Projeto Portugal 2030

Sumário

This research seeks to revolutionize the production of BC by tackling significant challenges and integrating novel concepts that surpass the current state of the art. The primary objectives are: 1- To advance the understanding of BC biosynthesis in K. sucrofermentans, by identifying key genes and pathways involved in BC production and optimizing their expression levels by studying this strain’s metabolic profile. We will refine our generated draft GSMM (fig.1) for this strain reflecting its metabolic flux under BC-producing conditions. Using CEB/UM proprietary algorithms, we will implement and experimentally validate in silico optimizations of the metabolic network and genetic manipulations, complementing with the respective analyses and biological interpretations. We aim to unveil innovative metabolic engineering strategies that will substantially increase BC yield. 2- To pioneer the genome edition CRISPR/Cas9 or Cas9n toolbox for K. sucrofermentans (fig.2), incorporating multiplexing [11] capabilities for efficient genome editing. The recent establishment of CRISPR interference [12] proved Cas9 ability to bind specific sites in the K. sucrofermentans genome. We will develop a CRISPR plugin for the easy and fast assembly of an all-in-one vector for simultaneous expression of the Cas9 enzyme and the single-guide RNA, using plasmids and genetic parts recently characterized [5, 6, 13]. Besides the gene editing targets to be identified in silico, this novel strategy builds upon our previous work [7], where a mutant unable to produce the major byproduct of BC fermentation (gluconic acid) displayed c.a. 6-fold increase in volumetric productivity despite its lower efficiency at assimilating glucose from the culture medium. We can further increase the yield by modulating the ethanol respiratory chain. Specifically we will overexpress the PQQ-dependent alcohol dehydrogenase, the PQQ biosynthetic operon and the terminal oxidases. Additionally, we plan to overexpress a glucose transporter and the direct polymerization pathway (glk, pgm, and celA genes) in the same modified strain. We will also express the E. coli phosphofructokinase gene in K. sucrofermentans, to complete the EMP pathway thus enhancing ATP supply. These novel strategies are aimed at creating a suite of genetically engineered strains with exceptional BC production efficiency. 3- To investigate a novel fermentation process using a nanobubble-based bioreactor system (fig. 4), for its potential to enhance BC production. Air/oxygen nanobubbles offer an innovative solution to improve oxygen transfer rates in aerobic fermentation, due to their very high surface areas, stability, and lack of buoyancy in solution. 4- Finally, to explore the integration of BC into various cosmetic formulations. We will assess the performance of BC dry formulations (capable of quick redispersion in water, under low energy mixing, fully recovering the properties of the never-dried material) as stabilizers and texturizing agents in surfactant-free cosmetic formulations (lotions, creams, shampoos, and shaving foams), aiming to offer a competitive edge to cosmetic brands in the growing market for natural and organic cosmetics. Collectively, these objectives position this proposal at the forefront of interdisciplinary innovation. The project's ambition lies in its comprehensive approach to improving BC production and application, setting new benchmarks for efficiency, sustainability, and market relevance.