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Carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals and there is growing interest in producing them through microbial processes. While many CAs are intermediates of microbial central carbon metabolism, and therefore envisaging their production in a host of choice is relatively straightforward; for other CAs this approach is difficult. This could be because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). In a review published in Journal of Fungi, Nuno Mira’s team (iBB) reviewed the synthetic biology approaches that have been made towards enabling the production of non-natural CAs in microbes, with a large emphasis on metabolic retrobiosynthesis methodologies. Additionally, the authors also reviewed the pathway prospecting studies towards microbial levulinic, poly-lactic and methacrylic acid production, as two case-studies where there is a need to bridge the gap between natural CAs and their non-natural industrial derivatives.

The synthesis of hydroxide [Ag(OH)L] (L =I VL, VL, VIL, VIIL), oxide [{AgL}2}(μ-O)] (L = IL, IIL, IIIL, VL, VIL) or chloride [AgIIL]Cl, [Ag(VIL)2]Cl complexes, obtained from reactions of mono- or bicamphorimine derivatives with Ag(OAc) or AgCl, appeared in a recent publication on the journal Antibiotics, resulting from a collaborative research involving the iBB members Jorge H. Leitão, Nuno Mira, Ana Rego and Sílvia Sousa; M. Fernanda Carvalho from CQE; and Fernanda Marques from C2TN. The new complexes were characterized by spectroscopic (NMR, FTIR) and elemental analysis. X-ray photoelectron spectroscopy (XPS), ESI mass spectra and conductivity measurements were undertaken to corroborate formulations. The antimicrobial activity of complexes and some ligands were evaluated towards Candida albicans and Candida glabrata, and strains of the bacterial species Escherichia coli, Burkholderia contaminans, Pseudomonas aeruginosa and Staphylococcus aureus based on the Minimum Inhibitory Concentrations (MIC). A significant feature of these redesigned complexes is their ability to sensitize C. albicans, a trait that was not found for the previously investigated [Ag(NO3)L] complexes. The MIC values of the complexes towards bacteria were in the range of those of [Ag(NO3)L] and well above those of the precursors Ag(OAc) or AgCl. The activity of the complexes towards normal fibroblasts V79 was evaluated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Results showed that the complexes have a significant cytotoxicity.

Ana Isabel de Vila-Santa Braga Campos will be defending her PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Friday the 16th of April 2021, at 15:00 am (https://videoconf-colibri.zoom.us/j/81314376867). During the last years, and under the supervision of Nuno Mira and Frederico Ferreira from iBB, and Kristala Prather from MIT, Ana Isabel investigated how pathways for the synthesis of levulinic and itaconic acids can be engineered in yeasts. The title of her thesis is “Synthetic biology approaches to foster yeasts as hosts for the production of carboxylic acids and their derivatives: emphasis on levulinic and itaconic acids”.

Levulinic acid is a versatile platform molecule with potential to be used in replacement of oil-derived catalysts explored by the chemical industry for the synthesis of many add-value products. Unlike other carboxylic acids of interest whose microbial production has been addressed, levulinic acid is not described to be produced by living systems which restrains its microbial production. Exploring the power of synthetic biology in settling new-to-nature pathways (through the association between metabolites and/or enzymes that are not found in nature), hundreds of potential combinations were screened exploring different metabolic retrobiosynthesis tools followed by extensive manual curation and genome scale metabolic modeling. Five complete synthetic pathways (with identified intermediates and enzymes) were identified as promising candidates to enable microbial production of levulinic acid. The results of this study, resulting from a collaboration between the iBB team led by Nuno P Mira and the team of Kristala Prather from MIT, were recently published in ACS Synthetic Biology. Not only does the herein described approach offer a platform for future implementation of microbial production of levulinic acid, but it also provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of add-value chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology. 

The effectiveness of Candida glabrata as an emerging human pathogen relies on its ability to acquire azole drug resistance. In a paper just published in Antimicrobial Agents and Chemotherapy, the first time-course evaluation of the global gene expression changes that lead a drug susceptible C. glabrata clinical isolate to step-wise acquisition of resistance to azole drugs was conducted. This work, which results from the collaboration of six different teams under the coordination of Miguel C Teixeira from BSRG-iBB, highlights the multifactorial nature of azole resistance acquisition, including the Epa3 adhesin as a new player, while providing fascinating clues on the underlying evolutionary path. This knowledge is of crucial importance to design more effective antifungal therapy.