Novel tools and technologies to optimise metabolic engineering in diatoms

近年来，人们对微藻作为生物技术宿主的兴趣迅速增加。与传统的代谢工程模式生物、细菌和酵母相比，微藻是生产高价值化合物（如萜类化合物）的可持续选择。由于其巨大的结构复杂性和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，生物技术方法生产萜类化合物作为一种商业上可行的方法被广泛探索。随着饮用水成为一种有限的资源，海洋微藻特别有吸引力，因为它们能够在盐和废水上生长。在三角藻等微藻中进行萜类化合物的工程改造需要引入萜类合成酶和细胞色素P450酶。目前在三角霉代谢工程中看到的挑战是转基因转录水平总体较低，以及随着时间的推移最终失去表达。我们假设代谢和表观遗传效应都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三角霉的转基因表达，从而提高其作为生物技术宿主的质量。利用产生二萜类氧化manoyl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缺失的分子原因。目前实验室中可用的菌株是通过将转基因表达盒随机插入基因组中产生的。这些菌株中转基因的确切基因组位置将被确定，以将着陆点与表达水平联系起来。由于染色质的特性，一些基因组位置可能比其他位置更适合插入异源序列。如果找到理想的常染色质位点，我们将使用CRISPR/Cas9技术在这些确切的位点上靶向额外的转基因，并测试这是否提高了表达水平和/或稳定性。除了基因组位置外，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因子的基因来评估，如DNA和组蛋白甲基转移酶以及组蛋白去乙酰化酶。具有较少表观遗传变异和基因沉默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，我们将确定一个潜在靶标列表，然后使用CRISPR/Cas9进行修改。除了对转化体的遗传和表观遗传影响外，代谢工程还可能对细胞施加压力，从而导致细胞代谢的重编程和碳通量的改变。我们将通过测量重要的代谢物来描述转化细胞的代谢状态，如类胡萝卜素和叶绿素色素，它们利用与二萜相同的前体。研究异源萜类化合物生产对三角蕨细胞稳态的影响，有助于在不影响细胞生长和活力的情况下，集中碳通量生产萜类化合物。最后，我们将通过设计一种能够精确激活转基因转录的启动子来扩展三角角霉的生物技术工具包。蛋氨酸合成酶（METE）基因的维生素b12抑制启动子将成为“开启开关”启动子生成的起始模板。通过打乱METE启动子的基序，我们希望能够为异源表达盒的转录激活创建一个可靠的b12独立开关。

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.