Curing the endogenous megaplasmid in Clostridium saccharoperbutylacetonicum N1-4 (HMT) using CRISPR-Cas9 and preliminary investigation of the role of the plasmid for the strain metabolism

Gu, Yanyan; Feng, Jun; Zhang, Zhong-Tian; Wang, Shaohua; Guo, Liang; Wang, Yifen

doi:10.1016/j.fuel.2018.09.030

Cited by 15 publications

(13 citation statements)

References 27 publications

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“…We hypothesized that different flux levels of these precursors within various clostridial strains would make a big difference for the specific type(s) of ester production. Therefore, we selected five strains (from four representative species) including C. tyrobutyricum Δ cat1::adhE1 12 , C. tyrobutyricum Δ cat1::adhE2 12 , C. pasteurianum SD-1 13 , C. saccharoperbutylacetonicum N1-4-C 14 and C. beijerinckii NCIMB 8052 15 to evaluate their capabilities for ester production through metabolic engineering (Table S1). We included both C. tyrobutyricum Δ cat1::adhE1 and Δ cat1::adhE2 here because they produce different levels (and thus ratios) of butanol and ethanol 12,16 .…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, control strains with the empty plasmid (pMTL82151 or pTJ1) also produced noticeable EA, BA and BB. This could be because: 1) the endogenous lipase in clostridia can catalyze ester production 14 ; 2) catP on pMTL82151 encoding a chloramphenicol acetyltransferase (belonging to the same class of enzymes as AATs) has AAT activities 5,24 .…”

Section: Resultsmentioning

confidence: 99%

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Renewable Fatty Acid Ester Production inClostridium

Feng

Zhang

Wang

et al. 2020

Preprint

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44Endproduct toxicity is a key bottleneck for biofuel/biochemical production. Biosynthesis of 45 high-value derivatives that can easily separate would alleviate such toxicity and thus enhance 46 bioproduction efficiency. As a proof of principle, biosynthesis (along with in-line extractive 47 recovery) of fatty acid esters using clostridia was investigated in this study. We hypothesize 48 that solventogenic clostridia are excellent platforms for ester production, because they co-49 produce acyl-CoAs (acetyl-CoA and butyryl-CoA), acids (acetate and butyrate), and alcohols 50 (ethanol and butanol). Through rational screening for host strains and genes (encoding alcohol 51 acyl transferases and lipase), systematic metabolic engineering, and elimination of putative 52 prophages, we obtained strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl 53 butyrate respectively, which were both historically highest levels in microbial hosts. Our 54 principle for engineering microorganisms to produce high-value and easy-recoverable 55 endproducts is highly applicable to other bioprocesses, and could lead to breakthroughs in 56 biofuel/biochemical production and general bioeconomy. 57 58 59 60 61 Keywords: clostridia, biofuels and biochemicals, fatty acid ester, butyl acetate, butyl butyrate, 62 ethyl acetate, CRISPR-Cas9, hydrolysates, prophages 63 64 4 Main 65 Although tremendous efforts have been invested for biofuel and biochemical research, it 66is still challenging to generate robust strains that can produce target products at desirable 67 levels 1 . One key bottleneck is the intrinsic toxicity of endproducts to host cells 2 . Therefore, the 68 production of high-value bioproducts which can be easily recovered from fermentation might 69 be a solution to tackle the bottleneck in bioproduction. Fatty acid esters, or mono-alkyl esters, 70 can be used as valuable fuels such as diesel components or specialty chemicals for food 71 flavoring, cosmetic and pharmaceutical industries 3 . It is projected that the US market demand 72 for fatty acid esters could reach $4.99 billion by 2025 4 . In addition, esters, with fatty acid and 73 alcohol moieties, are generally hydrophobic and can easily separate from fermentation; thus 74 the production of ester can help mitigate endproduct toxicity to host cells and efficient 75 bioproduction can be achieved. 76 Conventionally, esters are produced through Fischer esterification which involves high 77 temperature and inorganic catalysts 5,6 . The reaction consumes a large amount of energy and 78 generates tremendous wastes, and thus is not environmentally friendly 5 . On the other hand, 79 ester production through biological routes is becoming more and more attractive because it is 80 renewable and environmentally benign. There are two primary biological pathways for ester 81 production: one is through esterification of fatty acid and alcohol catalyzed by lipases 7 , and the 82 other is based on condensation of acyl-CoA and alcohol catalyzed by alcohol acyl transfer...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Renewable Fatty Acid Ester Production inClostridium

