Linking single cell phenotype to gene expression by single cell transcriptions

布景主题：工业生物技术和生物能源在这个项目中，最初的目标是从细菌培养物中纯化足够数量的抗真菌化合物，主要使用NMR方法进行完整的结构测定。一旦结构已知，生物合成的工作将开始。使martillamide编码聚酮合酶（PKS）和非核糖体肽合成酶（NRPS）蛋白的基因簇和部分结构预测可以基于酰基转移酶和腺苷酸化结构域的预测特异性进行。然而，仍有许多未知的，例如起始单位，一个独特的黄素单加氧酶结构域在NRPS中的作用，以及簇中其他酶的作用。这是可能的，在martillamide中的一些氨基酸残基将已被修改，我们将调查这些是否已被修改前或后NRPS通过饲料合成的前体选择的基因敲除菌株，看看这是否恢复生产martillamide。此外，簇中的其他酶（氨基转移酶、水解酶、氧化还原酶和脱氢酶）将过表达，以测试它们对推定底物（或合成类似物）的酶活性。基因工程、半合成和非天然前体的代谢掺入都将被尝试作为制备马替拉胺的修饰形式的方法，并且将测试这些化合物的抗真菌活性以开始建立抗真菌活性的结构-活性关系（SAR）。这些酶的底物特异性，结构和机制将在体外进行研究，以期工程改造的酶，更有效地使matillamide类似物。

Theme: Industrial Biotechnology and BioenergyIn this project the initial objective will be to purify the antifungal compound)(s) from the bacterial cultures in sufficient quantity for a full structure determination using mainly NMR methods. Once the structure is known work on the biosynthesis will begin. The gene cluster that makes matillamide encodes for both polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) proteins and a partial structure prediction can be made on the basis of the predicted specificities of the acyl transferase and adenylation domains. However much remains unknown, e.g. the starter unit, the role of a unique flavin mono-oxygenase domain in the NRPS, and the role of the other enzymes in the cluster. It is likely that some of the amino acid residues in matillamide will have been modified and we will investigate whether these have be modified pre- or post-NRPS by feeding synthesised precursors to selected gene knock-out strains to see if this restores production of matillamide. Additionally the additional enzymes in the cluster (an aminotransferase, a hydrolase, an oxidoreductase, and a dehydratase) will be over-expressed, to test their enzymic activity on their putative substrates (or synthetic analogues). Genetic engineering, semi-synthesis and metabolic incorporation of unnatural precursors will all be tried as ways to make modified versions of matillamide and the anti-fungal activity of these compounds will be tested to start to build up a structure-activity relationship (SAR) for the antifungal activity. The substrate specificity, structures and mechanisms of these enzymes will be investigated in vitro with a view to engineering modified enzymes to more efficiently make matillamide analogues.