Tylosin

„Improvement of tylosin A production yields by engineering of the producer strain”

01.05.2021 bis 31.10.2021

„Improvement of tylosin A production yields by engineering of the producer strain”

Abstract: Tylosin


Tylosin is a 16-membered macrolide antibiotic used as veterinary medicine to treat infections caused by Gram-positive bacteria. The antibiotic is produced by diverse Streptomyces species, including Streptomyces fradiae, Streptomyces rimosus, and Streptomyces hygroscopicus. The material enriched from fermentation cultures of a producer strain and the formulated products are composed of tylosin factor-A or tylosin-A (80–90%), and small amounts of factors B, C, and D (4). All components contribute to the potency of tylosin. However, tylosin A is the major agent (usually about 90% and not less than 80%), which provides the antibacterial activity. Although, several biosynthetic mutants of S. fradiae were obtained and the biosynthetic steps are relatively well studied, there are reactions of which mechanisms remain unexplored (e.g. the conversion of tylosin A to tylosin D). As tylosin A is the main component exhibiting the highest activity against Gram-positive pathogens, the optimization of tylosin A production and the investigation, and understanding of tylosin D biosynthesis are of particular interest for industrial manufacturing of tylosin.
The main objective of this project are the increase of tylosin A production and generation of strain-derivatives, in which the production of tylosin D is reduced or eliminated. These engineered mutants will be exploited for industrial production of tylosin.
The project „Improvement of tylosin A production yields by engineering of the producer strain” involves three phases (work packages WP1-WP10).
In the first phase (WP1-WP6), protocols for laboratory handling and genetic manipulation, a genome sequence, and first promising mutants of the parental tylosin producer strain were obtained. In addition, a transcriptome analysis of the parental tylosin producer strain was initiated.
In the second phase (WP7-WP9), mutants in which positive effects on tylosin production have been observed were combined in double mutants. The transcriptome analysis was completed. New mutants were designed based on the outcome of the transcriptome analysis. Furthermore, the routes for tylosin D biosynthesis were investigated.
Although the first and second phase of this project delivered promising engineering “targets” and mutants, there are variations in the productivity of the obtained clones. Furthermore, it is of particular interest to examine if the positive effects on tylosin production can be strengthened to boost even higher tylosin production. Therefore, in the third phase of the project, a reporter assay will be developed and used for the identification of strong promoters for S. fradiae. Depending on the strength of the promoter, constructs will be cloned for overexpression experiments. This strategy will be applied to combine positive effects on the tylosin production in S. fradiae mutant strains. This should lead to stabilization and further increase the tylosin production.