Invented by Lukasz Drewniak, Aleksandra Sklodowska, Monika Radlinska, Martyna Ciezkowska, Uniwersytet Warszawski

The market for bacterial strains and plasmids capable of chemolithotrophic oxidation is rapidly growing as industries and researchers recognize the potential of these organisms in various applications. Chemolithotrophic bacteria are unique as they can obtain energy by oxidizing inorganic compounds, such as minerals or gases, instead of organic matter. This ability makes them valuable in fields like bioremediation, bioleaching, and bioenergy production. One of the main methods of producing bacterial strains capable of chemolithotrophic oxidation is through isolation and screening. Scientists search for these bacteria in diverse environments, such as deep-sea hydrothermal vents, hot springs, or contaminated sites. Once a potential strain is identified, it undergoes rigorous testing to confirm its chemolithotrophic capabilities. This process involves analyzing the bacteria’s metabolic pathways, growth rates, and tolerance to different environmental conditions. Another method of producing these bacterial strains is through genetic engineering. Researchers can modify existing bacteria or introduce genes from other organisms to enhance their chemolithotrophic capabilities. This approach allows for the customization of bacterial strains to suit specific applications. For example, genes encoding enzymes involved in chemolithotrophic oxidation pathways can be inserted into bacteria to increase their efficiency or broaden their substrate range. Plasmids, small circular DNA molecules, play a crucial role in the production of bacterial strains with enhanced chemolithotrophic oxidation capabilities. These plasmids can carry genes encoding enzymes or regulatory elements that facilitate the oxidation of inorganic compounds. They can be easily manipulated in the laboratory and transferred between bacteria, making them valuable tools for genetic engineering. Plasmids also allow for the expression of multiple genes simultaneously, enabling the construction of complex metabolic pathways in bacteria. The market for bacterial strains and plasmids capable of chemolithotrophic oxidation is driven by various industries and research sectors. In the field of bioremediation, these organisms can be used to clean up contaminated environments by oxidizing pollutants, such as heavy metals or hydrocarbons. The mining industry also benefits from the use of chemolithotrophic bacteria in bioleaching processes, where they extract valuable metals from low-grade ores. Additionally, these bacteria hold promise in bioenergy production, as they can generate electricity or produce biofuels from inorganic compounds. The market for bacterial strains and plasmids is expected to witness significant growth in the coming years. Advances in genetic engineering techniques, such as CRISPR-Cas9, have made it easier to modify bacterial genomes and engineer complex metabolic pathways. This has opened up new possibilities for the development of highly efficient bacterial strains with enhanced chemolithotrophic oxidation capabilities. Furthermore, the increasing demand for sustainable and environmentally friendly solutions in various industries is driving the adoption of these bacteria. In conclusion, the market for bacterial strains and plasmids capable of chemolithotrophic oxidation is expanding rapidly due to their potential in bioremediation, bioleaching, and bioenergy production. The methods of producing these bacteria involve isolation and screening from diverse environments, as well as genetic engineering techniques. Plasmids play a crucial role in the construction of bacterial strains with enhanced capabilities. With advancements in genetic engineering and growing demand for sustainable solutions, the market for these organisms is expected to continue to grow in the future.

The Uniwersytet Warszawski invention works as follows

The invention also relates to the composition, comprising the novel bacterial strain or plasmid pSinA, and use of these novel strains, as well as a method for generating bacterial strains that are capable of chemolithotrophic oxidation. The invention also pertains to the composition containing the novel bacterial strain or plasmid, and the use thereof, as well the method of bioaugmentation in an arsenic-contaminated environment.

Background for Bacterial strains and plasmids: Methods of producing bacterial strains that are capable of chemolithotrophic oxidation of arsenites, and their uses

Arsenic, which is widely distributed on the Earth’s surface, is found in trace amounts in soils and minerals. Arsenic can also be released into the air and water by natural processes or human activities. Arsenic compounds present in drinking water can be harmful to both human and animal health. Arsenic has the most severe effects in Bangladesh and Western Bengali, India. According to the World Health Organization, over 50 millions people are exposed to drinking water that is contaminated by this toxic element.

