2,3-Butanediol (23BD) is a high-value chemical usually produced petrochemically but which can also be synthesized by some bacteria. To date, the best microbial 23BD production rates have been observed using pathogenic bacteria in fermentation systems that depend on sugars as the carbon and energy sources for product synthesis. Here we present evidence of 23BD production by three nonpathogenic acetogenic Clostridium species-Clostridium autoethanogenum, C. ljungdahlii, and C. ragsdalei-using carbon monoxide-containing industrial waste gases or syngas as the sole source of carbon and energy. Through an analysis of the C. ljungdahlii genome, the complete pathway from carbon monoxide to 23BD has been proposed. Homologues of the genes involved in this pathway were also confirmed for the other two species investigated. A gene expression study demonstrates a correlation between mRNA accumulation from 23BD biosynthetic genes and the onset of 23BD production, while a broader expression study of Wood-Ljungdahl pathway genes provides a transcription-level view of one of the oldest existing biochemical pathways.

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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.

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage‐gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4‐amino‐3‐oxo‐guanidinoheptane (ethyl ketone) by an unusual polyketide synthase‐like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC‐ESI‐HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled‐precursor feeding with [guanidino‐15N2] arginine and [1,2‐13C2] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.

Saxitoxins are neurotoxic alkaloids produced by cyanobacteria and dinoflagellates. Due to their antagonism of voltage‐gated sodium channels, they are desirable candidates for drug development as anaesthetics and muscle relaxants. We describe the heterologous expression and biochemical characterization of the first enzyme in the saxitoxin pathway, SxtA, in E. coli. This unusual polyketide synthase‐like enzyme is able to condense multiple acid‐CoA substrates with arginine and could potentially be exploited for the production of novel compounds of biomedical value. More information can be found in the communication by R. Kellmann, B. Neilan et al. Credits: Bloom photo courtesy of Dr. Nick Crosbie, Melbourne Water. Syringe image by Jupiter Images (sourced through Canva Pro).