Algal biofuels: novel approaches to strain improvement.

开发生物燃料作为化石燃料的替代品具有全球重要性。目前，人们对用更经济和生态上可接受的生物质来源取代基于农作物的生物燃料有相当大的兴趣。一个有希望的替代方案是微藻，因为这些光合生物具有非常高的生长速率，可以在不使用肥沃土地的情况下进行培养，并且具有将生物质生产与从工业烟道气中捕获CO2或废水处理相结合的潜力。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，但主要的生物挑战是开发具有大规模培养所需特性的合适菌株。这些包括高脂质含量，快速生长，耐高CO2和易于收获。这个学生的目标是开发新的遗传方法来改善应变。特别是生产在活跃生长期间具有高脂质含量的菌株。该项目建立在藻类分子生物学学术导师的专业知识和脂质生物学和分析的工业合作伙伴的基础上，以确保广泛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：遗传筛选，以分离高脂突变体，并确定参与碳分配的调控基因。藻类中储存脂质（三酰甘油酯，TAG）的产生在生长缓慢的营养胁迫条件下是最大的，并且是碳水化合物和TAG之间固定碳分配的转变的结果。破坏这种分配控制的突变应该允许分离在生长最大的非胁迫条件期间积累高水平TAG的藻类。此外，突变体的分子分析将提供有价值的洞察控制机制。通过诱变细胞群，然后使用亲脂性荧光染料筛选荧光增加的菌落，鉴定在活跃生长期间具有升高水平的储存脂质的突变菌落。在该筛选中将使用两个物种：将使用莱茵衣藻，因为基因组和分子遗传学工具已经可用于受影响基因的作图和表征，以及Neochloris oleoabundans，因为这种产油衣藻预期产生具有非常高的TAG含量的突变体，因此可能具有商业价值。（ii）。脂质生物合成的代谢工程。在藻类中，脂质生物合成仅发生在叶绿体中。由于衣原体叶绿体基因组的遗传工程是完善的，因此通过引入可能改善途径通量的生物合成酶的基因来上调TAG生物合成应该是可行的。珀顿实验室已经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中。将检查所得菌株的脂质谱，以确定这些遗传操作的影响。iii）通过原生质体融合的新型藻类杂交体。原生质体的细胞融合以产生异核体是已广泛用于产生植物和酵母的体细胞杂种系的成熟技术。然而，尽管对藻类的早期研究很有希望，但这种技术尚未被利用来创造新的藻类杂交体。该项目将开发藻类的原生质体融合技术，并测试我们是否可以创造出稳定的杂交种，将不同藻类的理想表型联合收割机结合在一起。分子标记将用于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界限和利用微藻中发现的多样性来创造新菌株，而不使用转基因技术。

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.