N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.

Department of Biomedical Engineering Faculty of Electrical Engineering and Communication Brno University of Technology Brno Czechia

Department of Biotechnology University of Chemistry and Technology Prague Prague Czechia

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Ahmed M., Borsch C. M., Taylor S. S., Vázquez-Laslop N., Neyfakh A. A. (1994). A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates. J. Biol. Chem. 269 28506–28513. PubMed

Alsaker K. V., Spitzer T. R., Papoutsakis E. T. (2004). Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell’s response to butanol stress. J. Bacteriol. 186 1959–1971. 10.1128/JB.186.7.1959-1971.2004 PubMed DOI PMC

Annous B. A., Blaschek H. P. (1991). Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity. Appl. Environ. Microbiol. 57 2544–2548. PubMed PMC

Baer S. H., Blaschek H. P., Smith T. L. (1987). Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol-tolerant Clostridium acetobutylicum. Appl. Environ. Microbiol. 53 2854–2861. PubMed PMC

Basler G., Thompson M., Tullman-Ercek D., Keasling J. (2018). A Pseudomonas putida efflux pump acts on short-chain alcohols. Biotechnol. Biofuels 11:136. 10.1186/s13068-018-1133-9 PubMed DOI PMC

Bohnert J. A., Schuster S., Fähnrich E., Trittler R., Kern W. V. (2007). Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF). J. Antimicrob. Chemother. 59 1216–1222. 10.1093/jac/dkl426 PubMed DOI

Bolger A. M., Lohse M., Usadel B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 2114–2120. 10.1093/bioinformatics/btu170 PubMed DOI PMC

Bowles L. K., Ellefson W. L. (1985). Effects of butanol on Clostridium acetobutylicum. Appl. Environ. Microbiol. 50 1165–1170. PubMed PMC

Branska B., Pechacova Z., Kolek J., Vasylkivska M., Patakova P. (2018). Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions. Biotechnol. Biofuels 11:99. 10.1186/s13068-018-1096-x PubMed DOI PMC

Bui L. M., Lee J. Y., Geraldi A., Rahman Z., Lee J. H., Kim S. C. (2015). Improved n-butanol tolerance in Escherichia coli by controlling membrane related functions. J. Biotechnol. 204 33–44. 10.1016/j.jbiotec.2015.03.025 PubMed DOI

Bush S. J., Foster D., Eyre D. W., Clark E. L., De Maio N., Shaw L. P., et al. (2020). Genomic diversity affects the accuracy of bacterial single-nucleotide polymorphism-calling pipelines. Gigascience 9:giaa007. 10.1093/gigascience/giaa007 PubMed DOI PMC

Chen C. K., Blaschek H. P. (1999). Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101. Appl. Microbiol. Biotechnol. 52 170–173. 10.1007/s002530051504 PubMed DOI

Chimalapati S., Cohen J. M., Camberlein E., MacDonald N., Durmort C., Vernet T., et al. (2012). Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene Lgt on ABC transporter function and on growth in vivo. PLoS One 7:e41393. 10.1371/journal.pone.0041393 PubMed DOI PMC

Drahokoupil M., Patáková P. (2020). Production of butyric acid at constant pH by a solventogenic strain of Clostridium beijerinckii. Czech J. Food Sci. 38 185–191. 10.17221/95/2020-CJFS DOI

Ewels P., Magnusson M., Lundin S., Kaller M. (2016). MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32 3047–3048. 10.1093/bioinformatics/btw354 PubMed DOI PMC

Fisher M. A., Boyarskiy S., Yamada M. R., Kong N., Bauer S., Tullman-Ercek D. (2014). Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol. ACS Synth. Biol. 3 30–40. 10.1021/sb400065q PubMed DOI

Fletcher E., Pilizota T., Davies P. R., McVey A., French C. E. (2016). Characterization of the effects of n-butanol on the cell envelope of E. coli. Appl. Microbiol. Biotechnol. 100 9653–9659. 10.1007/s00253-016-7771-6 PubMed DOI

Gagneur J., Toedling J., Bourgon R., Delhomme N. (2020). genomeIntervals: Operations on Genomic Intervals. R package version 1.44.2. Available online at: https://bioconductor.org/packages/release/bioc/html/genomeIntervals.html (accessed June 13, 2020).

