Flavin antibiotics

Title: Construction and properties of the recombinant yeast strains producing bacterial antibiotics roseoflavin and aminoriboflavin

Supervisor: prof. dr hab. Andriy Sybirnyy, e-mail: asybirnyy@ur.edu.pl

Short description:

The antibiotic roseoflavin (RoF), a natural structural analog of vitamin B2 (riboflavin), is produced by soil bacterium Streptomyces davaonensis and is active against many Gram-positive pathogens. The immediate biosynthetic precursor of RoF, aminoriboflavin (AF), is also active against Gram-positive bacteria being at the same time non-toxic toward mammalian cells. However, S. davaonensis synthesizes tiny amounts of RoF and no AF is accumulated. For many years, PI of this project works with riboflavin overproducing yeast Candida famata and possesses a unique overproducer of flavin mononucleotide (FMN), which serves as a precursor for AF and RoF biosynthesis. In this project, we plan to construct for the first time, yeast strains producing bacterial antibiotics. The FMN overproducing strain of C. famata will serve as a starting point to convert this nucleotide FMN to AF and RoF. For this, genes of S. davaonensis responsible for conversion of FMN to AF phosphate (rosB), AF phosphate hydrolysis to AF (rosC) and finally AF conversion to RoF (rosA) will be cloned and expressed in the mentioned FMN overproducing strain of C. famata under control of constitutive and regulatory promoters. To broaden the potential of yeast systems, the gene FMN1 coding for riboflavin kinase and rosB, rosC and rosA will be expressed in riboflavin overproducing strain of Komagataella phaffii (Pichia pastoris). Flavin substances accumulated in the cultural liquid of the yeast transformants will be identified, toxicity of AF and RoF to the recombinant yeast expressing genes for flavin antibiotic synthesis will be determined. Additional attention will be paid to properties of the genes responsible for the excretion of riboflavin and flavin antibiotics in the recombinant strains of C. famata and K. phaffii. They will be cloned and overexpressed and flavin antibiotics excretion and overall production will be determined. To realize this project, methods of microbial molecular genetics, physiology, biochemistry and analytical chemistry will be used. Genes of interest will be cloned from host organisms or alternatively, synthetic genes with adapted codons for expression in yeast host will be used. Cloned genes will be overexpressed under control of strong constitutive or regulatory promoters. Low molecular weight compounds accumulating in recombinant cultures will be purified and identified. This project has big importance as there are high demands for new and efficient antibiotics, non-toxic to mammalian organisms. The last new antibiotic, linezolid, was implemented into practice nearly 20 years ago. Construction of recombinant yeast strains producing AF and RoF could open quite new perspectives for production of bacterial antibiotics in technologically well-suited yeast cells. Structural and regulatory genes of riboflavin synthesis have been described (Abbas and Sibifny, 2011; Dmytruk and Sibirny, 2012). Attention should be paid on SEF1 gene involved as positive controller in regulation of riboflavin synthesis (Dmytruk et al., 2006; 2011; 2014). Besides, genes FMN1 coding for riboflavin kinase and FAD1 coding for FAD synthetase have been cloned and overexpressed resulting in construction of FMN and FAD overproducers (Yatsyshyn et al., 2009; 2014). Besides this, methods of molecular genetics for the flavinogenic yeast C. famata have been developed. Also systems of riboflavin transport have been identified by the group of A. Sibirny which include riboflavin uptake and excretion in the close flavinogenic species Meyerozyma guilliermondii (Sibirny et al., 1978; Sibirny and Shavlovsky, 1984) and excretion in C. famata (Tsyrulnyk et al., 2020; 2021). PI also has big experience working with the second model organism in this study, the yeast K. phaffii, especially on cell biology of this organism (Stasyk et al., 2006; Nazarko et al., 2011; Sibirny, 2016; Dmytruk et al., 2021).

Competences / skills and expectations:

• Master of Biology or Biotechnology,
• Strong motivation for scientific work,
• Analytical thinking,
• Interpersonal skills and the ability to work in a team,
• English, level at least B2.