Biological removal from contaminated areas appears to be necessary as a complement to many of the traditional chemical methods of remediation. As a result of coagulation and filtration, not only is arsenic removed from the treated environment but also any other element. The current studies on biological systems to remove arsenic, focus mainly on the use the potential of plants and microorganisms (Kostal, Tripathi, 2004).

Effective purification is achieved by removing both the inorganic and organic forms of arsenic, As III and V.” Arsenates are easily and selectively precipitated using strong adsorbents. (Pattanayak, et. al. 2000), but arsenites cannot be treated with selective oxidants. The microbial oxidation (As(III)) is therefore a viable alternative to chemical oxidation. Lievermont et al. Herminiimonas Arsenicoxidans (ULPAs1) bacteria was used to propose a two-step, efficient and low input technology for the removal of arsenic from water. The authors demonstrated that ULPAs1, immobilised onto alginate deposits, could efficiently oxidise 100 mg/L As (III). This strain can be used in technologies to remove arsenic where initial oxidation is required.

The only known bioremediation methods that use arsenite-oxidising bacterium are laboratory studies and ex situ techniques. In situ bioremediation methods are not effective in areas with arsenic contamination. This is because the bacteria introduced to the “new” environment cannot survive. The bacteria introduced into the?new? environment cannot survive under these new conditions. This is due to the fact that there are physicochemical conditions outside of the laboratory, and the competition between the microflora. “The proposed solution for this problem is biostimulation or use of genetically engineered organisms.

Yang et al. The 2010 paper relates to the lab-constructed vector, a derivative of plasmid BBR1MCS-5 that contains genes for both the large and the small subunits in arsenite oxidease. This vector contains a gene that confers resistance to gentamicin. Its use requires the application of selection pressure with gentamicin in concentrations of 60 mg/L. This means that introducing bacteria with such plasmids into the environment can lead to the spread of genes for resistance and instability. Yang et. al. vector The vector of Yang et al. (2010) is used to construct strains that are useful for bioremediation. However, it only works if it’s introduced into strains capable of arsenite-oxidation and only increases the existing process. This vector does NOT cause a new ability to be acquired, which is catalysing As (III).

The proposed use of genetically engineered organisms would introduce foreign genes, such as markers for antibiotic resistance, or genes encoding green fluorescent protein (Gfp), into the natural world, which was unacceptable for social and environmental reasons. It could also lead to the loss of plasmids if there is no selection pressure in the environment for the chosen markers.

The citation or identification of any document within this application does not mean that it is admissible as prior art for the present invention.

It is desirable that the microorganisms that are capable of arsenite-oxidation also demonstrate resistance to other heavy metals present in the environment.

Sinorhizobium sp. The M14 strain has been isolated from microbial mats in a gold mine near Zloty Stok. This strain can grow chemolithoautotrophically using arsenites as the source of energy and can mobilise arsenic from arsenopyrite (Drewniak et al. 2010). Strain M14 contains two megaplasmids, a 109-kbp named pSinA plasmid and a 300-kbp named pSinB. (Drewniak 2009). The partial sequence of plasmid M14 was found in GenBank NCBI under accession number GU990088.1. (The revealed sequence corresponded to nucleotides 2198 to 4849 of SEQ ID No: 1 according the present invention).

The present invention aims to overcome these inconveniences by providing novel bacterial plasmids and methods that enable the introduction of a new plasmid to a bacterial sys-tem, particularly an indigenous sys-tem, to produce stable and improved strains capable of arsenic oxidation and which are characterized furthermore by increased resistance to heavy metals. These strains can be stripped of unwanted marker genes such as anti-biotic resistance genes. “The invention aims to develop novel bacterial strains that are capable of arsenite-oxidation but do not accumulate arsenic. It also aims to develop compositions containing them and their use.

