Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0212, USA.
FEBS J. 2012 Apr;279(7):1243-51. doi: 10.1111/j.1742-4658.2012.08517.x. Epub 2012 Mar 1.
Filamentous marine cyanobacteria are extremely rich sources of bioactive natural products and often employ highly unusual biosynthetic enzymes in their assembly. However, the current lack of techniques for stable DNA transfer into these filamentous organisms, combined with the absence of heterologous expression strategies for nonribosomal cyanobacterial gene clusters, prohibit the creation of mutant strains or the heterologous production of these cyanobacterial compounds in other bacteria. In this study, we evaluated the capability of a derivative of the model actinomycete Streptomyces coelicolor A3(2) to express enzymes involved in the biosynthesis of the protein kinase C activator lyngbyatoxin A from a Hawaiian strain of Moorea producta (previously classified as Lyngbya majuscula). Despite large differences in GC content between these two bacteria and the presence of rare TTA/UUA leucine codons in lyngbyatoxin ORFs we were able to achieve expression of the cytochrome P450 monooxygenase LtxB and reverse prenyltransferase LtxC in S. coelicolor M512 and confirmed the in vitro functionality of S. coelicolor overexpressed LtxC. Attempts to express the entire lyngbyatoxin A gene cluster in S. coelicolor M512 were not successful because of transcript termination observed for the ltxA gene, which encodes a large nonribosomal peptide synthetase. However, these attempts did show a detectable level of cyanobacterial promoter recognition in Streptomyces. Successful expression of lyngbyatoxin A proteins in Streptomyces provides a new platform for biochemical investigation of natural product enzymes from Moorea strains.
丝状海洋蓝藻是生物活性天然产物的极其丰富来源，并且在其组装中经常采用非常不寻常的生物合成酶。然而，目前缺乏将稳定的 DNA 转移到这些丝状生物中的技术，再加上非核糖体蓝藻基因簇的异源表达策略的缺失，这就阻止了突变株的创建或这些蓝藻化合物在其他细菌中的异源生产。在这项研究中，我们评估了模式放线菌变铅青链霉菌 A3(2) 的衍生物表达参与从夏威夷 Moorea producta（以前归类为 Lyngbya majuscula）菌株生物合成蛋白激酶 C 激活剂 lyngbyatoxin A 的酶的能力。尽管这两种细菌的 GC 含量存在很大差异，并且在 lyngbyatoxin ORFs 中存在罕见的 TTA/UUA 亮氨酸密码子，但我们能够在变铅青链霉菌 M512 中实现细胞色素 P450 单加氧酶 LtxB 和反向 prenyltransferase LtxC 的表达，并证实了变铅青链霉菌过表达 LtxC 的体外功能。由于 ltxA 基因（编码一个大型非核糖体肽合酶）观察到转录终止，因此在变铅青链霉菌 M512 中表达整个 lyngbyatoxin A 基因簇的尝试并未成功。然而，这些尝试确实显示了链霉菌中可检测到的蓝藻启动子识别水平。在链霉菌中成功表达 lyngbyatoxin A 蛋白为来自 Moorea 菌株的天然产物酶的生化研究提供了一个新的平台。
