Enzyme prospecting and metabolic pathway optimization for vanillin production from sugar cane bagasse lignin

Abstract

Lignin, one of the most recalcitrant natural materials, consists in a three dimensional polymer composed of multiple aromatic rings stably connected, being present in combination with cellulose and hemicellulose in plant cell walls. Lignin is strongly associated with cell wall polysaccharides, which hampers their use and virtually prevents its isolation in an unchanged form. Therefore, most of the data on the structure of lignin is based on analysis of isolated fractions of monomers and dimers derived from its chemical degradation. The water-soluble products resulting from the oxidation of lignin are predominantly p-hydroxybenzoic acid, syringaldehyde and vanillin. Vanillin, one of the oxidation products of lignin, is obtained in the natural form from the pod of tropical orchids of the genus Vanilla. Because of its low productivity and difficulty of cultivation outside its natural habitat, the total production of natural vanillin is only 40 Ton/year, supplying only about 0.25% of total world demand. Historically, this demand has being met by chemical synthesis, primarily from eugenol and from lignin present in the black liquor, and later replaced by chemical synthesis from non-renewable resources such as guaicol and glyoxylic acid. Recently, natural fermentative pathways have been developed for the production of vanillin from renewable sources. A new biosynthetic pathway in S. pombe has been developed, producing about 45 mg/L of vanillin using glucose as source material. The company Rhodia developed a vanillin producing method by microbial bioconversion and sells its product under the trade name Rhovanil® Natural at $ 700/kg. Since it is derived from renewable sources, this product carries the "natural" label in the USA and European Union markets. We propose in this project the use of the soluble lignin fraction from sugarcane bagasse, which is a residue from the extraction of cellulose and hemicellulose and is currently discarded. This liquid has considerable amounts of phenolic compounds derived from lignin that can be used as precursors to other molecules, including vanillin. Our work plan for this is to establish a degrading microbial consortium by the enrichment of a soil sample from sugar cane plantation in a lignin containing medium. This consortium will be characterized for their ability to degrade lignin, and then we will start a metagenomics and metatranscriptomics approach, followed by bioinformatics data analysis, to search for new enzymes. The genes of these enzymes will be synthesized and characterized for their catalytic efficiency and later we will assemble them in a synthesis pathway, in which we will apply a technique developed by one of our international partners that will allow us to attenuate the co-expression levels of these enzymes using "molecular switches" named riboswitches. (AU)