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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 type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): October 01, 2018
Effective date (End): February 28, 2021
Field of knowledge:Biological Sciences - Genetics
Cooperation agreement: BBSRC, UKRI
Principal Investigator:Fábio Márcio Squina
Grantee:Victoria Ramos Sodré de Castro
Home 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 feluric acid to high value chemicals, AP.BIOEN.TEM

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
(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. Web of Science Citations: 1.

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