Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum

Xu, Tao; Li, Yongchao; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

doi:10.3389/fmicb.2017.01744

Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum

Journal Article · Thu Sep 07 00:00:00 EDT 2017 · Frontiers in Microbiology

DOI:https://doi.org/10.3389/fmicb.2017.01744· OSTI ID:1628159

Xu, Tao ^[1]; Li, Yongchao ^[1]; He, Zhili ^[1]; Van Nostrand, Joy D. ^[1]; Zhou, Jizhong ^[2]

Univ. of Oklahoma, Norman, OK (United States). Inst. for Environmental Genomics and Dept. of Microbiology and Plant Biology
Univ. of Oklahoma, Norman, OK (United States). Inst. for Environmental Genomics and Dept. of Microbiology and Plant Biology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Division; Tsinghua Univ., Beijing (China). School of Environment. Beijing Key Joint Lab. of Environment Simulation and Pollution Control

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibioticfree medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1628159

Journal Information:: Frontiers in Microbiology, Vol. 8; ISSN 1664-302X

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

References (40)

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields Hemme, Christopher L.; Fields, Matthew W.; He, Qiang Applied and Environmental Microbiology, Vol. 77, Issue 22 https://doi.org/10.1128/Aem.05677-11	journal	September 2011
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 152, Issue 5, p. 1173-1183 https://doi.org/10.1016/j.cell.2013.02.022	journal	February 2013
Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation Li, Yongchao; Xu, Tao; Tschaplinski, Timothy J. Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-25	journal	January 2014
Cellulose Catabolism by Clostridium cellulolyticum Growing in Batch Culture on Defined Medium Desvaux, M.; Guedon, E.; Petitdemange, H. Applied and Environmental Microbiology, Vol. 66, Issue 6 https://doi.org/10.1128/AEM.66.6.2461-2470.2000	journal	June 2000
Side effects of chaperone gene co-expression in recombinant protein production Martínez-Alonso, Mónica; García-Fruitós, Elena; Ferrer-Miralles, Neus Microbial Cell Factories, Vol. 9, Issue 1, Article No. 64 https://doi.org/10.1186/1475-2859-9-64	journal	January 2010
Essential genes as antimicrobial targets and cornerstones of synthetic biology Juhas, Mario; Eberl, Leo; Church, George M. Trends in Biotechnology, Vol. 30, Issue 11 https://doi.org/10.1016/j.tibtech.2012.08.002	journal	November 2012
Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs Na, Dokyun; Yoo, Seung Min; Chung, Hannah Nature Biotechnology, Vol. 31, Issue 2 https://doi.org/10.1038/nbt.2461	journal	January 2013
Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824 Lee, S. FEMS Microbiology Letters, Vol. 108, Issue 3 https://doi.org/10.1016/0378-1097(93)90562-G	journal	April 1993
Artificial trans -encoded small non-coding RNAs specifically silence the selected gene expression in bacteria Man, Shuai; Cheng, Rubin; Miao, Cuicui Nucleic Acids Research, Vol. 39, Issue 8 https://doi.org/10.1093/nar/gkr034	journal	February 2011
Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa Kung, Stephanie H.; Retchless, Adam C.; Kwan, Jessica Y. Applied and Environmental Microbiology, Vol. 79, Issue 5 https://doi.org/10.1128/AEM.03525-12	journal	January 2013
Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum Papanek, Beth; Biswas, Ranjita; Rydzak, Thomas Metabolic Engineering, Vol. 32 https://doi.org/10.1016/j.ymben.2015.09.002	journal	November 2015
Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations Li, Yongchao; Tschaplinski, Timothy J.; Engle, Nancy L. Biotechnology for Biofuels, Vol. 5, Issue 1 https://doi.org/10.1186/1754-6834-5-2	journal	January 2012
Consolidated bioprocessing of cellulosic biomass: an update Lynd, Lee R.; van Zyl, Willem H.; McBride, John E. Current Opinion in Biotechnology, Vol. 16, Issue 5, p. 577-583 https://doi.org/10.1016/j.copbio.2005.08.009	journal	October 2005
Construction and characterization ofpta gene-deleted mutant ofClostridium tyrobutyricum for enhanced butyric acid fermentation Zhu, Ying; Liu, Xiaoguang; Yang, Shang-Tian Biotechnology and Bioengineering, Vol. 90, Issue 2 https://doi.org/10.1002/bit.20354	journal	January 2005
Enzymes Are Enriched in Bacterial Essential Genes Gao, Feng; Zhang, Randy Ren PLoS ONE, Vol. 6, Issue 6 https://doi.org/10.1371/journal.pone.0021683	journal	June 2011
Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose Higashide, Wendy; Li, Yongchao; Yang, Yunfeng Applied and Environmental Microbiology, Vol. 77, Issue 8 https://doi.org/10.1128/AEM.02454-10	journal	March 2011
Clostridium cellulolyticum : model organism of mesophilic cellulolytic clostridia Desvaux, Mickaël FEMS Microbiology Reviews, Vol. 29, Issue 4 https://doi.org/10.1016/j.femsre.2004.11.003	journal	September 2005
Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker Heap, John T.; Ehsaan, Muhammad; Cooksley, Clare M. Nucleic Acids Research, Vol. 40, Issue 8 https://doi.org/10.1093/nar/gkr1321	journal	January 2012
Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum: asRNA downregulation of a C. cellulolyticum cellulase Perret, Stéphanie; Maamar, Hédia; Bélaich, Jean-Pierre Molecular Microbiology, Vol. 51, Issue 2 https://doi.org/10.1046/j.1365-2958.2003.03860.x	journal	January 2004
Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production Lee, Jun; Sung, Bong; Kim, Mi Microbial Cell Factories, Vol. 8, Issue 1 https://doi.org/10.1186/1475-2859-8-2	journal	January 2009
The Vienna RNA Websuite Gruber, A. R.; Lorenz, R.; Bernhart, S. H. Nucleic Acids Research, Vol. 36, Issue Web Server https://doi.org/10.1093/nar/gkn188	journal	May 2008
Bacterial Antisense RNAs: How Many Are There, and What Are They Doing? Thomason, Maureen Kiley; Storz, Gisela Annual Review of Genetics, Vol. 44, Issue 1 https://doi.org/10.1146/annurev-genet-102209-163523	journal	December 2010
Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system Bikard, David; Jiang, Wenyan; Samai, Poulami Nucleic Acids Research, Vol. 41, Issue 15 https://doi.org/10.1093/nar/gkt520	journal	June 2013
Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens Cheung, Jackie K.; Awad, Milena M.; McGowan, Sheena PLoS ONE, Vol. 4, Issue 6 https://doi.org/10.1371/journal.pone.0005849	journal	June 2009
Diversity of CRISPR-Cas immune systems and molecular machines Barrangou, Rodolphe Genome Biology, Vol. 16, Issue 1 https://doi.org/10.1186/s13059-015-0816-9	journal	November 2015
Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum Lin, Paul P.; Mi, Luo; Morioka, Amy H. Metabolic Engineering, Vol. 31 https://doi.org/10.1016/j.ymben.2015.07.001	journal	September 2015
Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield Shaw, A. J.; Podkaminer, K. K.; Desai, S. G. Proceedings of the National Academy of Sciences, Vol. 105, Issue 37, p. 13769-13774 https://doi.org/10.1073/pnas.0801266105	journal	September 2008
High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes Argyros, D. Aaron; Tripathi, Shital A.; Barrett, Trisha F. Applied and Environmental Microbiology, Vol. 77, Issue 23, p. 8288-8294 https://doi.org/10.1128/AEM.00646-11	journal	September 2011
Essential genes of a minimal bacterium Glass, J. I.; Assad-Garcia, N.; Alperovich, N. Proceedings of the National Academy of Sciences, Vol. 103, Issue 2 https://doi.org/10.1073/pnas.0510013103	journal	January 2006
A new mathematical model for relative quantification in real-time RT-PCR Pfaffl, M. W. Nucleic Acids Research, Vol. 29, Issue 9 https://doi.org/10.1093/nar/29.9.e45	journal	May 2001
Correction: Quantitative Characteristics of Gene Regulation by Small RNA Levine, Erel; Zhang, Zhongge; Kuhlman, Thomas PLoS Biology, Vol. 6, Issue 1 https://doi.org/10.1371/journal.pbio.0060005	journal	January 2008
Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum Brown, S. D.; Guss, A. M.; Karpinets, T. V. Proceedings of the National Academy of Sciences, Vol. 108, Issue 33 https://doi.org/10.1073/pnas.1102444108	journal	August 2011
Current and future prospects for CRISPR-based tools in bacteria: CRISPR-Based Tools in Bacteria Luo, Michelle L.; Leenay, Ryan T.; Beisel, Chase L. Biotechnology and Bioengineering, Vol. 113, Issue 5 https://doi.org/10.1002/bit.25851	journal	October 2015
Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase Xu, Tao; Li, Yongchao; Shi, Zhou Applied and Environmental Microbiology, Vol. 81, Issue 13 https://doi.org/10.1128/AEM.00873-15	journal	April 2015
Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum Desai, Ruchir P.; Papoutsakis, Eleftherios T. Applied and Environmental Microbiology, Vol. 65, Issue 3 https://doi.org/10.1128/AEM.65.3.936-945.1999	journal	March 1999
Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in Staphylococcus aureus Infection Ji, Yinduo; Marra, Andrea; Rosenberg, Martin Journal of Bacteriology, Vol. 181, Issue 21 https://doi.org/10.1128/JB.181.21.6585-6590.1999	journal	November 1999
Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum Andersch, Wolfram; Bahl, Hubert; Gottschalk, Gerhard European Journal of Applied Microbiology and Biotechnology, Vol. 18, Issue 6 https://doi.org/10.1007/bf00504740	journal	November 1983
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 184, Issue 3 https://doi.org/10.1016/j.cell.2021.01.019	journal	February 2021
Improvement of Cellulolytic Properties of Clostridium cellulolyticum by Metabolic Engineering Guedon, E.; Desvaux, M.; Petitdemange, H. Applied and Environmental Microbiology, Vol. 68, Issue 1 https://doi.org/10.1128/aem.68.1.53-58.2002	journal	January 2002
Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans Wang, Bing; Kuramitsu, Howard K. Infection and Immunity, Vol. 73, Issue 6 https://doi.org/10.1128/iai.73.6.3568-3576.2005	journal	June 2005

