Towards bacterial valorization of low molecular weight lignin

Sammanfattning: Lignin is one of the major constituents in lignocellulosic biomass and is the mostabundant source of renewable aromatics on terrestrial ecosystems. The workcarried out in this thesis concerns bacterial conversion of low molecular weightlignin. This thesis is divided into three major sections, with an initial emphasison screening and characterization of selected bacterial species on lignin modelcompounds, followed by testing their performance on treated lignin substrates,and finally progressing towards strain improvement via metabolic engineering. During screening for bacteria using samples from natural and man-madeenvironments, Pseudomonas species were found dominant. Some of theseisolates, and the well-known aromatic degrader – Pseudomonas putida KT2440– were cultivated on lignin model compounds. P. putida and Pseudomonas sp.isolate 9.1 attained specific growth rates of about 0.21-0.27 h-1 and 0.12-0.30 h-1 respectively, on several compounds from the coniferyl, p-coumaryl and benzoyl branches of the funnelling pathways. Meanwhile, a contaminant was found growing on syringate plates, and was later identified to be a bacterium belonging to the Microbacterium genus. This Gram-positive bacterium, named RG1, was able to consume syringate and syringaldehyde besides other aromatic compounds from the coniferyl and p-coumaryl branches. Due to its interesting abilities to assimilate syringyl compounds, the genome of this strain was sequenced to identify genes involved in a putative syringyl pathway.To assess the performance of selected bacteria on lignin substrates, cultivationswere performed using alkaline- and oxidatively-treated Kraft lignin. P. putida and P. fluorescens consumed 4-HBA, vanillin, and vanillate in the complex ligninmixture that likely contained various toxic products. In addition, Rhodococcusopacus and Sphingobium sp. SYK-6 assimilated guaiacol and acetovanillonerespectively, from the lignin mixture. Interestingly, P. fluorescens was able tobreak down the higher molecular weight lignin and produce several smallermolecules.P. putida was selected as a host organism for genetic engineering aimed atexpanding the range of substrates utilized. Heterologous expression of thecytochrome P450 and oxidoreductase genes from R. rhodochrous enabled P.putida to assimilate guaiacol – one of the major depolymerization products fromsoftwood lignin – as the sole carbon source. Furthermore, the identification anddeletion of an aldehyde reductase in a P. putida strain that converts ferulate tovanillin, increased the yield of vanillin by eliminating the formation of vanillylalcohol as by-product.