Gallardo R., Alves M., Rodrigues L. R. (2017). Influence of nutritional and operational parameters on the production of butanol or 1,3-propanediol from glycerol by a mutant Clostridium pasteurianum. N. Biotechnol. 34 59–67. 10.1016/j.nbt.2016.03.002 PubMed DOI

Gish W., States D. J. (1993). Identification of protein coding regions by database similarity search. Nat. Genet. 3 266–272. 10.1038/ng0393-266 PubMed DOI

Greenblum S., Carr R., Borenstein E. (2015). Extensive strain-level copy-number variation across human gut microbiome species. Cell 160 583–594. 10.1016/j.cell.2014.12.038 PubMed DOI PMC

Gutierrez N. A., Maddox I. S. (1987). Role of chemotaxis in solvent production by Clostridium acetobutylicum. Appl. Environ. Microbiol. 53 1924–1927. PubMed PMC

Hayashida S., Yoshino S. (1990). Degeneration of solventogenic Clostridium caused by a defect in NADH generation. Agric. Biol. Chem. 54 427–435. 10.1271/bbb1961.54.427 DOI

Hermann M., Fayolle F., Marchal R., Podvin L., Sebald M., Vandecasteele J. P. (1985). Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum. Appl. Environ. Microbiol. 50 1238–1243. PubMed PMC

Hocq R., Bouilloux-Lafont M., Lopes Ferreira N., Wasels F. (2019). σ 54 (σ L) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii. Sci. Rep. 9:7228. 10.1038/s41598-019-43822-2 PubMed DOI PMC

Ingram L. O. (1986). Microbial tolerance to alcohols: role of the cell membrane. Trends Biotechnol. 4 40–44. 10.1016/0167-7799(86)90152-6 DOI

Jain M. K., Beacom D., Datta R. (1994). Mutant strain of C. acetobutylicum and process for making butanol. Patent 12:242 10.1016/0734-9750(94)90895-8 DOI

Jiménez-Bonilla P., Zhang J., Wang Y., Blersch D., De-Bashan L.-E., Guo L., et al. (2020). Enhancing the tolerance of Clostridium saccharoperbutylacetonicum to lignocellulosic-biomass-derived inhibitors for efficient biobutanol production by overexpressing efflux pumps genes from Pseudomonas putida. Bioresour. Technol 312:123532. 10.1016/j.biortech.2020.123532 PubMed DOI

Jones A. J., Venkataramanan K. P., Papoutsakis T. (2016). Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum. FEMS Microbiol. Lett. 363:fnw063. 10.1093/femsle/fnw063 PubMed DOI

Jureckova K., Koscova P., Sedlar K., Kolek J., Patakova P., Provaznik I. (2018). “In silico prediction of genes coding efflux pumps in Clostridium beijerinckii NRRL B-598,” in Proceedings of the 6th International Conference on Chemical Technology, eds Vesely M., Hrdlicka Z., Hanika J., Lubojacky J. (Czech Republic: Czech Society of Industrial Chemistry; ), 86–90.

Knaus B. J., Grünwald N. J. (2017). vcfr: a package to manipulate and visualize variant call format data in R. Mol. Ecol. Resour. 17 44–53. 10.1111/1755-0998.12549 PubMed DOI

Kong X., He A., Zhao J., Wu H., Ma J., Wei C., et al. (2016). Efficient acetone-butanol-ethanol (ABE) production by a butanol-tolerant mutant of Clostridium beijerinckii in a fermentation-pervaporation coupled process. Biochem. Eng. J. 105 90–96. 10.1016/j.bej.2015.09.013 DOI

Lee J., Blaschek H. P. (2001). Glucose uptake in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101. Appl. Environ. Microbiol. 67 5025–5031. PubMed PMC

Lee J., Mitchell W. J., Tangney M., Blaschek H. P. (2005). Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101. Appl. Environ. Microbiol. 71 3384–3387. 10.1128/AEM.71.6.3384-3387.2005 PubMed DOI PMC

