Novel tools and technologies to optimise metabolic engineering in diatoms

博士项目战略主题：可再生资源和清洁生长的生物科学近年来，对微藻作为生物技术宿主的兴趣迅速增加。与代谢工程的经典模式生物细菌和酵母相比，微藻为生产高价值化合物（如萜类化合物）提供了可持续的替代品。由于它们巨大的结构复杂性和在自然界中有限的可用性，在工业规模上化学合成萜类化合物是无效的，并且生产萜类化合物的生物技术方法作为商业上可行的方法被广泛探索。随着饮用水成为一种受限制的资源，海洋微藻特别有吸引力，因为它们能够在盐和废水中生长。在微藻例如硅藻三角褐指藻中改造萜类化合物需要引入萜类脱氢酶和细胞色素P450酶。目前在三角褐指藻的代谢工程中看到的挑战是转基因转录的总体低水平以及随着时间的推移表达的最终丧失。我们假设代谢和表观遗传效应可能有助于降低转基因表达水平。该项目的目的是增强和稳定三角褐指藻中的转基因表达，从而提高其作为生物技术宿主的质量。使用产生二萜类manoyl氧化物的菌株作为监测转基因活性的模型，我们将探索转基因表达丧失的分子原因。目前实验室中可用的菌株是用随机插入基因组的转基因表达盒产生的。将鉴定这些菌株中转基因的确切基因组位置，以将着陆位点与表达水平相关联。由于染色质性质，一些基因组位置可能比其他位置更适合插入异源序列。如果找到了理想的常染色体位置，我们将使用CRISPR/Cas9技术在这些确切的位置靶向额外的转基因，并测试这是否会提高表达水平和/或稳定性。除了基因组位置，表观遗传标记对转基因表达的影响将通过突变编码表观遗传修饰剂的基因来评估，如DNA和组蛋白甲基转移酶以及组蛋白脱乙酰酶。表观遗传变异和基因沉默减少的菌株可能有利于转基因表达。基于文献研究和在其他物种中所做的工作，我们将确定一系列潜在的靶点，然后使用CRISPR/Cas9进行修饰。除了转化体中的遗传和表观遗传效应，代谢工程可能会对细胞施加压力，从而导致细胞代谢的重新编程并改变碳通量。我们将通过测量重要的代谢物，如类胡萝卜素和叶绿素色素，利用与二萜类化合物相同的前体，来确定转化细胞的代谢状态。研究三角褐指藻中异源萜类化合物的产生对细胞内稳态的影响可能有助于集中萜类化合物产生的碳通量，而不会对细胞的生长和活力产生负面影响。最后，我们的目标是通过设计一个允许精确激活转基因转录的启动子来扩展三角褐指藻的生物技术工具包。甲硫氨酸合酶（METE）基因的维生素B12阻遏型启动子将是产生“开关”启动子的起始模板。通过扰乱METE启动子的基序，我们希望能够创建用于异源表达盒的转录激活的可靠的B12非依赖性开关。

PhD project strategic theme: Bioscience for renewable resources and clean growthIn recent years, interest in microalgae as biotechnological hosts has increased rapidly. Compared to the classical model organisms for metabolic engineering, bacteria and yeast, microalgae present a sustainable alternative for producing high-value compounds, such as terpenoids. Because of their enormous structural complexity and limited availability in nature it is ineffective to chemically synthesise terpenoids on an industrial scale, and biotechnological approaches to produce terpenoids are widely explored as a commercially feasible method. With potable water becoming a restricted resource, marine microalgae are particularly attractive as they are able to grow on salt and waste water. Engineering of terpenoids in microalgae such as the diatom Phaeodactylum tricornutum requires the introduction of terpene synthases and cytochrome P450 enzymes. Current challenges seen in metabolic engineering of P. tricornutum are the overall low levels of transgene transcription as well as the eventual loss of expression over time. We hypothesise that both metabolic and epigenetic effects may contribute to decreasing transgene expression levels. The aim of this project will be to enhance and stabilise transgene expression in P. tricornutum and thus improve its quality as a biotechnological host. Using strains producing the diterpenoid manoyl oxide as a model for monitoring transgene activity, we will explore the molecular causes underlying the loss of transgene expression.Current strains available in the lab have been generated with the transgene expression cassette being inserted randomly into the genome. The exact genomic locations of the transgenes in these strains will be identified to correlate landing sites with expression levels. Some genomic locations may be preferable over others for inserting heterologous sequences, due to chromatin properties. If ideal euchromatic locations were found, we would use CRISPR/Cas9 technology to target additional transgenes at those exact locations and test whether this improves expression levels and/or stability. In addition to genomic locations, the impact of epigenetic marks on transgene expression will be assessed by mutating genes encoding epigenetic modifiers, such as DNA and histone methyl transferases, and histone deacetylases. Strains with reduced epigenetic variance and gene silencing might prove favourable for transgene expression. Based on literature research and work done in other species, we will identify a list of potential targets that will then be modified using CRISPR/Cas9.Besides genetic and epigenetic effects in transformants, metabolic engineering may impose a strain on the cells, thus resulting in reprogramming of the cells' metabolism and altering the carbon flux. We will characterise the metabolic status of transformed cells by measuring important metabolites, such as carotenoid and chlorophyll pigments, which utilise the same precursor as diterpenoids. Investigating the impact of heterologous terpenoid production in P. tricornutum on cell homeostasis may help focus the carbon flux for terpenoid production without affecting cell growth and viability negatively.Lastly, we will aim to expand the biotechnological toolkit for P. tricornutum by designing a promoter which allows precise activation of transgene transcription. The vitamin B12-repressible promoter of the methionine synthase (METE) gene will be the starting template for the generation of an 'on-switch' promoter. By scrambling the motifs of the METE promoter we hope to be able to create a reliable B12-independent switch for transcriptional activation of a heterologous expression cassette.