Characterization of acidity resistance in Pseudomonas putida using synthetic circuits

• Grant number: 19/18783-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2019
• End date: October 31, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: María Eugenia Guazzaroni
• Grantee: Caroline Moncaio Moda
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 15/04309-1 - Novel approaches to improve functional screening of biocatalysts in metagenomic libraries, AP.JP

Abstract

Microbial fermentation has been used for the benefit of society even before the very discovery of microorganisms, as for example in the of cheese and bread production, which are millennial processes (LEGRAS et al., 2007). In the context of organic acids production, one of the major problems is the medium acidification. For example, considering that pKa for most organic acids is in the range of 3 to 5, the pH of a batch should decrease to nearly 2.0 when an acid production of about 50 g/l is present. This is usually circumvented by adding large amounts of base to the culture medium to maintain the pH at tolerable levels for the host (CHEN; NIELSEN, 2016). However, choosing an acid-tolerant lineage as a host for the development of pH-lowering processes, such as the production of organic acids, is an excellent alternative to neutralizing the medium, precisely because it minimizes the consumption of bases for pH control and thus reducing both the environmental impact and the downstream processing cost (CHEN; NIELSEN, 2016). In our research group, an ongoing project aims to combine several translation levels of three acid resistance-related metagenomic genes, to obtain an optimized cluster of genes that confer low pH resistance in bacteria. In the present proposal, we intend to investigate the effect of these synthetic circuits on Pseudomonas putida, a bacterium of high industrial relevance. Thus, some of these gene clusters will be previously selected in Escherichia coli, seeking to have different resistance levels to be transferred to P. putida. Afterwards, growth experiments at different pH will be performed and the kinetic parameters will be calculated in order to identify the circuits that efficiently confer acidity resistance in this bacterium.