Metabolic engineering of d-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid

Xiao, Han; Li, Zhilin; Jiang, Yu; Yang, Yunliu; Jiang, Weihong; Gu, Yang; Yang, Sheng

doi:10.1016/j.ymben.2012.05.003

Cited by 100 publications

(85 citation statements)

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“…2). In contrast, previous studies exhibited typical sequential utilization, consuming glucose first and then xylose (9,14,24,36,37,38). For instance, when providing a mixture of glucose and xylose (30 g/liter each), strain G117 consumed all of the glucose in the first 48 h, and xylose utilization was initiated only after the glucose was completely consumed (after 48 h), resulting in a total of 6.5 g/liter butanol after 100 h of fermentation.…”

Section: Resultsmentioning

confidence: 90%

“…Therefore, among all of the reported solventogenic microorganisms, simultaneous utilization of glucose and xylose enables Clostridium sp. strain BOH3 to produce the largest amount of butanol (12.4 g/liter) from glucose and xylose mixtures under similar growth conditions (14,37,38). During the fermentation process, strain BOH3 coproduces riboflavin, which adds to the economic value of the process.…”

Section: Fig 5 Comparison Of Relative Xylose Isomerase Transcription mentioning

confidence: 99%

“…Good examples are the solventogenic Clostridium species (e.g., C. acetobutylicum, C. beijerinckii, and C. pasteurianum), which are among the few microorganisms able to ferment both pentose and hexose sugars (10). However, the solvents, including butanol, are still produced mainly from glucose, while small amounts are produced from xylose, as observed in cultures of C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052 when fermenting a mixture of glucose and xylose (14,17,38). Gu et al have shown that C. acetobutylicum ATCC 824 utilized 86% of glucose within 40 h, whereas only 6% of xylose was consumed even after an elongated incubation time (14).…”

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confidence: 99%

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Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

Xin

2014

Appl Environ Microbiol

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Cellulose and hemicellulose constitute the major components in sustainable feedstocks which could be used as substrates for biofuel generation. However, following hydrolysis to monomer sugars, the solventogenic Clostridium will preferentially consume glucose due to transcriptional repression of xylose utilization genes. This is one of the major barriers in optimizing lignocellulosic hydrolysates that produce butanol. Unlike studies on existing bacteria, this study demonstrates that newly reported Clostridium sp. strain BOH3 is capable of fermenting 60 g/liter of xylose to 14.9 g/liter butanol, which is similar to the 14.5 g/liter butanol produced from 60 g/liter of glucose. More importantly, strain BOH3 consumes glucose and xylose simultaneously, which is shown by its capability for generating 11.7 g/liter butanol from a horticultural waste cellulosic hydrolysate containing 39.8 g/liter glucose and 20.5 g/liter xylose, as well as producing 11.9 g/liter butanol from another horticultural waste hemicellulosic hydrolysate containing 58.3 g/liter xylose and 5.9 g/liter glucose. The high-xylose-utilization capability of strain BOH3 is attributed to its high xylose-isomerase (0.97 U/mg protein) and xylulokinase (1.16 U/mg protein) activities compared to the lowxylose-utilizing solventogenic strains, such as Clostridium sp. strain G117. Interestingly, strain BOH3 was also found to produce riboflavin at 110.5 mg/liter from xylose and 76.8 mg/liter from glucose during the fermentation process. In summary, Clostridium sp. strain BOH3 is an attractive candidate for application in efficiently converting lignocellulosic hydrolysates to biofuels and other value-added products, such as riboflavin.

