Isoeugenol monooxygenase characterization and whole-cell bioconversion systems towards vanillin production from plant-derived precursor isoeugenol

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2019This work is a response to the crescent interest by the biorefinery industries that drive research towards more cost-competitive and environmentally sustainable processes. New ways to value materials that would once be considered wasteful or valueless are researched. Lignin is one of such cases, a complex biopolymer that is considered by the paper and ethanol producing industries as waste. Most importantly, lignin is the only large-volume renewable feedstock that contains aromatic compounds, making it an attractive candidate for research efforts towards its valorization. In this study, isoeugenol, a compound resultant from lignin reductive catalytic fractionation (RCF) was used as substrate for vanillin production. This bioconversion was achieved resorting to an isoeugenol monooxygenase as biocatalyst. At start, the heterologous production of two isoeugenol monooxygenases, one from Pseudomonas putida (PpIEMO) and one from P. nitroreducens (PnIEMO) were compared regarding protein solubility and enzyme activity, in 50 mL scale cultures using different E. coli strains. The isoeugenol monooxygenase from P. nitroreducens was then picked to proceed to scale-up and characterization processes using E. coli Tuner ΔcueO as host. After production at 1L-scale, PnIEMO was purified by affinity chromatography techniques and biochemically characterized to assess its potential application in biotechnology. The evaluating of its properties and limitations will allow in the future, to proceed to enzyme evolution. Purification of PnIEMO from 1 L of growth medium yielded ≈ 1.8 mg of purified enzyme. The molecular mass determined was of 50.1 kDa. The optimum pH and temperature for this enzyme were pH 9 and 30 °C. Michaelis Menten parameters were calculated and resulted in Km and Km/Kcat of 0.13 mM and 118 mM-1 s-1, respectively. PnIEMO presented low stability towards temperature, with half-life of 7 min at 35 °C and a melting temperature of 32 °C (temperature at which 50 % of the molecules are unfolded), when following tryptophan fluorescence as protein denaturation indicator. This enzyme presented some tolerance towards guanidine hydrochloride, displaying a midpoint of the concentration of 1.3 M (where 50 % of molecules are unfolded). The techniques assessed to improve the yields of soluble enzyme, namely the use of hosts co-expressing chaperones, or to recover active enzyme from the inclusion bodies present in the cell pellets, by unfolding and refolding processes, did not show improvements. Whole-cell assays were performed using free and alginate immobilized E. coli cells expressing PnIEMO towards isoeugenol bioconversion to vanillin. Both approaches enabled ≈ 100 % vanillin production in 24 h, although using free whole-cells the reaction was faster than with alginate immobilized whole-cells. Summing up, this enzyme is an excellent candidate for use in biotechnological vanillin production processes, using isoeugenol resultant from lignin RCF

Plant Growth Promotion, Phytohormone Production and Genomics of the Rhizosphere-Associated Microalga, Micractinium rhizosphaerae sp. nov.

Funding Information: This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Funding Information: F.Q.-N. and P.R.B. acknowledge receiving a PhD fellowship from FCT (2022.10633.BD; 2021.07927.BD514, respectively). Publisher Copyright: © 2023 by the authors.Microalgae are important members of the soil and plant microbiomes, playing key roles in the maintenance of soil and plant health as well as in the promotion of plant growth. However, not much is understood regarding the potential of different microalgae strains in augmenting plant growth, or the mechanisms involved in such activities. In this work, the functional and genomic characterization of strain NFX-FRZ, a eukaryotic microalga belonging to the Micractinium genus that was isolated from the rhizosphere of a plant growing in a natural environment in Portugal, is presented and analyzed. The results obtained demonstrate that strain NFX-FRZ (i) belongs to a novel species, termed Micractinium rhizosphaerae sp. nov.; (ii) can effectively bind to tomato plant tissues and promote its growth; (iii) can synthesize a wide range of plant growth-promoting compounds, including phytohormones such as indole-3-acetic acid, salicylic acid, jasmonic acid and abscisic acid; and (iv) contains multiple genes involved in phytohormone biosynthesis and signaling. This study provides new insights regarding the relevance of eukaryotic microalgae as plant growth-promoting agents and helps to build a foundation for future studies regarding the origin and evolution of phytohormone biosynthesis and signaling, as well as other plant colonization and plant growth-promoting mechanisms in soil/plant-associated Micractinium.publishersversionpublishe

From Microalgae Growth Promotion to the Production of Secondary Metabolites

Funding Information: F.Q.-N. and P.R.B. acknowledge receiving a Ph.D. fellowship (2022.10633.BD; 2021.07927.BD514, respectively) funded by FCT/MCTES. Funding Information: This research was conducted in the scope of the project “PhycoµBiome: Understanding and harnessing the power of the microalgae microbiome aiming the maximization of marine microalgae productivity” funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal), grant number PTDC/BAA-BIO/1262/2020. The research was performed with the support of iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020) also funded by the FCT/MCTES. Publisher Copyright: © 2023 by the authors.Marine bacteria are a significant source of bioactive compounds for various biotechnological applications. Among these, actinomycetes have been found to produce a wide range of secondary metabolites of interest. Saccharopolyspora is one of the genera of actinomycetes that has been recognized as a potential source of these compounds. This study reports the characterization and genomic analysis of Saccharopolyspora sp. NFXS83, a marine bacterium isolated from seawater from the Sado estuary in Portugal. The NFXS83 strain produced multiple functional and stable extracellular enzymes under high-salt conditions, showed the ability to synthesize auxins such as indole-3-acetic acid, and produced diffusible secondary metabolites capable of inhibiting the growth of Staphylococcus aureus. Furthermore, when Phaeodactylum tricornutum was co-cultivated with strain NFXS83 a significant increase in microalgae cell count, cell size, auto-fluorescence, and fucoxanthin content was observed. Detailed analysis revealed the presence of clusters involved in the production of various secondary metabolites, including extracellular enzymes, antimicrobial compounds, terpenes, and carotenoids in the genome of strain NFXS83. Ultimately, these findings indicate that Saccharopolyspora sp. NFXS83 has a significant potential for a wide range of marine biotechnological applications.publishersversionpublishe