Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
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-prod...
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doaj-f6efaa531dbe48bab741be31dd8b5bd52020-11-24T22:56:06ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2017-09-01810.3389/fmicb.2017.01744285997Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticumTao Xu0Yongchao Li1Zhili He2Joy D. Van Nostrand3Jizhong Zhou4Jizhong Zhou5Jizhong Zhou6Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, NormanOK, United StatesInstitute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, NormanOK, United StatesInstitute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, NormanOK, United StatesInstitute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, NormanOK, United StatesInstitute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, NormanOK, United StatesEarth Sciences Division, Lawrence Berkeley National Laboratory, BerkeleyCA, United StatesState Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua UniversityBeijing, ChinaEssential 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 antibiotic-free 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.http://journal.frontiersin.org/article/10.3389/fmicb.2017.01744/fullessential genesgenome editinggene repressionmetabolic engineeringconsolidated bioprocessingClostridium cellulolyticum |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Tao Xu Yongchao Li Zhili He Joy D. Van Nostrand Jizhong Zhou Jizhong Zhou Jizhong Zhou |
spellingShingle |
Tao Xu Yongchao Li Zhili He Joy D. Van Nostrand Jizhong Zhou Jizhong Zhou Jizhong Zhou Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum Frontiers in Microbiology essential genes genome editing gene repression metabolic engineering consolidated bioprocessing Clostridium cellulolyticum |
author_facet |
Tao Xu Yongchao Li Zhili He Joy D. Van Nostrand Jizhong Zhou Jizhong Zhou Jizhong Zhou |
author_sort |
Tao Xu |
title |
Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum |
title_short |
Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum |
title_full |
Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum |
title_fullStr |
Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum |
title_full_unstemmed |
Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum |
title_sort |
cas9 nickase-assisted rna repression enables stable and efficient manipulation of essential metabolic genes in clostridium cellulolyticum |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2017-09-01 |
description |
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 antibiotic-free 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. |
topic |
essential genes genome editing gene repression metabolic engineering consolidated bioprocessing Clostridium cellulolyticum |
url |
http://journal.frontiersin.org/article/10.3389/fmicb.2017.01744/full |
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