Lee S. Y., Park J. H., Jang S. H., Nielsen L. K., Kim J., Jung K. S. (2008). Fermentative butanol production by clostridia. Biotechnol. Bioeng. 101 209–228. 10.1002/bit.22003 PubMed DOI

Lepage C., Fayolle F., Hermann M., Vandecasteele J. P. (1987). Changes in membrane lipid composition of Clostridium acetobutylicum during acetone-butanol fermentation: effects of solvents, growth temperature and pH. Microbiology 133 103–110. 10.1099/00221287-133-1-103 DOI

Li H., Durbin R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 1754–1760. 10.1093/bioinformatics/btp324 PubMed DOI PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., et al. (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25 2078–2079. 10.1093/bioinformatics/btp352 PubMed DOI PMC

Liao Z., Zhang Y., Luo S., Suo Y., Zhang S., Wang J. (2017). Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium. J. Biotechnol. 252 1–10. 10.1016/j.jbiotec.2017.04.031 PubMed DOI

Lin Y. L., Blaschek H. P. (1983). Butanol production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth. Appl. Environ. Microbiol. 45 966–973. 10.1128/aem.45.3.966-973.1983 PubMed DOI PMC

Liu J., Qi H., Wang C., Wen J. (2015). Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli. Biotechnol. Biofuels 8:108. PubMed PMC

Liu X. B., Gu Q. Y., Yu X. B. (2013). Repetitive domestication to enhance butanol tolerance and production in Clostridium acetobutylicum through artificial simulation of bio-evolution. Bioresour. Technol. 130 638–643. 10.1016/j.biortech.2012.12.121 PubMed DOI

Long Q., Rabanal F. A., Meng D., Huber C. D., Farlow A., Platzer A., et al. (2013). Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden. Nat. Genet. 45 884–890. 10.1038/ng.2678 PubMed DOI PMC

Lv J., Jiao S., Du R., Zhang R., Zhang Y., Han B. (2016). Proteomic analysis to elucidate degeneration of Clostridium beijerinckii NCIMB 8052 and role of Ca2+ in strain recovery from degeneration. J. Ind. Microbiol. Biotechnol. 43 741–750. 10.1007/s10295-016-1754-6 PubMed DOI

Maddox I. S., Steiner E., Hirsch S., Wessner S., Gutierrez N. A., Gapes J. R., et al. (2000). The cause of “acid crash” and “acidogenic fermentations” during the batch acetone-butanol-ethanol (ABE-) fermentation process. J. Mol. Microbiol. Biotechnol. 2 95–100. PubMed

Mann M. S., Dragovic Z., Schirrmacher G., Lutke-Eversloh T. (2012). Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress. Biotechnol. Lett. 34 1643–1649. 10.1007/s10529-012-0951-2 PubMed DOI

Mao S., Luo Y., Zhang T., Li J., Bao G., Zhu Y., et al. (2010). Proteome reference map and comparative proteomic analysis between a wild type Clostridium acetobutylicum DSM 1731 and its mutant with enhanced butanol tolerance and butanol yield. J. Proteome Res. 9 3046–3061. 10.1021/pr9012078 PubMed DOI

Martínez I., Zhu J., Lin H., Bennett G. N., San K. Y. (2008). Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab. Eng. 10 352–359. 10.1016/j.ymben.2008.09.001 PubMed DOI

Máté de Gérando H., Wasels F., Bisson A., Clement B., Bidard F., Jourdier E., et al. (2018). Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423. BMC Genom. 19:242. 10.1186/s12864-018-4636-7 PubMed DOI PMC

Matta-el-Ammouri G., Janati-Idrissi R., Rambourg J.-M., Petitdemange H., Gay R. (1986). Acetone butanol fermentation by a Clostridium acetobutylicum mutant with high solvent productivity. Biomass 10 109–119. 10.1016/0144-4565(86)90059-4 DOI

McKenna A., Hanna M., Banks E., Sivachenko A., Cibulskis K., Kernytsky A., et al. (2010). The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20 1297–1303. 10.1101/gr.107524.110 PubMed DOI PMC

