Improved FK506 production by the precursors and product-tolerant mutant of Streptomyces tsukubaensis based on genome shuffling and dynamic fed-batch strategies.
FK506, a secondary metabolite produced by Streptomyces tsukubaensis, is well known for its immunosuppressant properties to prevent rejection of transplanted organs and treat autoimmune diseases. However, the low titer of FK506 in the original producer strain limits the further industrialization efforts and restricts its clinical applications. To address this issue, a highly efficient method combined genome shuffling and dynamic fed-batch strategies was systematically performed in this work. Firstly, after five rounds of genome shuffling based on precursors and product resistances, a higher yielding strain TJ-P325 was successfully acquired, whose production reached 365.6 mg/L, 11-fold increase compared with the original strain. Then, the possible mechanism of different production capabilities between TJ-P325 and the wild type was explored through comparative gene expression analysis of key genes. Results showed that the transcription level of key genes was altered significantly in the mutant. Moreover, precursors addition enhanced the FK506 production by 28 %, as well as reduced the by-products biosynthesis. Finally, the disodium malonate and disodium methylmalonate dynamic fed-batch strategies dramatically led to the production of 514.5 mg/L in a 7.5-L bioreactor. These results demonstrated that genome shuffling and dynamic fed-batch strategies could be successfully applied to generate high-yield strains with value-added natural products during industrial microbial fermentation.
Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
Wenjie
W
Du
Di
D
Huang
Menglei
M
Xia
Jianping
J
Wen
Ming
M
Huang
D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D032621:DNA Shuffling; D005285:Fermentation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D042822:Genomic Instability / Q000235:genetics; D057166:High-Throughput Screening Assays; D007166:Immunosuppressive Agents / Q000378:metabolism; D008314:Malonates / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics; D013302:Streptomyces / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016559:Tacrolimus / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
D007166:Immunosuppressive Agents; D008314:Malonates; D016559:Tacrolimus