Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052
Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spa...
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Published in | Microbiology (Society for General Microbiology) Vol. 159; no. Pt 12; pp. 2558 - 2570 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
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Microbiology Society
01.12.2013
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Abstract | Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(-1) of butanol and 26.3 g l(-1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(-1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation. |
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AbstractList | Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on
Clostridium beijerinckii
NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis
d
-glucose and
d
-fructose and only
d
-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l
−1
of butanol and 26.3 g l
−1
of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g
−1
, respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in
d
-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase
cbs
_4400 and the solute binding protein
cbs
_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the
d
-fructose phosphotransferase transport system and delayed sporulation. Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(-1) of butanol and 26.3 g l(-1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(-1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation. |
Author | Chinn, Mari S Thon, Michael R Sandoval-Espinola, Walter J Azcárate-Peril, M Andrea Makwana, Satya T Bruno-Bárcena, José M |
Author_xml | – sequence: 1 givenname: Walter J surname: Sandoval-Espinola fullname: Sandoval-Espinola, Walter J organization: Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA – sequence: 2 givenname: Satya T surname: Makwana fullname: Makwana, Satya T organization: Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA – sequence: 3 givenname: Mari S surname: Chinn fullname: Chinn, Mari S organization: Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695-7615, USA – sequence: 4 givenname: Michael R surname: Thon fullname: Thon, Michael R organization: Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Departamento de Microbiología y Genética, Universidad de Salamanca, Calle Del Duero 12, Villamayor 37185, Spain – sequence: 5 givenname: M Andrea surname: Azcárate-Peril fullname: Azcárate-Peril, M Andrea organization: Department of Cell Biology and Physiology, School of Medicine and Microbiome Core Facility, Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC 27599-7545, USA – sequence: 6 givenname: José M surname: Bruno-Bárcena fullname: Bruno-Bárcena, José M organization: Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA |
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Snippet | Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome... |
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SubjectTerms | Butanols - metabolism Butanols - toxicity Carbon - metabolism Clostridium beijerinckii - drug effects Clostridium beijerinckii - genetics Clostridium beijerinckii - growth & development Clostridium beijerinckii - metabolism Culture Media - chemistry DNA, Bacterial - chemistry DNA, Bacterial - genetics Fructose - metabolism Genes and Genomes Genome, Bacterial Glucose - metabolism Growth Inhibitors - toxicity Metabolic Networks and Pathways - genetics Molecular Sequence Data Polymorphism, Genetic Sequence Analysis, DNA Solvents - metabolism Solvents - toxicity |
Title | Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052 |
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