Engineering of leucine-responsive regulatory protein improves spiramycin and bitespiramycin biosynthesis
Engineering of leucine-responsive regulatory protein improves spiramycin and bitespiramycin biosynthesis
复制标题
亮氨酸响应调节蛋白的工程改善了螺旋霉素和bitspiramycin的生物合成
DOI：
10.1186/s12934-019-1086-0
复制
发表时间：
2019-02-19
影响因子：
6.4
通讯作者：
He, Weiqing
中科院分区：
文献类型：
--
作者：
Lu, Zhili;Zhang, Xiaoting;He, Weiqing
bitspiramycin （BT）是由重组螺旋霉素（SP）产生菌株螺旋链霉菌（Streptomyces spiramyticus）产生的，该菌株含有异源的4- o -异戊基转移酶基因（清单）。外源l-亮氨酸（l-Leucine, l-Leu）可以促进BT的产生，从螺旋菌基因组序列中发现orf2基因编码亮氨酸应答调节蛋白（leucine-responsive regulatory protein, Lrp）家族调控因子SSP_Lrp。研究了SSP_Lrp和l-Leu在螺旋菌中螺旋霉素（SP）和BT合成中的作用。结果sssp_lrp是一个全局调控因子，直接影响SP或BT生物合成中3个阳性调控基因bsm23、bsm42和acyB2的表达。螺旋菌1941的SSP_Lrp基因失活导致SP产量小幅增加。而含有L - leu结合结构域SSP_Lrp缺失的SSP_Lrp-SP菌株的SP产量高于S. spiramyceticus 1941（分别为476.23.1g/L和313.3 +/- 25.2g/L），特别是SPⅲ显著增加。SSP_Lrp-BT菌株的BT产量是1941-BT的2倍以上。SSP_Lrp-SP细胞内支链氨基酸（BCAAs）浓度明显下降，表明BCAAs的分解代谢增加为SP的生物合成提供了更多的前体。对比分析SSP_Lrp-SP和S. spiramyceticus 1941的转录组谱，发现有12个基因表达有明显差异，其中上调基因6个，下调基因6个。上调基因与SP生物合成PKS基因、类异戊二烯生物合成基因、Sigma24家族因子、天冬氨酸、丙酮酸和酰基辅酶a代谢相关；下调的基因与核糖体蛋白、AcrR家族调节因子以及萜类、谷氨酸和谷氨酰胺的生物合成有关。结论螺旋菌>SSP_Lrp是参与SP和BT生物合成的负调控因子。SSP_Lrp假定的l-Leu结合域的缺失有利于BT和SP的产生，尤其是它们的III组分。
BackgroundBitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4-O-isovaleryltransferase gene (ist). Exogenous l-Leucine (l-Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and l-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus.ResultsSSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23, bsm42 and acyB2, in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the SSP_Lrp-SP strain containing an SSP_Lrp deficient of putative l-Leu binding domain was higher than that of S. spiramyceticus 1941 (476.23.1g/L versus 313.3 +/- 25.2g/L, respectively), especially SP III increased remarkably. The yield of BT in SSP_Lrp-BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the SSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the SSP_Lrp-SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine.Conclusion>SSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative l-Leu binding domain was advantageous for production of BT and SP, especially their III components.