13TSB_SynBio: Synthetic biology to improve antibiotic production

细菌感染是世界范围内死亡的主要原因，抗生素是控制细菌感染的一个关键资源。莫匹罗星是一种成功的抗生素，用于治疗革兰氏阳性细菌，特别是与医院和社区获得性感染相关的MRSA，并且对大多数现有抗生素具有耐药性。从医护人员的皮肤和鼻子上去除MRSA也是一种标准的治疗方法。这种抗生素的市场在中国和其他发展中国家正在增长，葛兰素史克希望在不扩大生产工厂/发酵能力或运营成本的情况下增加产量。托马斯小组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开启和控制的。因此，我们有明确的策略来通过操纵基因簇来增加产量。然而，编码制造莫匹罗星的蛋白质工厂的一组基因是复杂的，占据了大约75,000个碱基对的DNA片段。这个集群中有30多个基因，因此很难操纵它。合成生物学（从化学合成的DNA到我们自己的设计来构建基因）应该提供一个方便的方法来做到这一点，而这个项目提供了一个验证这个想法的机会。通过重建基因，我们可以改变代码，使其能够快速和增加蛋白质合成，同时我们可以将DNA分裂成方便的“生物砖”（合成生物学的构建块），可以按不同的顺序组装，并补充额外的DNA序列，以增加它们被打开的程度。目前的基因簇并不是配置基因的唯一有效方式，这一信心来自于我们的发现，即构成相关质粒硫代玛油醇的基因是以不同的顺序排列的。最重要的是，我们发现在现有的遗传组织中，增加激活子MupR的产量可以使产量增加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依赖MupR的方式促进这些基因表达的DNA，以提高每种细菌的生产力。我们将使用最先进的技术来评估这些变化的影响，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，这些变化将被纳入新基因的设计中。基因簇的另一个特点是基因的顺序不是很合乎逻辑——通常，基因簇中的基因是按照它们在生化途径中的工作方式排列的。因此，我们将对莫匹罗星基因（Biobrick）进行洗牌，以提高途径效率，我们将筛选衍生物以增加产量。我们将使用一种酶（Int）来做到这一点，这种酶会故意打乱细菌中的基因。我们将插入允许Int在生物砖之间工作的DNA，然后短暂地表达Int来洗牌基因，以产生许多基因顺序的排列。这种方法已经被其他人验证，并被证明可以提高大肠杆菌色氨酸生物合成操纵子的效率——一个被充分研究的模型系统。如上所述，将在实验室和发酵罐中评估细菌以增加产量。最后，为了探索如何进一步改善基因，我们将在通路的关键模块中添加额外的功能单元，以增加吞吐量并融合基因以创建新的多功能基因。基因融合可以确保蛋白质伴侣折叠在一起，从而更有效地催化连续的酶步骤，从而提高效率。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.