Development of E. coli-Rhodococcus CRISPR/Cas9 shuttle vector targeting ferulic acid accumulating Rhodococcus jostii RHA1 mutants

• Grant number: 17/05901-7
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2017
• End date: September 30, 2018
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Fábio Márcio Squina
• Grantee: Victoria Ramos Sodré de Castro
• Host Institution: Faculdade de Engenharia Química (FEQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 15/50590-4 - Lignin valorization in cellulosic ethanol plants: biocatalytic conversion via ferulic acid to high value chemicals, AP.BIOEN.TEM

Abstract

Lignocellulosic biomass is a cheap and abundant resource which arises as an attractive alternative to oil for conversion into fuels and chemicals. Up to a third of lignocellulosic biomass is composed of lignin, a highly recalcitrant aromatic polymer, which is disposed as waste or burned for power in biomass processing biorefineries and pulp/paper industries. These streams represent an untapped source of aromatic compounds that could be converted to high value molecules. Ferulic acid (FA) is one of the main components of lignin and present potent antioxidant capacities, which can be exploited by the pharmaceutical and cosmetic industries, and is also an important precursor for other molecules of aggregated interest, such as eugenol, vanillin and coniferyl alcohol. The lignin-degrading bacterium Rhodococcus jostii RHA1 is able to depolymerize lignin, using the peroxidase DypB, into lower molecular weight (LMW) aromatic compounds such as ferulic acid (FA), which are catabolized. Due to its robust and versatile metabolism, Rhodococcus species are of biotechnological interest; however, the paucity of genetic tools available hinders its exploitation. In this project, we intent to develop a E. coli-Rhodococcus shuttle CRISPR/Cas9 expression vector, which can promote quick and efficient gene targeting procedures. We expect to apply this system to knockout the CouM gene of p-hydroxycinnamate catabolism in R. jostii RHA1 and RHA045 ”vdh, leading to increased yields of ferulic acid when grown in sugarcane bagasse lignocellulose. It is expected that this project provide the establishment and first use of the CRISPR/Cas9 system in Rhodococcus, as well as to develop strains and vectors for microbial lignin valorization conversion steps into high-market molecules. We also expect the novel E. coli-Rhodococcus shuttle vector to be a tool of interest to other researchers in the field. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SODRE, VICTORIA; ARAUJO, JUSCEMACIA NASCIMENTO; GONCALVES, THIAGO AUGUSTO; VILELA, NATHALIA; KIMUS BRAZ, ANTONIO SERGIO; FRANCO, TELMA TEIXEIRA; NETO, MARIO DE OLIVEIRA; DE LIMA DAMASIO, ANDRE RICARDO; GARCIA, WANIUS; SQUINA, FABIO MARCIO. An alkaline active feruloyl-CoA synthetase from soil metagenome as a potential key enzyme for lignin valorization strategies. PLoS One, v. 14, n. 2, FEB 25 2019. (17/05901-7, 17/22669-0, 17/16976-8, 15/50612-8, 17/16089-1, 15/50590-4, 17/08166-6, 18/18101-1, 17/17275-3)