Abstract
The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype.
Highlights
Background strain No requiredSpecific issuesOff-target insertions can occur; Requires sensitivity to thiamphenicol/erythromycin or spectinomycinIntegration of genes at pyrE locus using Allele Coupled Exchange (ACE)Mariner transposon Reverse Forward Yes PotentiallyIn-frame deletion/point mutations N/A No
Tolerance of growth in the presence of 0.5% and 1% butanol of the 11 highest butanol-producing isolates were measured alongside a C. beijerinckii National Collection of Industrial and Marine Bacteria (NCIMB) 8052 control, with 3 of the isolates showing an inability to grow in broth containing 0.5% butanol (Figure S1, Supporting Information)
Strain 59B, which was isolated from garden soil by screening with media containing starch, showed good butanol production characteristics, sensitivity to a range of antibiotics and a level of butanol tolerance comparable to C. beijerinckii NCIMB 8052 (Figure S1 and Table S4, Supporting Information)
Summary
Clostridium spp. were cultivated at 37 C in an anaerobic cabinet (Don Whitely, UK) using CBM,[21] 2xYTG (16 g LÀ1 tryptone, 10 g LÀ1 yeast extract, 5 g LÀ1 NaCl, 5 g LÀ1 glucose) or CGM media,[22] supplemented with antibiotics when required. E. coli strains TOP10 and DH10B (Thermo, UK) were grown at either 30 C or 37 C using LB media, supplemented with antibiotics where necessary. For growth of pyrEÀ mutants, media were supplemented with 50 μg mLÀ1 uracil. After 24 hr incubation, growth was sub-cultured onto CBM xylose agar plates. Colonies were sub-cultured twice on CBM xylose agar to obtain pure colonies. Cultures that grew were checked for growth on 6% glucose (w/v) CBM agar plates. Products of metabolism were quantified using a Thermo Focus GC as described previously,[23] with the exception that 5 mL 10 M H2SO4 was added for acidification, propyl-propionate was used for extraction of solvents and 50 mM valerate was added as an internal standard
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