9-kb PCR product was amplified and cloned into pMD18-T (TaKaRa) to generate pJTU1201. Then, the 0.7-kb SfiI-AflII fragment from pJTU1201 was used to replace the 1.4-kb corresponding region in pHZ1904 to result in a dndB in-frame deletion vector, pJTU1202, in which a 729-bp DNA fragment was removed from dndB. Vector construction for dndC deletion: after pHZ1904 was digested with SmaI and XbaI, a 5.0-kb fragment carrying dndC-E was introduced into the corresponding sites of pUC18 to generate pJTU1205. Using pJTU1205 as template, and xtg3 (with introduced BglII site) and xtg4 as primers,

a 0.9-kb PCR product was amplified and cloned into pMD18-T to give pJTU1209. The 0.5-kb AflII-BglII fragment from pJTU1209 was used to replace the 1.3-kb corresponding region from pJTU1205 Adriamycin chemical structure to generate pJTU1210 with an 819-bp in-frame deletion in dndC. The 4.8-kb AflII-XbaI fragment of pHZ1904 was replaced by the 4.0-kb

AflII-XbaI fragment of pJTU1210 to generate pJTU1211, which carried dndC with an 819-bp in-frame deletion. Vector construction for dndD deletion: using pJTU1205 as template, and xtg5 (with introduced AgeI site) and xtg6 as primers, a 0.5-kb PCR product was amplified and cloned into pMD18-T Trichostatin A datasheet to give pJTU1212. The 0.4-kb BglII-AgeI fragment from pJTU1212 was used to replace the 2.1-kb corresponding region of pJTU1205 for generation of pJTU1213 with a 1704-bp in-frame deletion in dndD. The 4.8-kb AflII-XbaI fragment of pHZ1904 was replaced by the 3.1-kb AflII-XbaI fragment of pJTU1213 to generat pJTU1214, which carried dndD with a 1704-bp in-frame deletion. Vector construction for dndE deletion: using pJTU1205 as template, and xtg7 and xtg8 (with introduced AgeI and AvrII sites) as primers, a 0.7-kb PCR product was amplified and cloned into pMD18-T to give pJTU1215. The 0.6-kb AgeI-MluI fragment from pJTU1215 was used to replace a 1.0-kb corresponding region of pJTU1205 to generate pJTU1217 with a 0.4-kb deletion Ku-0059436 cost traversing dndD and dndE. Using pJTU1205 as template, and xtg9 (with introduced

AvrII site) and xtg10 as primers, a 1.0-kb PCR product was amplified and cloned into pMD18-T to give pJTU1216. The engineered 0.9-kb BstXI-AvrII fragment from pJTU1216 was used to replace a 0.7-kb corresponding region of pJTU1217 to generate pJTU1218 with a 216-bp in-frame deletion Phospholipase D1 in dndE only. The 4.8-kb AflII-XbaI fragment of pHZ1904 was replaced by the 4.6-kb fragment corresponding fragment of pJTU1218 for to generate pJTU1219, which carried dndE with 216-bp in-frame deletion. pHZ2862, pJTU1202, pJTU1211, pJTU1214, pJTU1219 were introduced into HXY6 by conjugation from E. coli ET12567 carrying pUZ8002 [25]. Construction of the expression vectors used in Streptomyces each carrying an independent dnd gene dndA expression vector: a 1.2-kb engineered NdeI-BamHI fragment carrying dndA from pHZ882 was inserted into the corresponding sites of pHZ1272 to give pJTU2001.