## 3 Discussion  
The wild-type _S. xiamenensis_ strain M1-94P, an affined strain of _S. xiamenensis_ 318, was isolated from deep-sea sediments and used as a biosource in the search for new benzopyran compounds. The possibility of discovering novel chemical structures may be higher when shifting to an alien niche, such as deep-sea derived microbial strains, from the view of chemical ecology, but the production of secondary metabolites in the deep-sea derived microbes is normally quite low, due to physiological stress, when they are cultivated under the usual laboratory conditions. Therefore, the use of a rational strain development approach such as ribosomal engineering may be useful to increase the production of secondary metabolites in the laboratory. Improving the production of the secondary metabolites by bacteria by modulating the ribosome components as well as other translating factors or RNAP [15] has several advantages, including the ability to screen for drug-resistant mutations by simple selection on drug-containing plates and without prior genetic mutagenesis [19]. It was reported that the introduction of multiple drug resistant mutations had the cumulative effect of increasing the production of secondary metabolites, which led to a dramatic increase in the production of the bioactive compounds. Rifampicin and streptomycin is a pair of drugs widely used in ribosomal engineering to screen potential high producers among drug-resistant mutants. In our case, the introduction of combined drug resistance to rifampicin and streptomycin in _S. xiamenensis_ M1-94P was a successful method to improve the production of benzopyran compounds for chemical isolation, although the detailed mechanism of the enhancement of the specific natural product compound remains unknown. Because spontaneous rifampicin resistance mutants always have point mutations clustered in the so-called Rif domain on the _rpoB_ gene (encoding the \(\beta\)-subunit of RNA polymerase) and could mimic the ppGpp binding effect on the mutated RNAP [20], we decided to screen for a Rif resistant mutant as the first step in the strain development. We have sequenced the _rpoB_ gene fragment of nine Rif mutants including six Rif mutants from \(5\times\mathrm{MIC}\) and 3 Rif mutants from \(10\times\mathrm{MIC}\). All of the mutants have a point mutation in the Rif domain in the _rpoB_ gene. There are two types of streptomycin resistance that could effectively alter ribosomal function. Determination of the mutation in the ribosomal RNA that engenders a low level of streptomycin resistance is not easy, but the introduction of a mutation into the highly conserved ribosomal protein S12 is conveniently achieved by site-directed mutagenesis using PCR (polymerase chain reaction). Furthermore, certain _rpsL_ mutations, resulting in the amino acid substitution of either K88E or L90K, that confer resistance to Str can increase the production of secondary metabolites in _Streptomyces_[21, 22, 23, 24, 25]. Thus, it is more rational to engineer ribosomal protein S12 as the second step in the strain development; thus, we introduced a mutated _rpsL_ gene to confer streptomycin resistance to the Rif mutant M1-5R22 and to interfere with its ribosomal function. As shown in the HPLC profiles, the production of the target compounds, xiamenmycin increased drastically in the Rif-Str double resistance strains M5 and M6. Xiamenmycin, xiamenmycin C (**1**) and D (**2**), with highly similar chemical structures, were all isolated from mutant M6 of deep-sea derived _S. xiamenensis_ M1-94P. Compared with xiamenmycin, obtained from our previous study [11], xiamenmycin C had the same benzopyran skeleton and isoprene side chain. The only difference in the structures is in the amino acid moiety (on position 1'). Compound **1** is the possible precursor of xiamenmycin during biosynthesis. Compound **2** is methylester of xiamenmycin, which may be synthesized in the tailing step of the biosynthetic pathway.