Page 51 - Demo

SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 55herboxidiene polyketide chain by the last module is tailored by KR and DH domains to finally result in enoyl function at 2 - 3 position. However, the mutant strain of S. chromofuscuswith site specific mutagenesis of active site of DH domain for functional inactivation results in 3-OH function in place of 2 - 3 enoyl function which escapes the proposed ring formation mechanism of herboxidiene through 3 - 7 cyclization for the formation of THP ring [4]. The S. chromofuscus is capable of producing a number of herboxidiene analogs which could complicate the analysis of the desired linear analog due to the presence of a similar number of linear analogs as the original ones. Moreover, three tailoring enzymes involved in biosynthesis of herboxidiene may or may not tailor the linear analog of herboxidiene leaving the room for a number of possibilities. The construction of the domain deactivated mutant heavily relies on prediction of domain boundaries by comparing to wellstudied PKS system from online PKS analysis tool such as antiSMASH. Thus, in case of less studied PKS systems, the defined boundaries might not be exactly same as the well-studied PKS system, and resulting mutant suffers loss in the catalytic efficiency as compared to wild type [18]. For example, the nonfunctional DH domain was found to be present in nystatin biosynthesis pathway and thought to be present just for protein structural integrity [19].Inactivation of only DH domain present on fourth module of erythromycin PKS lead to complete loss in its function upon mutation of active site histidine (H) [18]. However, in case of herboxidiene PKS surprisingly, S. chromofuscus %u0394M8DH with mutated module 8 DH domain still shows the chromatogram