New host-vector system in solvent-producing Clostridium saccharoperbutylacetonicum strain N1-4

Nakayama, Shunichi; Irie, Ryouichi; Kosaka, Tomoyuki; Matsuura, Kentaro; Yoshino, Sadazo; Furukawa, Koichi

doi:10.2323/jgam.53.53

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…The C. beijerinckii filamentous phage CAK1's origin of replication has been used in C. beijerinckii strains. 187 Additionally, the development of a replicon specific for C. saccharoperbutylacetonicum N1−4 was reported in 2007, 188 this replicon is identical to the origin of the endogenous plasmid from C. saccharoperbutylacetonicum N1−504. 15 A thermosensitive origin pWV01ts derived from Lactococcus lactis cremoris 189 has been shown to work in both C. ljungdahlii and C. acetobutylicum.…”

Section: ■ Replicationmentioning

confidence: 98%

“…The C. beijerinckii filamentous phage CAK1’s origin of replication has been used in C. beijerinckii strains . Additionally, the development of a replicon specific for C. saccharoperbutylacetonicum N1–4 was reported in 2007, this replicon is identical to the origin of the endogenous plasmid from C. saccharoperbutylacetonicum N1–504 . A thermosensitive origin pWV01ts derived from Lactococcus lactis cremoris has been shown to work in both C. ljungdahlii and C. acetobutylicum .…”

Section: Replicationmentioning

confidence: 99%

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Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

et al. 2017

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The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

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Section: ■ Replicationmentioning

confidence: 98%

Section: Replicationmentioning

confidence: 99%

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

et al. 2017

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“…In addition, there were rare reports concerning attempts at metabolic engineering of this species. So far, to our best knowledge, there are only two reports related to genetic engineering in this strain; one is about the development of a host-vector system and the expression of an amylase gene in C. saccharoperbutylacetonicum N1-4 (19), and the other is about the downregulation of the hydrogenase gene cluster using the antisense RNA strategy (20). Both reports were published about 10 years ago from the same research group.…”

mentioning

confidence: 99%

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System

Wang

Dong

Wang

et al. 2017

Appl Environ Microbiol

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N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for was evaluated in by targeting and, two essential genes for acetate and butyrate production, respectively. and single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P , we achieved a mutation rate of 75% for deletion without serial subculturing as suggested previously for Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. An efficient CRISPR-Cas9 genome engineering system was developed for N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.

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“…Without an established transformation method, stable host/vector system, and efficient gene disruption strategy, the types of advances made using rational metabolic engineering in C. acetobutylicum and C. beijerinckii are not possible for C. saccharoperbutylacetonicum without cumbersome and tedious screening of traditional mutagenesis libraries. To our knowledge, only one report from 2007 has detailed a transformation method for any C. saccharoperbutylacetonicum strain (strain N1-4 ATCC 13564) (32). Other than this report and a single follow-up study by the same group in 2008 (33), we were unable to find any reports demonstrating transformation methods or heterologous gene expression for any of the C. saccharoperbutylacetonicum strains [which include strain N1-4 ATCC 13564 and its two derivatives, strain N1-4 (HMT) ATCC 27021 and strain N1-504 ATCC 27022 (9)].…”

mentioning

confidence: 99%

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

Herman

Bedi

et al. 2017

Appl Environ Microbiol

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While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 ϫ 10 6 CFU/g DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombinationmediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5-and 11.8-fold increases (respectively) in batch ethanol production.IMPORTANCE This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.KEYWORDS ABE fermentation, Clostridium, biofuel, genetics

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New host-vector system in solvent-producing Clostridium saccharoperbutylacetonicum strain N1-4

Cited by 8 publications

References 25 publications

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

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