Enhancing tylosin production by combinatorial overexpression of efflux, SAM biosynthesis, and regulatory genes in hyperproducing strain.

Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas. To improve the fermentation titer of tylosin in the hyperproducing strain TL01, we sequenced its whole genome and identified the biosynthetic gene cluster therein. Overexpression of the tylosin efflux gene , the luster-ituated -adenosyl methionine (SAM) synthetase gene , the SAM biosynthetic genes -, or the pathway-specific activator gene enhanced tylosin production by 18%, 12%, 11%, and 11% in the respective engineered strains TLPH08-2, TLPH09, TLPH10, and TLPH12. Co-overexpression of and - as two transcripts increased tylosin production by 22% in the resultant strain TLPH11 compared to that in TL01. Furthermore, combinational overexpression of , , -, and as four transcripts increased tylosin production by 23% (10.93g/L) in the resultant strain TLPH17 compared to that in TL01. However, a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2. Transcription analyses indicated that the expression of and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11. Based on this observation, the five genes were rearranged into one or two operons to coordinate their overexpression, yielding two engineered strains TLPH23 and TLPH24, and leading to further enhancement of tylosin production over TLPH17. In particular, the production of TLPH23 reached 11.35 g/L. These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.