2009). Article  PubMed  PubMed Central  CAS  Google Scholar  * Ramos, J. L. et al. Mechanisms for solvent tolerance in bacteria. _J. Biol. Chem._ 272, 3887–3890

(1997). Article  CAS  PubMed  Google Scholar  * Rojas, A. et al. Three efflux pumps are required to provide efficient tolerance to toluene in _Pseudomonas putida_ DOT-T1E. _J. Bacteriol._

183, 3967–3973 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Terán, W. et al. Complexity in efflux pump control: cross-regulation by the paralogues TtgV and TtgT. _Mol.

Microbiol._ 66, 1416–1428 (2007). PubMed  Google Scholar  * Segura, A. et al. Proteomic analysis reveals the participation of energy- and stress-related proteins in the response of

_Pseudomonas putida_ DOT-T1E to toluene. _J. Bacteriol._ 187, 5937–5945 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Dunlop, M. J. et al. Engineering microbial biofuel

tolerance and export using efflux pumps. _Mol. Syst. Biol._ 7, 487 (2011). Article  PubMed  PubMed Central  Google Scholar  * Tan, Z., Yoon, J. M., Nielsen, D. R., Shanks, J. V. &

Jarboe, L. R. Membrane engineering via trans unsaturated fatty acids production improves _Escherichia coli_ robustness and production of biorenewables. _Metab. Eng._ 35, 105–113 (2016).

Article  CAS  PubMed  Google Scholar  * Zingaro, K. A. & Terry Papoutsakis, E. GroESL overexpression imparts _Escherichia coli_ tolerance to i-, _n_-, and 2-butanol, 1,2,4-butanetriol

and ethanol with complex and unpredictable patterns. _Metab. Eng._ 15, 196–205 (2013). Article  CAS  PubMed  Google Scholar  * Cook, T. B. et al. Genetic tools for reliable gene expression

and recombineering in _Pseudomonas putida_. _J. Ind. Microbiol. Biotechnol._ 45, 517–527 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gong, T. et al. Metabolic engineering

of _Pseudomonas putida_ KT2440 for complete mineralization of methyl parathion and γ-hexachlorocyclohexane. _ACS Synth. Biol._ 5, 434–442 (2016). Article  CAS  PubMed  Google Scholar  *

Nikel, P. I. & de Lorenzo, V. _Pseudomonas putida_ as a functional chassis for industrial biocatalysis: from native biochemistry to trans-metabolism. _Metab. Eng._ 50, 142–155 (2018).

Article  CAS  PubMed  Google Scholar  * Bormann, S. et al. Engineering _Clostridium acetobutylicum_ for production of kerosene and diesel blendstock precursors. _Metab. Eng._ 25, 124–130

(2014). Article  CAS  PubMed  Google Scholar  * Anbarasan, P. et al. Integration of chemical catalysis with extractive fermentation to produce fuels. _Nature_ 491, 235–239 (2012). Article 

CAS  PubMed  Google Scholar  * Zhang, J., Wu, C. D., Du, G. C. & Chen, J. Enhanced acid tolerance in Lactobacillus casei by adaptive evolution and compared stress response during acid

stress. _Biotechnol. Bioprocess Eng._ 17, 283–289 (2012). Article  CAS  Google Scholar  * Zhou, L. et al. Improvement of d-lactate productivity in recombinant _Escherichia coli_ by coupling

production with growth. _Biotechnol. Lett._ 34, 1123–1130 (2012). Article  CAS  PubMed  Google Scholar  * Lee, J. Y., Kang, C. D., Lee, S. H., Park, Y. K. & Cho, K. M. Engineering

cellular redox balance in _Saccharomyces cerevisiae_ for improved production of l-lactic acid. _Biotechnol. Bioeng._ 112, 751–758 (2015). Article  CAS  PubMed  Google Scholar  * Yan, D. et

al. Construction of reductive pathway in _Saccharomyces cerevisiae_ for effective succinic acid fermentation at low pH value. _Bioresour. Technol._ 156, 232–239 (2014). Article  CAS  PubMed

  Google Scholar  * Kwon, Y. D., Kim, S., Lee, S. Y. & Kim, P. Long-term continuous adaptation of _Escherichia coli_ to high succinate stress and transcriptome analysis of the tolerant

strain. _J. Biosci. Bioeng._ 111, 26–30 (2011). Article  CAS  PubMed  Google Scholar  * Wright, J. et al. Batch and continuous culture-based selection strategies for acetic acid tolerance in

xylose-fermenting _Saccharomyces cerevisiae_. _FEMS Yeast Res._ 11, 299–306 (2011). Article  CAS  PubMed  Google Scholar  * Sandoval, N. R., Mills, T. Y., Zhang, M. & Gill, R. T.

