Genome Organisation for Optimising Synthetic Secondary Metabolism

该项目将研究基因组组织，特别是如何改变酵母基因组中代谢酶基因的位置和排列可以改变它们产生的代谢物的量，在这种情况下，抗生素青霉素。抗生素只是地球上各种生物自然进化产生的一类复杂化学物质。由生命产生的化学物质被称为代谢，而专门细胞产生的更复杂的高价值化学物质（例如在植物中）被称为次级代谢物，这些包括今天已知的大多数治疗分子。细胞中次级代谢产物的产生需要特定的酶，这些酶通常由严格控制（调节）的基因编码。随着我们对生物学的理解通过许多基础研究突破而得到改善，科学家们现在正在寻求重新设计次级代谢，以在细胞系统中产生易于使用的有价值的化合物。像啤酒酵母这样的微生物是完美的，因为它们易于培养，因此可以从糖等可再生资源中廉价地生产高产量的有价值的化合物。执行这种“代谢工程”最有希望的方法是使用所谓的合成生物学方法，其中基因及其控制器被视为具有明确定义的行为的模块化组件，然后以基于合理设计的方式组合。到目前为止，代谢工程的合成生物学方法已经成功地生产出抗疟疾药、化妆品和生物燃料等化合物，方法是提取植物和外来微生物中发现的酶的基因，并将这些基因结合到酵母等工业微生物中。在这些微生物中实现高产的关键是对每个细胞中酶的精确水平进行精细控制。目前科学家尚未探索的一种调节酶水平的方法是这些酶的基因在细胞基因组中的物理排列。众所周知，基因组中的基因定位和方向在所有生命形式的基因表达中起着重要作用。最近，还已经确定，在天然进行次级代谢的细胞（例如植物细胞）中，构成途径的基因的位置通常是紧密保守的，通常发生在被称为“亚端粒区域”的区域中的基因“簇”中。显然，如果自然和进化是正确的，那么路径基因添加到基因组中的位置一定会影响酶的水平，从而影响路径的输出。本项目旨在测试这样一种假设，即改变基因组中路径基因的位置可以提高高价值次级代谢产物的产量，在这种情况下，青霉素。编码青霉素途径的基因将被添加到实验室酵母菌株的基因组中，并选择产生最大量青霉素的细胞。然后将在一系列实验中监测产生的数量，其中途径基因在基因组中的五个不同位置周围系统地重排。这将为基因组中基因的排列如何影响该途径提供有价值的信息。最后，这些途径基因将被放置在一种经过工程改造的实验室菌株中，这种菌株是专门设计的，可以在进化压力下改变其基因组的某些部分。该菌株将与细菌竞争，并自动重新排列途径基因，以产生最多的青霉素。因此，该项目将为代谢工程提供重要的新合成生物学方法，并揭示基因组和基因组进化基础科学的宝贵新信息。

This project will research genome organisation and in particular how changing the location and arrangement of metabolic enzyme genes within a yeast genome can alter the amount of the metabolite they produce, in this case the antibiotic penicillin. Antibiotics are just one class of complex chemicals that the diverse array of organisms on Earth has naturally evolved to produce. The production of chemicals by life is known as metabolism and the more complex high-value chemicals that specialist cells produce (e.g. in plants) are called secondary metabolites and these include most therapeutic molecules known today. Production of secondary metabolites in cells requires specific enzymes which are encoded by genes usually under strict control (regulation). As our understanding of biology improves through many fundamental research breakthroughs, scientists are now looking to re-engineer secondary metabolism to produce valuable compounds in cellular systems that are easy to work with. Microbes like brewer's yeast are perfect as they are easy to culture and so could cheaply produce high yields of valuable compounds from renewable resources like sugar.The most promising way to perform this 'metabolic engineering' is to use what is known as a synthetic biology approach, where genes and their controllers are treated as modular components with well-defined behaviours and then combined in a rational design-based manner. So far the synthetic biology approach to metabolic engineering has been successful in producing compounds useful as anti-malarials, cosmetics and biofuels by taking genes for enzymes found in plants and exotic microbes and combining these inside industrially-used microbes such as yeast. Crucial for achieving high yields of production in these microbes is fine-control over the precise levels of enzymes in each cell.One method of tuning enzyme levels that is currently unexplored by scientists is how the genes for these enzymes are physically arranged within a cell's genome. It is already known that gene location and orientation within a genome plays an important role in gene expression in all forms of life. Recently, it has also been established that in cells that naturally perform secondary metabolism, such as plant cells, the location of genes that make up a pathway is often tightly conserved, usually in occurring in gene 'clusters' in areas known as 'sub-telomeric regions'. Clearly, if nature and evolution are correct, then the location of where pathway genes are added to a genome must affect the enzyme levels and therefore the pathway output.This project seeks to test the hypothesis that modifying pathway gene location within a genome can result in improved yields of a high-value secondary metabolite, in this case penicillin. The genes encoding the penicillin pathway will be added to the genome of a lab yeast strain and cells will be selected that produce the greatest amounts of penicillin. The amount produced will then be monitored in a series of experiments where the pathway genes are systematically rearranged around five different places in the genome. This will give valuable information on how the arrangement of genes in the genome affects the pathway. Finally, the pathway genes will be placed in an engineered lab strain specifically designed to shuffle parts of its genome when under an evolutionary pressure. The strain will be grown to compete against bacteria and in doing will automatically rearrange pathway genes to produce the most penicillin. This project will therefore provide an important new synthetic biology approach to metabolic engineering, and also uncover valuable new information on the fundamental science of genomes and genome evolution.