Prospecção de enzimas e vias metabólicas para a biossíntese de vanilina e valorização da lignina do bagaço de cana-de-açúcar

Author(s)

Eduardo Cruz Moraes

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2017-12-14

Examining board members

Fabio Marcio Squina; Fabiano Jares Contesini; Richard John Ward; Fernanda Mandelli

• Advisor: Thabata Maria Alvarez; Fabio Marcio Squina

Abstract

Lignin, one of nature's most recalcitrant materials, consists of a three-dimensional polymer composed of various aromatic rings bound by ester-like bonds, being present in combination with cellulose and hemicellulose in the plant cell wall. Lignin is strongly associated with polysaccharides of the cell wall, which makes difficult its use and isolation in an unchanged form. Vanillin is one of the products of lignin oxidation, and is obtained in the natural form from the pod of tropical orchids of the genus Vanilla. Due to its low productivity and the difficulty of growing outside its natural habitat, the world demand for this product has been met through chemical synthesis from non-renewable resources such as guaicol and glyoxylic acid. Recently, natural fermentative routes have been developed for the production of vanillin from renewable sources. Given the commercial importance of the vanillin molecule, associated to the demand for the substitution of non-renewable raw materials for low cost renewable raw materials, it is proposed in this work the establishment and characterization of a consortium of lignin degrading microorganisms as a strategy for the valorization of this sub-product. This consortium was created through the cultivation of microorganisms from sugar cane fields in a culture medium containing a solution of compounds from lignin. This solution is obtained through the process of delignification of sugarcane bagasse, and is composed mainly of phenolic compounds of low molecular weight. With the metagenomic sequencing of the consortium, it was possible to have access to the enzymes and metabolic pathways involved in the bioconversion processes of these phenolic compounds. As a method to validate this strategy for the prospection of lignolytic enzymes, feruloil-CoA synthetase (fcs) and enoyl-CoA hydratase / aldolase (ech) enzymes were selected and evaluated for the conversion capacity of ferulic acid to vanillin. The characterization of the consortium revealed the ability of the present microorganisms to degrade low molecular weight phenolic compounds from the culture medium. A subsequent taxonomic analysis indicated the enrichment of the consortium, with the decrease of the diversity of microorganisms in relation to the original soil sample, and the increase of species described in the literature as related to the degradation of aromatic compounds. In this way, the specialization of the consortium can be observed through the selection of microorganisms able to degrade the lignin present in the medium. The analysis of the metagenome of the consortium also allowed the identification of enzymes involved in the degradation of lignin and aromatic compounds, such as laccases, peroxidases and other enzymes involved in polycyclic aromatic hydrocarbon metabolism (PAHs). From the metagenomic sequencing data, three enzymes feruloil-CoA synthetase (fcs) and two enoyl-Coa hydratase / aldolase (ech) were performed in silico by sequence similarity analysis. Capillary electrophoresis and mass spectrometry analyzes showed the production of vanillin from ferulic acid in enzymatic assays with the selected enzymes. In parallel, microorganisms capable of growing independently in media containing lignin and with possible lignolytic activity were isolated. In particular, the isolated strain of Paenarthrobacter sp. (referred to as HW13) was shown to be capable of growing in culture medium containing only kraft lignin as the carbon source. This strain was also tested for growth / tolerability to various phenolic compounds of recognized toxicity, as well as having its genome sequenced and compared to an Paenarthrobacter genome obtained from the metagenomic sequencing data of the consortium, revealing a high degree of similarity. Therefore, it was shown in this work that the strategy of establishing a lignin degrading consortium, together with the access of lignolytic enzymes of the microorganisms by their isolation and by metagenomic tools, consists of effective ways of obtaining bioconversion routes of aromatic compounds derived from lignin. The obtaining of enzymes present in these routes has the potential of valorization of the sugarcane bagasse lignin through the conversion of this into molecules of greater added value (AU)

• FAPESP's process: 14/26152-4 - Enzyme prospecting and metabolic pathway optimization for vanillin production from sugar cane bagasse lignin
• Grantee: Eduardo Cruz Moraes
• Support Opportunities: Scholarships in Brazil - Doctorate