ANTIBIOTIC REGULATORY GENES AND METABOLIC ENGINEERING

在第一阶段，我们建议在红糖多孢子菌基因组中创建一个标记敲除突变文库，并发现至少一个影响红霉素生物合成的新突变。在第二阶段，我们计划对文库进行全面筛选，以发现所有影响红霉素生产的突变。在第三阶段，将开发代谢工程策略来创造改良菌株。虽然已经克隆并鉴定了60kb的红霉素生物合成基因簇，但尚未发现其中的调控基因。由于这一簇占基因组的不到1%，我们建议对基因组的剩余部分进行全面的搜索，以寻找调节突变。根据在密切相关的生物体中发现的结果，我们假设会发现许多影响生产的基因。传统的突变和筛选方法已经成功地在Sac中产生了这些突变。但目前还没有一个菌株改良基因通过这种方法被鉴定出来。相反，我们提出的研究应该能够鉴定出影响红霉素产生的一整套基因。这些基因的合理代谢工程有可能产生比现有菌株更好的菌株。拟议的商业应用：用于生产原料药红霉素的商业菌株负责生产一种全球每年市场价值超过6亿美元的化合物。因此，任何明显优于现有菌株的菌株都具有重要的经济价值。新的第一代和第二代红霉素衍生物的发展意味着红霉素作为化学中间体的市场将在下个世纪继续增长。

In Phase I we propose to create a library of tagged knock-out mutations in the Saccharopolyspora erythraea genome, and to find at least one new mutation that affects erythromycin biosynthesis. In Phase II we plan a comprehensive screen of the library to find all mutations that affect erythromycin production. In Phase III metabolic engineering strategies will be developed to create improved strains. Although the 60 kb erythromycin biosynthetic gene cluster has already been cloned and characterized, no regulatory genes have been found within it. Since this cluster makes up less than 1% of the genome, we propose a comprehensive search of the remaining portion of the genome for regulatory mutations. We hypothesize based on results in closely related organisms, that many genes affecting production will be found. Traditional mutate-and-screen methods have been successful in producing these mutations in Sac. erythraea, but not a single strain improvement gene has yet been identified using this approach. In contrast, the proposed study should lead to the identification of the complete set of genes affecting erythromycin production. Rational metabolic engineering of these genes has the potential of generating strains that are much better than those currently available. PROPOSED COMMERCIAL APPLICATIONS: Commercial strains for the production of the bulk pharmaceutical erythromycin are responsible for the production of a compound with a market value of more than $600 million per year, world-wide. Any strain that is significantly superior to existing strains would therefore be of significant economic value. The development of new first and second generation erythromycin derivatives means the market for erythromycin as a chemical intermediate will continue to grow well into the next century.