Mukhopadhyay A. (2015). Tolerance engineering in bacteria for the production of advanced biofuels and chemicals. Trends Microbiol. 23 498–508. 10.1016/j.tim.2015.04.008 PubMed DOI

Nielsen J. B. K., Lampen J. O. (1982). Membrane-bound penicillinases in gram-positive bacteria. J. Biol. Chem. 257 4490–4495. PubMed

Ohta T., Tokishita S. I., Yamagata H. (2001). Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 492 91–97. 10.1016/S1383-5718(01)00155-3 PubMed DOI

Pagès H., Aboyoun P., Gentleman R., DebRoy S. (2020). Biostrings: Efficient Manipulation of Biological Strings. R package version 2.56.0. Available online at: https://bioconductor.org/packages/release/bioc/html/Biostrings.html (accessed June 13, 2020).

Paixao L., Rodrigues L., Couto I., Martins M., Fernandes P., de Carvalho C. C. C. R., et al. (2009). Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. J. Biol. Eng. 3:18. 10.1186/1754-1611-3-18 PubMed DOI PMC

Patakova P., Kolek J., Sedlar K., Koscova P., Branska B., Kupkova K., et al. (2018). Comparative analysis of high butanol tolerance and production in clostridia. Biotechnol. Adv. 36 721–738. 10.1016/j.biotechadv.2017.12.004 PubMed DOI

Patel D., Kosmidis C., Seo S. M., Kaatz G. W. (2010). Ethidium bromide MIC screening for enhanced efflux pump gene expression or efflux activity in Staphylococcus aureus. Antimicrob. Agents Chemother. 54 5070–5073. 10.1128/AAC.01058-10 PubMed DOI PMC

Qureshi N., Blaschek H. P. (2001). Recent advances in ABE fermentation: Hyper-butanol producing Clostridium beijerinckii BA101. J. Ind. Microbiol. Biotechnol. 27 287–291. 10.1038/sj.jim.7000114 PubMed DOI

Reyes L. H., Almario M. P., Kao K. C. (2011). Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli. PLoS One 6:e17678. 10.1371/journal.pone.0017678 PubMed DOI PMC

Sandoval N. R., Venkataramanan K. P., Groth T. S., Papoutsakis E. T. (2015). Whole-genome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth. Biotechnol. Biofuels 8:227. 10.1186/s13068-015-0408-7 PubMed DOI PMC

Sandoval-Espinola W. J., Makwana S. T., Chinn M. S., Thon M. R., Andrea Azcárate-Peril M., Bruno-Bárcena J. M. (2013). Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052. Microbiology 159 2558–2570. 10.1099/mic.0.069534-0 PubMed DOI PMC

Schindler B. D., Kaatz G. W. (2016). Multidrug efflux pumps of Gram-positive bacteria. Drug Resist. Updat. 27 1–13. 10.1016/j.drup.2016.04.003 PubMed DOI

Schwarz K. M., Kuit W., Grimmler C., Ehrenreich A., Kengen S. W. M. (2012). A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum - Cellular behavior in adaptation to n-butanol. J. Biotechnol. 161 366–377. 10.1016/j.jbiotec.2012.03.018 PubMed DOI

Sedlar K., Kolek J., Gruber M., Jureckova K., Branska B., Csaba G., et al. (2019). A transcriptional response of Clostridium beijerinckii NRRL B-598 to a butanol shock. Biotechnol. Biofuels 12:243. 10.1186/s13068-019-1584-7 PubMed DOI PMC

Sedlar K., Kolek J., Provaznik I., Patakova P. (2017). Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598. J. Biotechnol. 244 1–3. 10.1016/j.jbiotec.2017.01.003 PubMed DOI

Sedlar K., Koscova P., Vasylkivska M., Branska B., Kolek J., Kupkova K., et al. (2018). Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq. BMC Genom. 19:415. 10.1186/s12864-018-4805-8 PubMed DOI PMC

Segura A., Molina L., Fillet S., Krell T., Bernal P., Muñoz-Rojas J., et al. (2012). Solvent tolerance in Gram-negative bacteria. Curr. Opin. Biotechnol. 23 415–421. 10.1016/j.copbio.2011.11.015 PubMed DOI