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Section: Resultsmentioning

confidence: 90%

Section: Fig 5 Comparison Of Relative Xylose Isomerase Transcription mentioning

confidence: 99%

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confidence: 99%

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Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

Xin

2014

Appl Environ Microbiol

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“…This is in stark contrast to C. acetobutylicum and C. beijerinckii, for which transformation methods have existed for more than 20 years (25,26). Building on these methods, rational metabolic engineering of these two species has enabled significant progress in expanding substrate utilization, improving oxygen tolerance, eliminating sporulation, increasing solvent titers/productivities, and enabling the generation of valuable end products beyond ABE (27)(28)(29)(30)(31). 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.…”

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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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“…Among solventogenic microbes, the Gram-positive anaerobe Clostridium beijerinckii NCIMB 8052 is capable of utilizing pentose and hexose sugars to produce acetone, butanol, and ethanol (ABE) without the "glucose repression" effect, which gives it an attractive advantage over another well-known solventogenic organism, Clostridium acetobutylicum (10). Recently, a newly discovered isolate, Clostridium beijerinckii G117, was distinguished as generating acetone and butanol (AB) but negligible ethanol from fermentation of glucose (1a).…”

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Draft Genome Sequence of Butanol-Acetone-Producing Clostridium beijerinckii Strain G117

Yang

et al. 2012

J Bacteriol

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A recently discovered wild-type strain, Clostridium beijerinckii G117, is unique in producing butanol and acetone but negligible amounts of ethanol, unlike previously identified acetone-butanol-ethanol (ABE)-generating microbes. Here we report the draft genome sequence of strain G117 (5,806,675 bp; GC content, 29.7%) and the novel findings obtained from its genome annotations.T he solventogenic Clostridium species have attracted renewed attention in the scientific field during the past few years due to the search for sustainable sources of energy (5). Among solventogenic microbes, the Gram-positive anaerobe Clostridium beijerinckii NCIMB 8052 is capable of utilizing pentose and hexose sugars to produce acetone, butanol, and ethanol (ABE) without the "glucose repression" effect, which gives it an attractive advantage over another well-known solventogenic organism, Clostridium acetobutylicum (10). Recently, a newly discovered isolate, Clostridium beijerinckii G117, was distinguished as generating acetone and butanol (AB) but negligible ethanol from fermentation of glucose (1a). The 16S rRNA gene of strain G117 shows 99% identity to that of C. beijerinckii NCIMB 8052. The genome sequencing data for C. beijerinckii NCIMB 8052 will facilitate our understanding of the "omics" (e.g., genomics, transcriptomics, and metabolomics) of solventogenic microbes (5, 9). To further our understanding of solventogenesis in this organism, we present a draft genome sequence of C. beijerinckii G117.The genome of C. beijerinckii G117 was sequenced by a wholegenome shotgun strategy using a high-throughput Illumina HiSeq 2000 at the Beijing Genomics Institute (BGI, Shenzhen, China). A total of 1,766,892 reads with a 296-bp insert size, counting up to 523 Mbp, were obtained, providing 90-fold coverage. Genome sequences were assembled by using the SOAPdenovo program (version 1.05) (6), resulting in 178 contigs with an N 50 of 95,461 bp and a total length of 5,806,675 bp for the whole genome. These contigs were assembled into 89 scaffolds with a maximum length of 271,884 bp. Putative protein coding sequences were identified by Glimmer (version 3.02) (2) and analyzed by BLASTP. The functions of predicted protein-coding genes were annotated through comparisons with Kyoto Encyclopedia of Genes and Genomes (KEGG) (3), Clusters of Orthologous Groups (COG) (8), and Swiss-Prot (1) databases. tRNA and rRNA were annotated with tRNAscan-SE 1.21 (7) and rRNAmmer 1.2 (4), respectively.The draft genome includes 5,806,675 bp with a low GC content of 29.7%, and the average nucleotide identity between strain G117 and NCIMB 8052 was determined to be 97.39%. The genome contains 5,262 predicted genes, 5,111 protein-coding sequences (CDSs), 54 tRNA genes, and 20 rRNAs (including 11 5S rRNAs, 3 16S rRNAs, and 6 23S rRNAs). Among the predicted CDSs, 2,687, 2,937, and 2,102 proteins were functionally annotated in the COG, KEGG, and Swiss-Prot databases, respectively. The annotations indicate that a total of 198 CDSs are involved in energy production and conversi...

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Metabolic engineering of d-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid

Cited by 100 publications

References 57 publications

Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

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

Draft Genome Sequence of Butanol-Acetone-Producing Clostridium beijerinckii Strain G117

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