Enhancement of geldanamycin production by pH shock implementation and enhancement of pH shock tolerance and geldanamycin secretion by heterologous expression of $Na^+ /H^+$ antiporter

Abstract

An acidic pH shock implementation for the enhancement of geldanamycin production and enhancement of acidic-pH shock tolerance and geldanamycin secretion by heterologous expression of a putative $Na^+/H^+$ antiporter have been studied. A preliminary study on effects of different pH-shock modes on secondary metabolism in S. coelicolor A3(2) showed that only acidic-pH shock affected secondary metabolism positively. With this evidence, various ways of acidic-pH shock implementation were tried for geldanamycin production in batch culture of S. hygroscopicus JCM4427. About two-fold enhancement of geldanamycin production was achieved with an abrupt pH shift from 6.1 to 5.0. Excessively strong or frequent acidic-pH shocks, however, caused cell damage resulting in extremely low geldanamycin production. This was considered to be potentially a major obstacle in applying pH shock technique. To overcome this problem, the development of pH shock tolerant strains was considered to be indispansable. $Na^+/H^+$ antiporters were expected to give a solution to this problem of overcoming the adverse effects of pH shock application. It was anticipated that when heterologously expressed, it could enhance the pH shock tolerance of the host by adding to the system its capability of pH homeostasis. In order to confirm its potentials in enhancing pH shock tolerance, a putative $Na^+/H^+$ antiporter of SCO3185 (designated as sha4) originated from S. coelicolor A3(2) was expressed in cation/$H^+$ antiporter-deficient E. coli strains and S. hygroscopicus JCM4427. Recovery of cation efflux activity, and enhancement of acidic-pH shock tolerance and salt tolerance were observed when sha4 was expressed in these E. coli strains. The sha4-transformed S. hygroscopicus JCM4427 showed a higher enhanced cell growth and geldanamycin production than the host strain under $K^+$ stress. Under $Na^+$ stress no significant differences were observed. No significant enhancement of geldanamycin production du...