Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum

Annan, Florence J.; Al-Sinawi, Bakir; Humphreys, Christopher M.; Norman, Rupert; Winzer; K�pke, Michael; Simpson, Sean D.; Minton, Nigel P.; Henstra, Anne M.

Abstract

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.

Citation

Annan, F. J., Al-Sinawi, B., Humphreys, C. M., Norman, R., Winzer, Köpke, M., Simpson, S. D., Minton, N. P., & Henstra, A. M. (2019). Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum. Applied Microbiology and Biotechnology, 103(11), 4633–4648. https://doi.org/10.1007/s00253-019-09763-6

• Journal Article Type: Article
• Acceptance Date: Mar 12, 2019
• Online Publication Date: Apr 10, 2019
• Publication Date: 2019-06
• Deposit Date: Apr 2, 2019
• Publicly Available Date: Apr 2, 2019
• Journal: Applied Microbiology and Biotechnology
• Print ISSN: 0175-7598
• Electronic ISSN: 1432-0614
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 103
• Issue: 11
• Pages: 4633–4648
• DOI: https://doi.org/10.1007/s00253-019-09763-6
• Keywords: Biotechnology; Applied Microbiology and Biotechnology; General Medicine
• Public URL: https://nottingham-repository.worktribe.com/output/1729914
• Publisher URL: https://link.springer.com/article/10.1007/s00253-019-09763-6
• Additional Information: Received: 26 September 2018; Revised: 19 February 2019; Accepted: 12 March 2019; First Online: 10 April 2019; : ; : FJA declares that she has no conflict of interest.BAS declares that he has no conflict of interest.CMH declares that he has no conflict of interest.RN declares that he has no conflict of interest.KW declares that he has no conflict of interest.KM is employed by LanzaTech. LanzaTech has commercial interest in gas fermentation.SDS is employed by LanzaTech. LanzaTech has commercial interest in gas fermentation.NPM declares that he has no conflict of interest.AMH declares that he has no conflict of interest.; : This article does not contain any studies with human participants or animals performed by any of the authors.
• Contract Date: Apr 2, 2019