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Team: IIT-Madras/ADaPtat1on

Description

ADaPtat1on

Motivations:

Acinetobacter baylyi ADP1 possesses catabolic pathways to degrade aromatic compounds. These pathways can be re-engineered to produce biofuels from Lignin monomers which is abundant in plant biomass. It can also be useful to produce bio-surfactants and lubricants such as Wax-Ester (Kannisto et al. 2016, Journal of Industrial Microbiology and Biotechnology). These pathways are not present in many organisms. Some work has been done on degrading aromatic compounds using Pseudomonas as chassis but even it has its own limitations. Acinetobacter baylyi ADP1 complements abilities and applications of Pseudomonas.
For routine model organisms like E. coli and L. lactis, they are not naturally competent and so we need to artificially make them competent. We found many ‘Naturally Competent’ microorganisms. However, most of them are pathogenic and known to cause harm to humans. Hence, it is not possible work with them in Biosafety level 1 lab. For example Streptococcus pneumoniae, Neisseria gonorrhoeae, Bacillus subtilis and Haemophilus influenzae.

Background:

We found from the literature that Acinetobacter baylyi ADP1 to be a good chassis for our project because of its ability to degrade aromatic compounds and it is naturally competent. It is also non-pathogenic and belongs to risk group 1^[1]. Also, recent work has been done to produce wax ester in Acinetobacter baylyi ADP1 ^[2]. Strains of Acinetobacter baylyi have been constructed that can utilize Gluconate and Glucose better than the existing strain ^[3]. However, a big shortcoming of using this organism is that it does not many tools for gene manipulation. For example, usually, T5 and T7 are the only two standard promoters used in engineering this organism.

Project:
Our objective was to create a T5 based synthetic promoter library for Acinetobacter baylyi ADP1. A reporter protein was required to measure the strength of promoter using fluorometry. We had approached GenScript for codon optimized GFP for Acinetobacter baylyi ADP1. They did not have the codon usage table data for Acinetobacter baylyi. .

Hence, we made a free use online tool called CUTE (codon usage table easy) that can generate codon usage table by taking into consideration the protein-coding annotation. This tool can be used for any other organism whose coding regions are annotated. Cute can be found on the cute.chassidex.org
Using this tool, we generated the codon usage table from the protein annotation data of Acinetobacter baylyi ADP1 which is available on the NCBI website. Using this table, we codon optimized GFP (which was codon optimized previously for E. coli).
Following this, we generated a T5 promoter-based library for Acinetobacter baylyi ADP1. Since these promoters are T5 based, they could potentially work in other gram-negative organisms like E. coli strains, Cornybacterium etc.

References:

https://www.dsmz.de/catalogues/details/culture/DSM-24193.html?tx_dsmzresources_pi5%5BreturnPid%5D=304
Suvi Santala, Elena Efimova, Perttu Koskinen, Matti Tapani Karp, and Ville Santala ACS Synthetic Biology 2014 3 (3), 145-151 DOI: 10.1021/sb4000788
Kannisto, Matti et al. “Metabolic Engineering of Acinetobacter Baylyi ADP1 for Improved Growth on Gluconate and Glucose.” Ed. S.-J. Liu. Applied and Environmental Microbiology 80.22 (2014): 7021–7027. PMC. Web. 17 Oct. 2018

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