Development of the CRISPR/Cas9 system in Rhodococcus jostii RHA1 for production of high-value chemicals from lignin streams

• Grant number: 18/18101-1
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: October 01, 2018
• End date: December 31, 2021
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Agreement: BBSRC, UKRI
• Principal Investigator: Fábio Márcio Squina
• Grantee: Victoria Ramos Sodré de Castro
• Host Institution: Pró-Reitoria de Pós-Graduação, Pesquisa, Extensão e Inovação. Universidade de Sorocaba (UNISO). Sorocaba , 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
• Associated scholarship(s): 20/05281-1 - Genetic engineering of Rhodococcus jostii RHA1 for lignin valorization strategies, BE.EP.DD

Abstract

In the context of lignin valorization, the use of whole-cell biocatalysts for deconstruction and conversion of lignin is a promising alternative to chemical catalysis that could render more cost-effective and environmentally-friendly results. The species Rhodococcus jostii RHA1 is of particular biotechnological interest due to its ability to catabolize a wide array of aromatic compounds, notably lignin. Due to this capacity, allied to the presence of catabolic routes for degradation of several aromatic molecules, RHA1 could be exploited to produce high-value molecules from lignin; however, this potential is hindered by a lack of genetic tools available. In the present project, we aim to develop the necessary tools for implementation of the CRISPR/Cas9 system in this species and apply the system to introduce novel pathways for lignin valorization. Initially, the proof-of-concept will be to knockout the CouM gene of p-hydroxycinnamate metabolism, leading to accumulation of Ferulic Acid (FA) by mutant strains grown in biorefinery-derived lignin streams. Due to its application in the cosmetic and pharmaceutical industries, FA represents a high-value compound itself, besides being convertible to other compounds of aggregated interest, for instance Coniferyl Alcohol (CA). The latter can be obtained from FA via a 2-step enzymatic reaction catalyzed sequentially by a carboxylic acid reductase (CAR) and an Aldo-Keto Reductase (AKR). In addition to characterization of CARs from diverse sources, our group has described a novel AKR isolated from the lower termite Coptotermes gestroi (CgAKR-1), which showed high catalytic values on 2-nitrobenzaldehyde (Km = 0.15 mmol/L, kcat = 7.5 s1) and promiscuous activity towards different aliphatic and aromatic aldehydes, including coniferyl aldehyde. We intend to introduce CAR and CgAKR-1 genes in R. jostii for production of CA in FA- accumulating strains. Besides being a high-value compound itself, CA is also a platform molecule, which can be further converted into other products of commercial interest. Among these, fragrances such as eugenol and isoeugenol can be enzymatically obtained from coniferyl alcohol by two sequential reactions, which we expect to express in CA-accumulating RHA1 strains as well. We expect that our work will not only contribute to the generation of novel genetic tools in this species, but also amplify the array of products that can be biologically obtained from lignin streams. (AU)

Scientific publications

(6)

(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; VILELA, NATHALIA; TRAMONTINA, ROBSON; SQUINA, FABIO MARCIO. icroorganisms as bioabatement agents in biomass to bioproducts application. BIOMASS & BIOENERGY, v. 151, AUG 2021. (16/07926-4, 18/18101-1, 15/50590-4, 17/08166-6)

ALVAREDO PAIXAO, DOUGLAS ANTONIO; TOMAZETTO, GEIZECLER; SODRE, VICTORIA RAMOS; GONCALVES, THIAGO A.; UCHIMA, CRISTIANE AKEMI; BUCHLI, FERNANDA; ALVAREZ, THABATA MARIA; PERSINOTI, GABRIELA FELIX; DA SILVA, MARCIO JOSE; BRAGATTO, JULIANO; et al. Microbial enrichment and meta-omics analysis identify CAZymes from mangrove sediments with unique properties. Enzyme and Microbial Technology, v. 148, AUG 2021. (12/19040-0, 15/50590-4, 14/04105-4, 16/09950-0, 15/23279-6, 18/18101-1, 17/16089-1)

DOS SANTOS, OTAVIO AUGUSTO LEITAO; GONCALVES, THIAGO AUGUSTO; SODRE, VICTORIA; VILELA, NATHALIA; TOMAZETTO, GEIZECLER; SQUINA, FABIO M.; GARCIA, WANIUS. Recombinant expression, purification and characterization of an active bacterial feruloyl-CoA synthase with potential for application in vanillin production. Protein Expression and Purification, v. 197, p. 7-pg., 2022-05-11. (17/16089-1, 15/50590-4, 18/18101-1, 17/08166-6, 20/15595-3, 20/05784-3, 17/17275-3)

TRAMONTINA, ROBSON; BRENELLI, LIVIA BEATRIZ; SODRE, VICTORIA; FRANCO CAIRO, JOAO PAULO; TRAVALIA, BEATRIZ MEDEIROS; EGAWA, VIVIANE YOSHIMI; GOLDBECK, ROSANA; SQUINA, FABIO MARCIO. Enzymatic removal of inhibitory compounds from lignocellulosic hydrolysates for biomass to bioproducts applications. WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, v. 36, n. 11, OCT 1 2020. (20/03051-9, 15/50590-4, 16/07926-4, 17/15477-8, 18/18101-1)

GONCALVES, THIAGO AUGUSTO; SODRE, VICTORIA; DA SILVA, STEPHANIE NEMESIO; VILELA, NATHALIA; TOMAZETTO, GEIZECLER; ARAUJO, JUSCEMACIA NASCIMENTO; MUNIZ, JOAO RENATO C.; FILL, TAICIA PACHECO; DAMASIO, ANDRE; GARCIA, WANIUS; et al. Applying biochemical and structural characterization of hydroxycinnamate catabolic enzymes from soil metagenome for lignin valorization strategies. Applied Microbiology and Biotechnology, v. 106, n. 7, p. 14-pg., 2022-03-30. (17/16976-8, 20/05784-3, 18/18101-1, 17/17275-3, 17/22669-0, 15/50612-8, 15/50590-4, 15/23279-6)

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)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CASTRO, Victoria Ramos Sodré de. Estratégias para valorização biológica de lignina e hidrolisados lignocelulósicos. 2021. Doctoral Thesis - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.