Hydrogen oxidising bacteria for production of single-cell protein and other food and feed ingredients.

Using hydrogen oxidising bacteria to produce protein and other food and feed ingredients is a form of industrial biotechnology that is gaining traction. The technology fixes carbon dioxide into products without the light requirements of agriculture and biotech that rely on primary producers such as plants and algae while promising higher growth rates, drastically less land, fresh water, and mineral requirements. The significant body of scientific knowledge on hydrogen oxidising bacteria continues to grow and genetic engineering tools are well developed for specific species. The scale-up success of other types of gas- fermentation using carbon monoxide or methane has paved the way for scale-up of a process that uses a mix of hydrogen, oxygen, and carbon dioxide to produce bacteria as a food and feed ingredients in a highly sustainable fashion.

The carbonic anhydrase of Clostridium autoethanogenum represents a new subclass of ß-carbonic anhydrases.

Carbonic anhydrase catalyses the interconversion of carbon dioxide and water to bicarbonate and protons. It was unknown if the industrial-relevant acetogen Clostridium autoethanogenum possesses these enzymes. We identified two putative carbonic anhydrase genes in its genome, one of the ß class and one of the γ class. Carbonic anhydrase activity was found for the purified ß class enzyme, but not the γ class candidate. Functional complementation of an Escherichia coli carbonic anhydrase knock-out mutant showed that the ß class carbonic anhydrase could complement this activity, but not the γ class candidate gene. Phylogenetic analysis showed that the ß class carbonic anhydrase of Clostridium autoethanogenum represents a novel sub-class of ß class carbonic anhydrases that form the F-clade. The members of this clade have the shortest primary structure of any known carbonic anhydrase.

Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium.

BACKGROUND: Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence. RESULTS: We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism's ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions. CONCLUSIONS: We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.