IDENTIFICATION AND CHARACTERIZATION OF

BIOLOGICAL ACTIVE MOLECULES- BioActive

A1.

ArthroBiotech
Developing an Arthrobacter nicotinovorans biotechnology for neuro-pharmaceuticals production
PN-III-P1-1.1-TE-2016-0367

Research project financed by the Ministry of Education and Scientific Research - Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI), contract no. 105/02.05.2018
Funding amount - 207995 lei (aprox. 44 300 euros);
Funding period - 16 months (01.01.2019 - 30.04.2020).

Project summary:

6-hidroxy-nicotine (6HNic), a naturally occurring metabolite of Arthrobacter nicotinovorans was shown to bind the nicotinic acetylcholine receptors and to modulate their function. Experimental tests using laboratory rats have shown the ability of 6HNic to cope with the acetylcholine depletion and to restore normal brain functions.
6HNic has been thereby identified as a high impact bio-pharmaceutical with application in the therapy of
neurodegenerative disorders associated with low acetylcholine levels.
The scope of the project is to develop a technology for the production of the neuroprotective agent 6HNic in an industrial-like environment and to test it at a pilot scale. For this, a genetically engineered Arthrobacter strain has been created. The mutant strain has been accommodated to a bio-reactor and the conditions for optimum 6HNic production are to be evaluated. The final goal is to formulate a microbial based biotechnology for the production of 6HNic ready to be transferred to the industrial environment.

Project members:

Romania
Marius Mihăşan, PhD	Project coordinator, Biochemistry, Molec. Biol.	More details...
Marius Ştefan, PostDoc	Microbiology, Immunology	More details...
Lucian Hritcu, researcher	Neurosciences, Behaviour	More details...
Researcher -vacant	Neurosciences, Behaviour

PhDs and students
Cornelia Babii	Microbiology
Boiangiu Răzvan	Molecular Biology, Biochemistry and Behavior

Researcher position on offer - details to fallow.

Project's objectives and activities:

General objective - Establish the optimum conditions for growing the Arthrobacter strains in the bioreactor for improved conversion of nicotine to 6-hidroxy-nicotine.

Activities in 2019:

A.1.1 - Evaluation of agitation and aeration levels on the accumulation of 6HNic.

A.1.2 - Studying the relation between pH and the accumulation of 6HNic into the growth medium.

A.1.3 - Establish the influence of nicotine concentration on the growth rate and accumulation of 6HNic.

Activities in 2020:

A.2.1 - The influence of some inhibitors for metabolic eflux pumps on the accumulation of of 6HNic in the growth medium.

A.2.2 - The impact of C and N sources on the nicotine conversion to 6HNic.

The complete project plan in Romanian is available as a pdf file.

List of publications supported by the project:
Published:

R. Brandsch, M. Mihasan A soil bacterial catabolic pathway on the move: Transfer of nicotine catabolic genes between Arthrobacter genus megaplasmids and invasion by mobile elements. (2020) J. Biosci. 45, 1–12. [Free FullText]
R. S. Boiangiu, M. Mihasan, D. L. Gorgan, B. A. Stache, B. A. Petre, L. Hritcu Cotinine and 6-Hydroxy-L-Nicotine Reverses Memory Deficits and Reduces Oxidative Stress in Aβ25-35-Induced Rat Model of Alzheimer’s Disease. (2020) Antioxidants. 9, 768. [Free FullText]
E. J. Dupree, M. Jayathirtha, H. Yorkey, M. Mihasan, B. A. Petre, C. C. Darie A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field. (2020) Proteomes. 8, 14 [Free FullText].

Submitted or under review

Mihasan, M.; Babii, C.; Aslebagh, R.; Channaveerappa, D.; Dupree, E. & Darie, C. C., Time-dependent analysis of Paenarthrobacter nicotinovorans pAO1 nicotine-related proteome, Submitted to Proteomics on 23.10.2019, pmic.201900356, Currently status: Article Received.

Conferences and posters - full list as pdf file is available here and here

Scientific report in brief:

At the end of 2019

The impact of the agitation and aeration levels, as well as pH and nicotine concentration in the medium on the accumulation of 6HNic was evaluated. We have established that the optimum agitation level is 5 Hz and that the pH and the pO2 are good indicators that can be used to indirectly monitor the the accumulation of 6HNic in the medium. Specific data on the best conditions to be used are subject of a patent applications and can not be disclosed. The full scientific report at the end of 2019 is available in Romanian as a pdf file.

Growing P. nicotinovorans in a fermenter

At inoculation 10 hours after inoculation 72 hours after inoculation

At the end of the project (October 2020)

Fermentor levels experiments continued in 2020 with tests involving the effect of nigericin, a ionofore known to inhibit the eflux pumps exporting metabolites resulting from the nicotine catabolism in Arthrobacter. Aldough the oxigen consumption and the pH of the medium during the growth of P. nicotinovorans on nicotine containing media are influenced by this inhibitor, no clear influence was observed in terms of nicotine consumption or 6HNic accumulation rate.

Moreover, as we have concluded that just trickling the growth conditions is not enough to improve the 6HNic yield, we decided that a better solution would be to use CRISPR to inactivate the downstream metabolic pathway. The plasmid pJYS3_ΔcrtYf from Corynebacterium harboring the CRISPR-Cpf1 system was acquired and electroporated into Paenarthrobacter nicotinovorans (former Arthrobacter nicotinovorans) competent cells. No colonies harboring the plasmid were obtained, indicating that pJYS3_ΔcrtYf can not replicate or is not compatible with P. nicotinovorans. In order to clear this out, we decided to clone the CRISPR-Cpf1 genes into a plasmid known to replicate in this strain, namely pART2. A fragment of 4288 bp containing the CRISPR-Cpf1(namely placM, FnCpf1, rRnBTer, J17spac, crRNA) was amplified by PCR, digested with two restriction enzymes and ligated into a linearized pART2 vector. After ligation, the mixture was used to transform E. coli XL1 Blue competent cells and 32 colonies were obtained. We are currently assessing the colonies and transform P. nicotinovorans competent cells with the recombinant plasmid. The full scientific report at the end of the project pdf file.

BIOLOGY FACULTY ALEXANDRU IOAN CUZA UNIVERSITY of IAŞI, Romania