Elucidating acetate tolerance in _E. coli_ using a genome-wide approach. _Metab. Eng._ 13, 214–224 (2011). Article  CAS  PubMed  Google Scholar  * Suominen, P. _et al_. Genetically modified

yeast of the species _Issatchenkia Orientalis_ and closely relates species, and fermentation processes using same (Cargill Inc., 2012). * Valdés, J. et al. _Acidithiobacillus ferrooxidans_

metabolism: from genome sequence to industrial applications. _BMC Genomics_ 9, 597 (2008). Article  PubMed  PubMed Central  CAS  Google Scholar  * Kernan, T. et al. Engineering the

iron-oxidizing chemolithoautotroph _Acidithiobacillus ferrooxidans_ for biochemical production. _Biotechnol. Bioeng._ 113, 189–197 (2016). Article  CAS  PubMed  Google Scholar  * Inaba, Y.,

Banerjee, I., Kernan, T. & Banta, S. Transposase-mediated chromosomal integration of exogenous genes in _Acidithiobacillus ferrooxidans_. _Appl. Environ. Microbiol._ 84, e01381–18

(2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Maezato, Y., Johnson, T., McCarthy, S., Dana, K. & Blum, P. Metal resistance and lithoautotrophy in the extreme

thermoacidophile _Metallosphaera sedula_. _J. Bacteriol._ 194, 6856–6863 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Auernik, K. S., Maezato, Y., Blum, P. H. & Kelly,

R. M. The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon _Metallosphaera sedula_ provides insights into bioleaching-associated metabolism. _Appl. Environ.

Microbiol._ 74, 682–692 (2008). Article  CAS  PubMed  Google Scholar  * Zeldes, B. M. et al. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels

and industrial chemicals. _Front. Microbiol._ 6, 1209 (2015). Article  PubMed  PubMed Central  Google Scholar  * Feist, A. M. et al. A genome-scale metabolic reconstruction for _Escherichia

coli_ K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. _Mol. Syst. Biol._ 3, 121 (2007). Article  PubMed  PubMed Central  CAS  Google Scholar  * Förster, J., Famili,

I., Fu, P., Palsson, B. O. & Nielsen, J. Genome-scale reconstruction of the _Saccharomyces cerevisiae_ metabolic network. _Genome Res._ 13, 244–253 (2003). Article  PubMed  PubMed

Central  CAS  Google Scholar  * Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. _Nature_ 460, 894–898 (2009). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Warner, J. R., Reeder, P. J., Karimpour-Fard, A., Woodruff, L. B. & Gill, R. T. Rapid profiling of a microbial genome using mixtures of barcoded

oligonucleotides. _Nat. Biotechnol._ 28, 856–862 (2010). Article  CAS  PubMed  Google Scholar  * Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A highly characterized yeast

toolkit for modular, multipart assembly. _ACS Synth. Biol._ 4, 975–986 (2015). Article  CAS  PubMed  Google Scholar  * Xu, P., Vansiri, A., Bhan, N. & Koffas, M. A. ePathBrick: a

synthetic biology platform for engineering metabolic pathways in _E. coli_. _ACS Synth. Biol._ 1, 256–266 (2012). Article  CAS  PubMed  Google Scholar  * Löbs, A. K., Schwartz, C. &

Wheeldon, I. Genome and metabolic engineering in non-conventional yeasts: current advances and applications. _Synth. Syst. Biotechnol._ 2, 198–207 (2017). Article  PubMed  PubMed Central 

Google Scholar  * Schwartz, C., Frogue, K., Ramesh, A., Misa, J. & Wheeldon, I. CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous

recombination in _Yarrowia lipolytica_. _Biotechnol. Bioeng._ 114, 2896–2906 (2017). Article  CAS  PubMed  Google Scholar  * Cao, M., Gao, M., Ploessl, D., Song, C. & Shao, Z.

