Algal biofuels: novel approaches to strain improvement.

发展生物燃料作为化石燃料的替代品在世界范围内具有重要意义。目前，人们有相当大的兴趣用更经济和更生态可接受的生物质来源来取代以农作物为基础的生物燃料。一种有希望的替代方案是微藻，因为这些光合作用生物具有非常高的生长率，可以在不使用肥沃土地的情况下进行培养，并有可能将生物质生产与从工业废气中捕获二氧化碳或用于废水处理相结合。虽然在藻类生物燃料成为商业现实之前，需要解决许多工程和下游加工问题，但主要的生物学挑战是开发具有大规模养殖所需特性的合适菌株。这些因素包括高脂肪含量、快速生长、对高二氧化碳的耐受性以及易于收获。这个项目的目标是开发新的遗传方法来改良菌株。特别是，在活跃的生长过程中产生高脂肪含量的菌株。该项目建立在藻类分子生物学学术导师和脂类生物学和分析行业合作伙伴的专业知识基础上，以确保广泛和有效的学生培训和成功的项目。该项目的三个关键方面是：1：分离高脂突变体的基因筛查，并识别参与碳分配的调控基因。在生长缓慢的营养胁迫条件下，藻类中储存类脂(三酰甘油酯，TAG)的产量最大，这是碳水化合物和TAG之间固定碳分配转移的结果。破坏这种分割控制的突变应该可以分离出在生长最快的非应激条件下积累高水平标签的藻类。此外，对突变体的分子分析将为控制机制提供有价值的见解。在活跃生长过程中储存脂质水平升高的突变菌落将通过诱变细胞群体来识别，然后使用亲脂荧光染料筛选荧光增强的菌落。在这一筛选中将使用两种：莱茵衣藻，因为基因组和分子遗传学工具已经可用于绘制和表征受影响的基因，以及新氯藻，因为这种油性藻类预计会产生具有非常高标签含量的突变体，因此可能具有商业价值。Ii)。脂类生物合成代谢工程。在藻类中，脂类的生物合成只发生在叶绿体中。由于衣藻叶绿体基因组的基因工程已经建立，通过引入生物合成酶的基因来上调TAG的生物合成应该是可行的，这可能会改善途径的通量。珀顿实验室开发了一系列叶绿体表达载体，这些载体将用于将各种基因组合插入叶绿体基因组。结果菌株的脂谱将被检查，以确定这些遗传操作的效果。3)通过原生质体融合的新型藻类杂交种。利用原生质体的细胞融合来产生异核体是一种成熟的技术，已被广泛用于生产植物和酵母的体细胞杂交系。然而，尽管对藻类的早期研究很有希望，但这项技术还没有被用于创造新的藻类杂交品种。该项目将为藻类开发原生质融合，并测试我们是否可以创造出稳定的杂交品种，将不同藻类物种的理想表型结合在一起。分子标记将被用来评估异核体的遗传状态和稳定性。这种体细胞育种方法特别有吸引力，因为它允许通过跨越物种边界和利用微藻中发现的多样性来创造新的菌株，而不需要使用转基因技术。

The development of biofuels as an alternative to fossil fuels is of world-wide importance. Currently there is considerable interest in replacing biofuels based on crop plants with more economic and ecologically acceptable sources of biomass. One promising alternative is microalgae since these photosynthetic organisms have very high growth rates, can be cultured without using fertile land and have the potential for coupling biomass production to CO2 capture from industrial flue gas, or to wastewater treatment. Whilst there are many engineering and downstream processing issues that need to be addressed before algal biofuels become a commercial reality, the major biological challenge is the development of suitable strains that have the necessary characteristics for mass culture. These include high lipid content, rapid growth, tolerance to high CO2, and ease of harvesting. The goal of this studentship is to develop novel genetic approaches to strain improvement. In particular, to produce strains with high lipid content during active growth. The project builds on the expertise of the academic supervisor in algal molecular biology, and the industrial partner in lipid biology and analysis to ensure a broad and effective student training and a successful project. The three key aspects of the project are: 1: A genetic screen to isolate high-lipid mutants and to identify regulatory genes involved in carbon partitioning. The production of storage lipids (triacylglycerides, TAGs) in algae is maximal under conditions of nutrient stress where growth is slow, and is the result of a shift in the partitioning of fixed carbon between carbohydrates and TAGs. Mutations that disrupt this partitioning control should allow the isolation of algae that accumulate high levels of TAGs during non-stress conditions where growth is maximal. Furthermore, molecular analysis of the mutants will provide valuable insight into the control mechanism. Mutant colonies that have elevated levels of storage lipids during active growth will be identified by mutagenising a cell population, and then screening the colonies for those with increased fluorescence using a lipophilic fluorescent dye. Two species will be used in this screen: Chlamydomonas reinhardtii will be used since genomic and molecular-genetic tools are already available for the mapping and characterisation of the affected genes, and Neochloris oleoabundans since this oleaginous alga is expected to yield mutants with very high TAG content, and may therefore be of commercial value. ii). Metabolic engineering of lipid biosynthesis. In algae, lipid biosynthesis takes place exclusively in the chloroplast. Since the genetically engineering of the Chlamydomonas chloroplast genome is well-established, it should be feasible to up-regulate TAG biosynthesis by introducing genes for biosynthetic enzymes likely to improve the flux of the pathway. The Purton lab has developed a series of chloroplast expression vectors and these will be used to insert various gene combinations into the chloroplast genome. The lipid profile of resulting strains will be examined to determine the effect of these genetic manipulations. iii) Novel algal hybrids via protoplast fusion. Cell fusion of protoplasts to create heterokaryons is a well-established technique that has been widely used to produce somatic hybrid lines of plants and yeasts. However, despite promising early research on algae, this technique has not been exploited to create novel algal hybrids. The project will develop protoplast fusion for algae and test whether we can create stable hybrids that combine desirable phenotypes from different algal species. Molecular markers will be used to assess the genetic state and stability of the heterokaryon. This somatic breeding approach is particularly attractive since it allows the creation of novel strains by crossing species boundaries and exploiting the diversity found amongst the microalgae, without using GM technology.