Seo S.-O., Janssen H., Magis A., Wang Y., Lu T., Price N. D., et al. (2017). Genomic, transcriptional, and phenotypic analysis of the glucose derepressed Clostridium beijerinckii mutant exhibiting acid crash phenotype. Biotechnol. J. 12 1700182. 10.1002/biot.201700182 PubMed DOI

Sharma P., Haycocks J. R. J., Middlemiss A. D., Kettles R. A., Sellars L. E., Ricci V., et al. (2017). The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity. Nat. Commun. 8:1444. 10.1038/s41467-017-01405-7 PubMed DOI PMC

Soucaille P., Joliff G., Izard A., Goma G. (1987). Butanol tolerance and autobacteriocin production by Clostridium acetobutylicum. Curr. Microbiol. 14 295–299. 10.1007/BF01568139 DOI

Tanaka Y., Kasahara K., Hirose Y., Morimoto Y., Izawa M., Ochi K. (2017). Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance. J. Biosci. Bioeng. 124 400–407. 10.1016/j.jbiosc.2017.05.003 PubMed DOI

Tomas C. A., Welker N. E., Papoutsakis E. T. (2003). Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program. Appl. Environ. Microbiol. 69 4951–4965. 10.1128/AEM.69.8.4951-4965.2003 PubMed DOI PMC

Vasylkivska M., Jureckova K., Branska B., Sedlar K., Kolek J., Provaznik I., et al. (2019). Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol-producing Clostridium beijerinckii NRRL B-598. PLoS One 14:e0224560. 10.1371/journal.pone.0224560 PubMed DOI PMC

Vasylkivska M., Patakova P. (2020). Role of efflux in enhancing butanol tolerance of bacteria. J. Biotechnol. 320 17–27. 10.1016/j.jbiotec.2020.06.008 PubMed DOI

Větrovský T., Baldrian P. (2013). The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS One 8:e57923. 10.1371/journal.pone.0057923 PubMed DOI PMC

Walker B. J., Abeel T., Shea T., Priest M., Abouelliel A., Sakthikumar S., et al. (2014). Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. 10.1371/journal.pone.0112963 PubMed DOI PMC

Wickham H. (2009). ggplot2. New York, NY: Springer, 10.1007/978-0-387-98141-3 DOI

Xu M., Zhao J., Yu L., Yang S. T. (2017). Comparative genomic analysis of Clostridium acetobutylicum for understanding the mutations contributing to enhanced butanol tolerance and production. J. Biotechnol. 263 36–44. 10.1016/j.jbiotec.2017.10.010 PubMed DOI

Xue C., Zhao J., Lu C., Yang S. T., Bai F., Tang I. C. (2012). High-titer n-butanol production by Clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping. Biotechnol. Bioeng. 109 2746–2756. 10.1002/bit.24563 PubMed DOI

Yang K. M., Woo J. M., Lee S. M., Park J. B. (2013). Improving ethanol tolerance of Saccharomyces cerevisiae by overexpressing an ATP-binding cassette efflux pump. Chem. Eng. Sci. 103 74–78. 10.1016/j.ces.2012.09.015 DOI

Yang S.-T., Zhao J. (2013). Adaptive engineering of Clostridium for increased butanol production. U.S. Patent No US8450093B1. Washington, DC: U.S. Patent and Trademark Office.

Zhang Y., Dong R., Zhang M., Gao H. (2018). Native efflux pumps of Escherichia coli responsible for short and medium chain alcohol. Biochem. Eng. J. 133 149–156. 10.1016/j.bej.2018.02.009 DOI

Zhao K., Liu M., Burgess R. R. (2007). Adaptation in bacterial flagellar and motility systems: from regulon members to ‘foraging’-like behavior in E. coli. Nucleic Acids Res. 35 4441–4452. 10.1093/nar/gkm456 PubMed DOI PMC

Zhao Y., Hindorff L. A., Chuang A., Monroe-Augustus M., Lyristis M., Harrison M. L., et al. (2003). Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 69 2831–2841. 10.1128/AEM.69.5.2831-2841.2003 PubMed DOI PMC