Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.

Several hundred fungi species are found to be pathogenic to humans. Fungi-associated diseases vary from allergies to cutaneous, subcutaneous, and/or systemic infections. Some clinically important mycoses exhibit high morbidity and mortality rates, associated, among others, to the improvement of individuals’ life expectancy, due to HIV/AIDS and cancer treatment-associated immunosuppression, the use of immunomodulatory drugs, and misuse of antimicrobial drugs. Additionally, the high cost associated with fungal infection treatment, the toxicity of available drugs, and the increase in drug resistance are a concern for mycosis since there are only a few classes of antimicrobial treatments available. Therefore the establishment and use of CRISPR-Cas methodologies recently applied on pathogenic fungi has been discussed as a cornerstone to investigate molecular mechanisms regarding virulence and antifungal tolerance and develop new and more powerful therapies and antifungals. In this chapter, we report some of the CRISPR-Cas-based systems applied to the most common human fungal pathogens.

The Fungi kingdom plays an important role in human life from human health to biotechnological industrial applications. Despite its already great relevance in the production of enzymes, biofuels, and secondary metabolites and, regardless of the availability of databases comprising more than 1000 complete fungi genomes, and the existence of bioinformatics and genetic engineering tools for fungi manipulation, further studies are still needed to better understand fungi biology and expand its use to solve industrial challenges. In this context, although CRISPR-Cas systems are not the only tool available for fungi gene editing, until now it is the most characterized and applied to produce industrial-engineered fungal strains. The range of CRISPR-Cas systems available for fungi industrial application is diverse in their molecular parts, genetic transformation methodologies, and CRISPR system assembly, as a single vector, dual vector, or ribonucleoprotein complexes, which are detailed and discussed in this chapter.

The methylotrophic yeast Komagataella phaffii is one of the most important microbial platforms to produce recombinant proteins. Despite its importance in the context of industrial biotechnology, the use of synthetic biology approaches in K. phaffii is hampered by the fact that few genetic tools are available for precise control of gene expression in this system. In this work, we used an RNA aptamer activated by tetracycline to modulate protein production at the translational level. Using lacZ as gene reporter, we have demonstrated significant reduction of the heterologous protein upon addition of tetracycline. Furthermore, this genetic control device was applied for the control of Ku70p. This protein is involved in non-homologous recombination and the control of its production paves the way for the development of strains exhibiting higher rates of homologous recombination. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-023-01637-5.

The diagnosis of SARS-CoV-2 by real-time detection of the viral genome using RT-qPCR (Reverse Transcriptase quantitative Polymerase Chain Reaction) and serological methods were essential to control its rapid spread during the pandemic. However, RT-qPCR, which is the gold standard method to identify SARS-CoV-2 at the initial phase of infection, has high costs and other limitations such as refrigeration and specialized equipment and professionals. Therefore, a sensitive, not time consuming, not costly, not laborious, and free of refrigeration and specialized equipment is of utmost importance in view of the threat of future epidemics. Here, we propose a detection method where SARS-CoV-2 RNA is recognized and cleaved by ribozymes releasing an initiator fragment. This fragment triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores which leads to a Fluorescence Resonant Energy Transfer (FRET) interaction. First, a consensus SARS-CoV-2 RNA sequence was identified. This target viral RNA fragment and ribozymes were in vitro transcribed, followed by cleavage reactions. DNA hairpins with Cy3/Cy5 fluorophores were designed and synthesized for FRET-HCR assays that were performed to target the RNA fragment sequences obtained after ribozyme cleavage. Our results indicated that two out of the three designed ribozymes cleaved the target RNA. Furthermore, DNA hairpins with Cy3/Cy5 pairs were efficient in target RNA detection and in triggering FRET-HCR detectable reactions. Altogether, the results of this study laid the cornerstone, as a proof of concept, for the joint use of ribozymes and DNA hairpins with Cy3/Cy5 in FRET-HCR reactions to detect SARS-CoV-2 virus.