Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum

Autor:	Anne M. Henstra, Rupert Norman, Florence J. Annan, Sean Dennis Simpson, Klaus Winzer, Michael Köpke, Bakir Al-Sinawi, Nigel P. Minton, Christopher M. Humphreys
Rok vydání:	2019
Předmět:	Clostridium acetobutylicum Operon Auxotrophy Gene Expression Biotin Allele coupled exchange Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound Plasmid Bioenergy and Biofuels Clostridium autoethanogenum Pantothenate Thiamine 030304 developmental biology Clostridium 2. Zero hunger 0303 health sciences biology 030306 microbiology Vitamins General Medicine biology.organism_classification Recombinant Proteins Culture Media Biosynthetic pathway Metabolic Engineering chemistry Biochemistry Desulfotomaculum Genes Bacterial Gas fermentation Clostridium ljungdahlii Metabolic Networks and Pathways Biotechnology
Zdroj:	Applied Microbiology and Biotechnology
ISSN:	1432-0614 0175-7598
Popis:	Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens. Electronic supplementary material The online version of this article (10.1007/s00253-019-09763-6) contains supplementary material, which is available to authorized users.
Databáze:	OpenAIRE
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