The invention’s essence is based on the unexpected discovery that it is possible for Sinorhizobium species to utilize a natural plasmid, pSinA. The M14 is used to produce stable strains of bacteria capable of arsenite-oxidation and preferably without any unwanted marker genes. It also focuses on developing a method of producing novel strains using this plasmid, or plasmid SinA. It was surprising to find that plasmids pSinA were introduced into strains of bacteria and species other than Sinorhizobium. is fully functional and stably maintained in them and enables such bacteria to chemolithotrophically oxidize arsenites. It was also found that, unlike Sinorhizobium species, the plasmid-containing strains of bacteria did not accumulate arsenic in their cells. The M14 strain and the new strains containing the plasmid did not accumulate arsenic in their cells but allowed it to be processed. This led to biomass that was free of arsenic.

It should be noted that terms such as ‘comprises? or?comprised?” are used in this disclosure, and in particular in the claims and/or paragraphs. The meaning of terms like?comprises?, or?comprised? can be attributed to them in U.S. Patent Law. ?consists essentially? They have the same meaning as U.S. Patent Law, e.g. they allow elements not explicitly recited but exclude elements found in prior art or that impact a fundamental or novel characteristic of the invention.

These and other embodiments can be disclosed or made obvious by the following detailed description.

DEPOSITS

The present invention relates to the novel Agrobacterium tumefaciens strain (D10) deposited on 30 March under the number KKP2039p. The present invention relates to the novel strains Agrobacterium tumefaciens (D10) deposited on 30 March 2012 under the number KKP2039p and Paracoccus alcaliphilus C10 deposited on 30 March 2012. The IAFB Collection of Industrial Microorganisms of Warsaw’s Institute of Agricultural and Food Biotechnology, Poland and their functional derivatives and variations thereof were deposited on 30 March 2012.

The term “variant (derivative),” as used in this invention, is a mutant or strain that is obtained by culturing either the deposited strains or the strains created by the method of the invention. This strain may contain the plasmid shown at SEQ ID No: 1, and it is capable of chemolithotrophic oxidation of arsenate.

Furthermore the invention relates either to the isolated plasmid, pSinA, shown in SEQ NO: 1, or its functional derivative.

The term “derivative of plasmid” is used. “The term ‘derivative of the plasmid’ or ‘functional derivative of plasmid” The plasmids may have a nucleotide code for open reading frames and encode products containing an amino acid or nucleotide that is identical or highly similar to the sequences coding by the original plasmid, e.g. The coding sequences of pSinA or other plasmids have been altered e.g. By substitution, replacement or deletion, it is possible to maintain the functional features of the original plasmid, e.g. pSinA, such as the ability to chemolithotrophically oxidize arsenites and the resistance to arsenates [As(V)] and arsenites [As(III)]. Highly homologous means the sequence is homologous and preferably identical to at least 70% of the sequence, or 80% if you prefer, 90% if you prefer, the most preferred, at least 95%.

The invention relates to novel strains Agrobacterium tumefaciens KKP2039p and Paracoccus sp. KKP2040p that harbour the natural plasmid pSinA from Sinorhizobium sp. The use of pSinA from Sinorhizobium species. M14 alone or its functional derivative, carrying: (i) all the genes necessary for chemolithoautotrophic arsenite oxidation, (ii) heavy metal resistance genes, and (iii) genes coding for the replication-stabilization system (with partitioning-active separation), multimer resolution system, and addiction toxin-antitoxin system providing stable maintenance of the plasmid in bacterial cells, for constructing bacterial strains capable of chemolithotrophic oxidation of arsenites. These strains, or the plasmid they carry, are useful for bioremediation. This includes the direct application of the plasmid in the process to bioaugmentation the microflora in an arsenic contaminated environment. Such strains may also be used to produce other strains capable of chemolithoautotrophic oxidation of arsenites or to improve the strains that already possess such a characteristic. The sequence of plasmid SinA from Sinorhizobium species is shown below. The sequence ID number for M14 is 1. The solution presented allows the creation of strains that are useful in removing arsenic contamination from contaminated environments without using genetic manipulations or introducing common risk genes. Resistance to antibiotics is circulating in the environment. The invention allows the introduction of the plasmid, pSinA, to cells of indigenous strains that are isolated from a given environment. This can be used to create stable strains of arsenite-oxidizing bacteria. The invention also allows the conduct of a method to select and monitor the strains that harbour the pSinA plasmid.

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