Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

中文翻译：

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隐藻酰胺在蓝细菌宿主中的异源表达

丝状海洋蓝藻产生多种生物活性分子，这些分子由聚酮化合物合酶、非核糖体肽合成酶和由大型生物合成基因簇编码的杂合途径产生。这些蓝藻天然产物代表了潜在的药物先导；然而，由于通常可从天然生物中获得的化合物数量有限，彻底的药理学研究受到阻碍。此外，由于无法在本地生产者中进行基因操作，对生物合成基因簇和酶促途径的研究一直很困难。在这里，我们报告了一组用于在蓝藻细长聚球藻PCC 7942 和鱼腥藻中异源表达生物合成基因簇的遗传工具( Nostoc ) PCC 7120。为了促进两种菌株中基因簇的转移，我们设计了一种鱼腥藻菌株，该菌株含有用于在中性位点进行染色体重组的S. elongatus同源序列，并设计了一种基于 CRISPR 的策略来有效地获得分离的双重组鱼腥藻的克隆。这些遗传工具被用来表达来自海洋蓝藻大28.7 kb的cryptomaldamide生物合成基因簇Moorena（莫雷阿岛）producens JHB在两个模型菌株。S. elongatus不产生隐马尔酰胺；然而，在鱼腥藻中获得了高滴度的隐马尔酰胺. 本研究中开发的方法将促进从海洋蓝藻和复杂宏基因组样本中分离的生物合成基因簇的异源表达。