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Team: IIT-Madras/ADaPtat1on

Description

ADaPtat1on

Motivations:

Acinetobacter baylyi ADP1 has catabolic pathways utilised in degrading aromatic compounds. These pathways can be re-engineered to produce biofuels from lignin monomers which are abundant in plant biomass. It can also be useful in producing 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 other organisms. Some work has been done with regard to degrading aromatic compounds using Pseudomonas as the chassis but this has its limitations. Acinetobacter baylyi ADP1 complements the abilities and applications of Pseudomonas.
Routine model organisms like E. coli and L. lactis are not naturally competent. We have found many other ‘Naturally Competent’ microorganisms like Streptococcus pneumoniae, Neisseria gonorrhoeae, Bacillus subtilis and Haemophilus influenzae. However, most of them are pathogenic and known to harm humans. Thus it is not possible work with them in a Biosafety level 1 laboratory.

Background:

Literature studies indicate that Acinetobacter baylyi ADP1 is a good chassis for our project because of its ability to degrade aromatic compounds and its naturally competency. It is non-pathogenic and belongs to risk group 1^[1]. Recent work has been done to produce wax ester in Acinetobacter baylyi ADP1 ^[2]. Strains of Acinetobacter baylyi have been engineered that can utilize Gluconate and Glucose more efficiently than existing strains. ^[3]. However, a major shortcoming of this organism is that not many tools are available for genetic engineering. For example, typically only T5 and T7 are the 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 to use online tool called CUTE (codon usage table easy) that can generate a 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 at cute.chassidex.org
Using this tool, we generated a 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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