Cited By (3)

Metabolic engineering strategies for consolidated production of lactic acid from lignocellulosic biomass Mazzoli, Roberto Biotechnology and Applied Biochemistry, Vol. 67, Issue 1 https://doi.org/10.1002/bab.1869	journal	January 2020
DNA targeting by Clostridium cellulolyticum CRISPR–Cas9 Type II-C system Fedorova, Iana; Arseniev, Anatolii; Selkova, Polina Nucleic Acids Research, Vol. 48, Issue 4 https://doi.org/10.1093/nar/gkz1225	journal	January 2020
CRISPR Genome Editing Systems in the Genus Clostridium : a Timely Advancement McAllister, Kathleen N.; Sorg, Joseph A. Journal of Bacteriology, Vol. 201, Issue 16 https://doi.org/10.1128/jb.00219-19	journal	May 2019

Figures / Tables (1)

Table S1(p. 1)

Similar Records

Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

Journal Article · Sun Jan 01 00:00:00 EST 2012 · Biotechnology for Biofuels · OSTI ID:1628159

Li, Yongchao; Tschaplinski, Timothy J; Engle, Nancy L; +6 more

Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation

Journal Article · Wed Jan 01 00:00:00 EST 2014 · Biotechnology for Biofuels · OSTI ID:1628159

Li, Yongchao; Xu, Tao; Tschaplinski, Timothy J; +4 more

A multi-purpose toolkit to enable advanced genome engineering in plants

Journal Article · Thu May 18 00:00:00 EDT 2017 · The Plant Cell · OSTI ID:1628159

Cermak, Tomas; Curtin, Shaun J.; Gil-Humanes, Javier; +8 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Microbiology
essential genes
genome editing
gene repression
metabolic engineering
consolidated bioprocessing
Clostridium cellulolyticum