Christopher M. Humphreys

Bio: Christopher M. Humphreys is an academic researcher from University of Nottingham. The author has contributed to research in topics: Clostridium autoethanogenum & Medicine. The author has an hindex of 7, co-authored 9 publications receiving 211 citations.

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

Abstract: 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. 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. 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.

Syngas Biorefinery and Syngas Utilization

Abstract: Autotrophic acetogenic bacteria are able to capture carbon (CO or CO2) through gas fermentation, allowing them to grow on a spectrum of waste gases from industry (e.g., steel manufacture and oil refining, coal, and natural gas) and to produce ethanol. They can also consume syn(thesis) gas (CO and H2) made from the gasification of renewable/sustainable resources, such as biomass and domestic/agricultural waste. Acetogenic gas fermentation can, therefore, produce ethanol in any geographic region without competing for food or land. The commercialization of the process is now at an advanced stage. The real potential of acetogens, however, resides in their capacity to produce chemicals and fuels other than ethanol. This requires the redesign and implementation of more efficient metabolic pathways, adapting them to high performing manufacturing processes. Respective species, their bioenergetics, the genetic tools developed for their metabolic engineering, culture techniques and fermenter set-ups, as well as the commercialization, are comprehensively described and discussed in this chapter.

Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum.

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.

Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks

Abstract: There is an immediate need to drastically reduce the emissions associated with global fossil fuel consumption in order to limit climate change. However, carbon-based materials, chemicals, and transportation fuels are predominantly made from fossil sources and currently there is no alternative source available to adequately displace them. Gas-fermenting microorganisms that fix carbon dioxide (CO2) and carbon monoxide (CO) can break this dependence as they are capable of converting gaseous carbon to fuels and chemicals. As such, the technology can utilize a wide range of feedstocks including gasified organic matter of any sort (e.g., municipal solid waste, industrial waste, biomass, and agricultural waste residues) or industrial off-gases (e.g., from steel mills or processing plants). Gas fermentation has matured to the point that large-scale production of ethanol from gas has been demonstrated by two companies. This review gives an overview of the gas fermentation process, focusing specifically on anaerobic acetogens. Applications of synthetic biology and coupling gas fermentation to additional processes are discussed in detail. Both of these strategies, demonstrated at bench-scale, have abundant potential to rapidly expand the commercial product spectrum of gas fermentation and further improve efficiencies and yields.

Third-generation biorefineries as the means to produce fuels and chemicals from CO2

Abstract: Concerns regarding petroleum depletion and global climate change caused by greenhouse gas emissions have spurred interest in renewable alternatives to fossil fuels. Third-generation (3G) biorefineries aim to utilize microbial cell factories to convert renewable energies and atmospheric CO2 into fuels and chemicals, and hence represent a route for assessing fuels and chemicals in a carbon-neutral manner. However, to establish processes competitive with the petroleum industry, it is important to clarify/evaluate/identify the most promising CO2 fixation pathways, the most appropriate CO2 utilization models and the necessary productivity levels. Here, we discuss the latest advances in 3G biorefineries. Following an overview of applications of CO2 feedstocks, mainly from flue gas and waste gasification, we review prominent opportunities and barriers in CO2 fixation and energy capture. We then summarize reported CO2-based products and industries, and describe trends and key challenges for future advancement of 3G biorefineries. A shift from sugar-based feedstocks and biomass to the use of atmospheric CO2 for the bioproduction of fuels and chemicals is desirable. This Review describes how microorganisms can be engineered for CO2 fixation and industrial valorization of this key molecule.