Feng

Zhang

Wang

et al. 2020

Preprint

Self Cite

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“…The use of genetic engineering techniques contributes to a better understanding of genetic information. Therefore, the strain C. saccharoperbutylacetonicum N1-4C demonstrated better plasmid stability and a slight increase in ABE production, probably due to the improvement in the efficiency of acids produced assimilation during the acidogenic phase [60]. The silencing of the pta and the buk genes, responsible for the expression of phosphotransacetylase and butyrate kinase, respectively, did not eliminate the formation of acetyl or butyrate.…”

Section: Clostridium Saccharoperbutylacetonicummentioning

confidence: 94%

Clostridium sp. as Bio-Catalyst for Fuels and Chemicals Production in a Biorefinery Context

et al. 2019

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Clostridium sp. is a genus of anaerobic bacteria capable of metabolizing several substrates (monoglycerides, diglycerides, glycerol, carbon monoxide, cellulose, and more), into valuable products. Biofuels, such as ethanol and butanol, and several chemicals, such as acetone, 1,3-propanediol, and butyric acid, can be produced by these organisms through fermentation processes. Among the most well-known species, Clostridium carboxidivorans, C. ragsdalei, and C. ljungdahlii can be highlighted for their ability to use gaseous feedstocks (as syngas), obtained from the gasification or pyrolysis of waste material, to produce ethanol and butanol. C. beijerinckii is an important species for the production of isopropanol and butanol, with the advantage of using hydrolysate lignocellulosic material, which is produced in large amounts by first-generation ethanol industries. High yields of 1,3 propanediol by C. butyricum are reported with the use of another by-product from fuel industries, glycerol. In this context, several Clostridium wild species are good candidates to be used as biocatalysts in biochemical or hybrid processes. In this review, literature data showing the technical viability of these processes are presented, evidencing the opportunity to investigate them in a biorefinery context.

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“…The 5 kb fragment proved to be more difficult to integrate and although 35% of colonies tested indicated integration had occurred, all of them showed a smaller fragment of the lambda phage DNA than anticipated which may be due to instability of the relatively large (12.7 kb) plasmid (Gu et al . 2019). In addition a reduction in transformation efficiencies was observed compared with the empty vector as the insert size increased; empty vector = 63.7 cfu/pmol, 1 kb insert = 51.6 cfu/pmol, 3 kb insert = 19.2 cfu/pmol, 5 kb insert = 13.9 cfu/pmol (N.B.…”

Section: Resultsmentioning

confidence: 99%

“…Recently Gu et al used CRISPR-cas9 editing to cure C. saccharoperbutylacetonicum N1-4(HMT) of its endogenous megaplasmid (Gu et al . 2019). The megaplasmid-cured mutant was shown to have improved transformation efficiencies over the wild type, and, to some extent, the deletion mitigated instability of non-endogenous plasmids.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT)

Atmadjaja

Holby

Harding

et al. 2019

FEMS Microbiology Letters

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The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and ‘green’ chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

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Curing the endogenous megaplasmid in Clostridium saccharoperbutylacetonicum N1-4 (HMT) using CRISPR-Cas9 and preliminary investigation of the role of the plasmid for the strain metabolism

Cited by 15 publications

References 27 publications

Renewable Fatty Acid Ester Production inClostridium

Renewable Fatty Acid Ester Production inClostridium

Clostridium sp. as Bio-Catalyst for Fuels and Chemicals Production in a Biorefinery Context

CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT)

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