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 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.
Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA.
Walter J
WJ
Sandoval-Espinola
Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA.
Satya T
ST
Makwana
Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695-7615, USA.
Mari S
MS
Chinn
Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Departamento de Microbiología y Genética, Universidad de Salamanca, Calle Del Duero 12, Villamayor 37185, Spain.
Michael R
MR
Thon
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.
M Andrea
MA
Azcárate-Peril
Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA.
José M
JM
Bruno-Bárcena
D000440:Butanols / Q000378:metabolism* / Q000633:toxicity*; D002244:Carbon / Q000378:metabolism; D046970:Clostridium beijerinckii / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D005632:Fructose / Q000378:metabolism; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism; D006131:Growth Inhibitors / Q000633:toxicity; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D012997:Solvents / Q000378:metabolism / Q000633:toxicity
D000440:Butanols; D003470:Culture Media; D004269:DNA, Bacterial; D006131:Growth Inhibitors; D012997:Solvents; D005632:Fructose; D002244:Carbon; D005947:Glucose