CRISPR-mediated genome editing and gene repression in _Scheffersomyces stipitis_. _Biotechnol. J._ 13, e1700598 (2018). Article  PubMed  CAS  Google Scholar  * Schwartz, C., Shabbir-Hussain,

M., Frogue, K., Blenner, M. & Wheeldon, I. Standardized markerless gene integration for pathway engineering in _Yarrowia lipolytica_. _ACS Synth. Biol._ 6, 402–409 (2017). Article  CAS

  PubMed  Google Scholar  * Schwartz, C. et al. Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast _Yarrowia lipolytica_. _Metab. Eng._ 55, 102–110

(2019). Article  CAS  PubMed  Google Scholar  * Schwartz, C. M., Hussain, M. S., Blenner, M. & Wheeldon, I. Synthetic RNA polymerase III promoters facilitate high-efficiency

CRISPR-Cas9-mediated genome editing in _Yarrowia lipolytica_. _ACS Synth. Biol._ 5, 356–359 (2016). Article  CAS  PubMed  Google Scholar  * Cao, M. et al. Centromeric DNA facilitates

nonconventional yeast genetic engineering. _ACS Synth. Biol._ 6, 1545–1553 (2017). Article  CAS  PubMed  Google Scholar  * Lewis, N. E., Nagarajan, H. & Palsson, B. O. Constraining the

metabolic genotype-phenotype relationship using a phylogeny of in silico methods. _Nat. Rev. Microbiol._ 10, 291–305 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Schwalen,

C. J., Hudson, G. A., Kille, B. & Mitchell, D. A. Bioinformatic expansion and discovery of thiopeptide antibiotics. _J. Am. Chem. Soc._ 140, 9494–9501 (2018). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Brophy, J. A. N. et al. Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria. _Nat.

Microbiol._ 3, 1043–1053 (2018). THIS WORK ENGINEERS THE INTEGRATIVE AND CONJUGATIVE ELEMENTS OF _B__ACILLUS SUBTILIS_ TO CREATE A STRAIN TECHNOLOGY CALLED XPORT THAT CAN BE USED TO RAPIDLY

ENGINEER NOVEL BACTERIAL ISOLATES. Article  CAS  PubMed  Google Scholar  * Schwartz, C., Curtis, N., Löbs, A. K. & Wheeldon, I. Multiplexed CRISPR activation of cryptic sugar metabolism

enables _Yarrowia lipolytica_ growth on cellobiose. _Biotechnol. J._ 13, e1700584 (2018). Article  PubMed  CAS  Google Scholar  * Boundy-Mills, K. L. et al. Yeast culture collections in the

twenty-first century: new opportunities and challenges. _Yeast_ 33, 243–260 (2016). Article  CAS  PubMed  Google Scholar  * Solomon, K. V. et al. Early-branching gut fungi possess a large,

comprehensive array of biomass-degrading enzymes. _Science_ 351, 1192–1195 (2016). THIS WORK USES ‘OMICS’-LEVEL ANALYSES AND BIOCHEMICAL ASSAYS TO DISCOVER NEW BIOTECHNOLOGY-RELEVANT

ENZYMES, INCLUDING CELLULASES FOR THE BREAKDOWN OF LIGNOCELLULOSIC BIOMASS IN NEW ISOLATES OF ANAEROBIC GUT FUNGI. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references

ACKNOWLEDGEMENTS This material is based upon work supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program under

Award Number DE-SC0019093, Air Force Office of Scientific Research award FA9550-17-1-0270, Army Research Office MURI award W911NF1410263, and National Science Foundation award NSF-CBET

1706545 for funding. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, CA, USA Sarah

Thorwall, Cory Schwartz & Ian Wheeldon * Department of Bioengineering, University of California Riverside, Riverside, CA, USA Justin W. Chartron * Center for Industrial Biotechnology,

Bourns College of Engineering, University of California Riverside, Riverside, CA, USA Ian Wheeldon Authors * Sarah Thorwall View author publications You can also search for this author

inPubMed Google Scholar * Cory Schwartz View author publications You can also search for this author inPubMed Google Scholar * Justin W. Chartron View author publications You can also search

for this author inPubMed Google Scholar * Ian Wheeldon View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Ian

Wheeldon. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

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Chartron, J.W. _et al._ Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis. _Nat Chem Biol_ 16, 113–121 (2020). https://doi.org/10.1038/s41589-019-0452-x

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