13TSB_SynBio: Synthetic biology to improve antibiotic production

细菌感染是全球范围内的主要死亡原因，抗生素提供了控制它们的关键资源。莫匹罗星是一种成功的抗生素，用于抗革兰氏阳性细菌，特别是MRSA，它与医院和社区获得性感染有关，对目前可用的大多数抗生素都有耐药性。它也是一种标准的治疗方法，可以从医护人员的皮肤和鼻子中去除MRSA。抗生素在中国和其他发展中国家的市场正在增长，葛兰素史克希望在不扩大工厂/发酵产能或运营成本的情况下增加产量。Thomas团队对土壤细菌荧光假单胞菌中的莫匹罗星生物合成簇进行了广泛的研究，并研究了这些基因是如何启动和控制的。结果，我们有了通过操纵基因簇来增加产量的明确策略。然而，制造莫匹罗星的蛋白质工厂的编码基因集是复杂的，占据了大约75,000个碱基对的DNA片段。该簇中有30多个基因，因此很难操作。合成生物学(从化学制造的DNA构建基因到我们自己的设计)应该提供一种方便的方法来做到这一点，这个项目提供了一个验证这一想法的机会。通过重建基因，我们可以改变密码，使其优化以快速和增加蛋白质合成，同时我们可以将DNA分割成方便的“生物砖”(合成生物学的构件)，这些生物砖可以按不同的顺序组装，并补充额外的DNA序列，以增加它们被开启的程度。相信当前的基因簇并不是配置基因的唯一有效方法，因为我们发现，构成相关质粒硫代马利诺的基因以不同的顺序排列。最重要的是，我们发现，在现有的遗传组织中，增加激活剂MupR的生产可以使产量增加20倍。因此，我们将首先引入产生更多MupR的突变，然后我们将系统地插入以MupR依赖的方式促进这些基因表达的DNA，以提高每个细菌的生产率。我们将利用技术技术来评估这些变化的影响，并看看它如何影响摇瓶中的抗生素生产，然后在发酵罐中进行更大规模的生产。如果成功，这些变化将被纳入到新基因的设计中。基因簇的另一个特征是基因的顺序不是很合逻辑--通常一个簇中的基因按照它们在生化途径中的工作方式排列在一起。因此，我们将对mupirocin基因(BioBrick)进行重组，以提高途径效率，并筛选用于提高产量的衍生品。我们将使用一种酶(Int)来实现这一点，这种酶可以故意改组细菌中的基因。我们将插入DNA，允许Int在BioBricks之间工作，然后瞬时表达Int，以改组基因，产生许多基因顺序的排列。这种方法已经被其他方法验证，并被证明提高了大肠杆菌色氨酸生物合成操纵子的效率-一个研究得很好的模型系统。细菌将在实验室和发酵罐中进行增产评估。最后，为了探索如何进一步改进基因，我们将在途径的关键模块中添加额外的功能单元，以提高吞吐能力并融合基因以创造新的多功能基因。基因融合可以通过确保蛋白质伴侣折叠在一起，随后更有效地催化连续的酶步骤来提高效率。这两种变化的例子都可以在其他生物合成工厂找到。

Bacterial infections are a major cause of death world wide and antibiotics provide one key resource for controlling them.Mupirocin is a successful antibiotic used against Gram positive bacteria, particularly MRSA which is associated with bothhospital- and community-acquired infections and is resistant to most currently available antibiotics. It is also a standardtreatment to remove MRSA from the skin and nose of healthcare workers. The market for the antibiotic is growing in Chinaand other parts of the developing world and GSK wish to increase production without expanding production-plant/fermentercapacity or running costs. The Thomas group have carried out extensive research on the mupirocin biosynthetic cluster inthe soil bacterium Pseudomonas fluorescens and have studied how the genes are switched on and controlled. As a result,we have clear strategies for increasing production by manipulating the gene cluster.However, the set of genes coding for the protein factory that makes mupirocin is complex and occupies a segment of about75,000 base pairs of DNA. There are more than 30 genes in the cluster so that it is difficult to manipulate it. Syntheticbiology (building the genes from chemcially made DNA to our own design) should provide a convenient way to do this andthis project gives an opportunity to validate that idea. By rebuilding the genes we can change the code so that it isoptimised for fast and increased protein synthesis and at the same time we can split the DNA into convenient "Biobricks"(the building blocks for Synthetic Biology) which can be assembled in different orders and supplemented with additionalDNA sequences that increase the extent to which they are switched on.Confidence that the current gene cluster is not the only efficient way to configure the genes comes from our discovery thatthe genes that make a related plasmid called thiomarinol are arranged in a different order. On top of that we have foundthat increased production of the activator MupR, in the existing genetic organisation, can increase production up to 20-fold.We will therefore first introduce mutations that produce more MupR and we will then systematically insert DNA thatpromotes expression of these genes in a MupR-dependent way, to increase the productivity of each bacterium. We will usestate of the art techniques to assess the effect of these changes and see how it affects antibiotic production in shake flasksand then on a larger scale in fermenters. If successful, these changes will be incorporated into the design of the newgenes.Another feature of the gene cluster is that the order of genes is not very logical - often genes in a cluster are lined up in theway they work in the biochemical pathway. We will therefore shuffle the mupirocin gene (Biobrick) order to increasepathway efficiency and we will screen derivatives for increased production. We will do this using an enzyme (Int) thatdeliberately shuffles genes in bacteria. We will insert DNA that allows Int to work between Biobricks and then transientlyexpress Int to shuffle the genes to produce many permutations of gene order. This approach has been validated by othersand shown to improve the efficiency of the E. coli tryptophan biosynthetic operon - a well studied model system. Bacteriawill be assessed for increased production in the lab and in fermenters as above.Finally, to explore how the genes can be further improved we will add extra functional units to key modules of the pathwayto increase throughput capacity and to fuse genes to create new multifunctional genes. Gene fusions may increaseefficiency by ensuring that protein partners fold together and subsequently catalyse successive enzymic steps moreefficiently. Examples of both of these sorts of changes are found in other biosynthetic factories.