Linking single cell phenotype to gene expression by single cell transcriptions

在这个项目中，最初的目标是从细菌培养物中纯化出足够数量的抗真菌化合物，以便主要使用核磁共振方法进行全结构测定。一旦知道了结构，生物合成的工作就开始了。合成matillamide的基因簇编码多酮合成酶（PKS）和非核糖体肽合成酶（NRPS）蛋白，可以根据预测的酰基转移酶和腺苷化结构域的特异性进行部分结构预测。然而，仍有许多未知，例如启动单元，NRPS中独特的黄素单加氧酶结构域的作用，以及簇中其他酶的作用。很可能玛蒂拉胺中的一些氨基酸残基已经被修饰，我们将通过将合成的前体喂给选定的基因敲除了菌株来研究这些氨基酸残基是否在nrps前或nrps后被修饰，看看这是否能恢复玛蒂拉胺的生产。此外，簇中的其他酶（转氨酶、水解酶、氧化还原酶和脱水酶）将被过度表达，以测试它们在假定底物（或合成类似物）上的酶活性。基因工程、半合成和非自然前体的代谢结合都将被尝试作为制造改良版本的玛蒂拉胺的方法，这些化合物的抗真菌活性将被测试，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，以期工程修饰酶更有效地制造玛蒂拉胺类似物。

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.