In order to determine the role of PKS I in the biosynthesis of acyltetramic acid antibiotic, Gene fragments of thioesterase II, 3-oxacyl-ACP synthase II, S- adenosylmethion ine synthetase and cytochrome P450 of Streptomyces lydicus AS 4.2501 were amplified by PCR. The function of these genes in streptolydigin biosynthesis were evaluated after the metabolites of disruption mutant strains of these genes were analyzed by LC/MS. For estimating the biomarker of Streptomyces lydicus for producing streptolydigin and the method for high-throughput screening the mutant strain, FTIR was used to detect the mycelium,which were calculated for different time, and the data were analyzed by PCA, HCA and ANN. The obtained results are as follows:As the GC content of its genomic DNA is more than 70%, the normal PCR method is not suitable for Streptomyces, and the PCR system for Streptomyces lydicus AS 4.2501 was optimized.Gene fragments of thioesterase II, 3-oxacyl-ACP synthase II, S- adenosylmethionine synthetase and cytochrome P450 of Streptomyces lydicus AS 4.2501 were firstly cloned by the optimized PCR in our laboratory. Functions of these genes were determined after the fermentation products of these genes mutant strains were analyzed by LC/MS. Thioesterase II, 3-oxacyl-ACP synthase II and S- adenosylmethionine synthetase took part in the biosynthesis of streptolydigin, whereas the cloned cytochrome P450 had no role in streptolydigin biosynthesis. Thioesterase take part in the biosynthesis of acyltetramic acid antibiotic in Streptomyces, which is different from the biosynthesis of fusarin C and equisetin being found in fungi up to now.The effects on streptolydigin of different kinds of L-Amino acid and its mechanism were investigated by adding 1 mmol/L of amino acids into fermentation culture media respectively. The results showed that streptolydigin contents were significantly decreased when L-methionine and L-argenine were added, meanwhile, L-lysine, L-isoleucine and L-tryptophane had faint inhibition role on streptolydigin biosynthesis. All amino acids used in the experiment significantly decreased S-