Directed evolution for optimization of industrially-relevant protists

鱼虾被认为是omega-3多不饱和脂肪酸（PUFAs）的重要生产者，特别是二十二碳六烯酸（DHA）和二十碳五烯酸（EPA），它们已被证明对动物和人类的健康有各种有益的影响。特别是DHA的消费被证明可以改善大脑和心脏功能，并对视网膜和免疫系统的发育产生积极影响，并有助于预防心血管疾病，因为它是膜流动性、细胞相互作用和细胞信号传导的关键因素。因此，thraustochytrids正在成为人类和动物饲料的可持续替代品，特别是在水产养殖业中。它们巨大的生物技术潜力还在于它们能够生产其他生物活性化合物，如类胡萝卜素色素、角鲨烯、胞外多糖和细胞外酶。本项目旨在利用这一生物技术潜力，通过定向进化过程增强这些原生生物的表型，从而生产出与工业相关的化合物。适应性实验室进化（ALE）迄今已被证明在特定进化压力下对细菌、酵母和微藻的菌株优化是有效的[10]。该方法的一些潜在生物技术应用包括改善生物量生产，增强菌株对工业加工中通常发生的胁迫的耐受性，诱导潜在途径的激活以提高产品耐受性和非天然化合物的生产，以及确定菌株适应的基本遗传基础bbb。与基因工程相比，定向进化策略允许多种有益突变同时发生在各种基因和调控基因网络中。此外，根据选择压力的不同，ALE可以调节许多不同的进化轨迹。根据所选择的压力因素和实验环境，项目本身预计会产生许多结果。分子生物学工具将用于描述所选菌株的生物化学特征，并阐明各种代谢途径以及它们如何相互连接以产生所需化合物。下一代测序技术和转录组分析将用于分析参与脂肪酸生产的关键基因的表达。该项目将研究多不饱和脂肪酸代谢途径的多样性，并旨在开发一种具有增加omega-3 PUFAs产量的突变菌株。为了促进ALE实验，筛选必须优化以识别具有所需功能的变体。此外，进化的thraustochytrid菌株的生长特性，脂肪酸定量和化学成分分析将通过分析化学方法进行研究，包括气相色谱法和尼罗红脂可视化结合光学显微镜。该项目是与MiAlgae有限公司合作的，MiAlgae是一家初创公司，通过回收工业副产品来培育富含Omega-3的微藻，该公司在专门设计的发酵容器中为微藻的最佳生长量身定制。通过生产对人类和动物健康有益的藻类油，MiAlgae有助于减少全球对鱼类作为人类和动物消费的Omega-3 PUFAs来源的过度依赖。由于该项目是与一个工业伙伴合作的，所选的突变菌株可能具有工业相关性。

Thraustochytrids are being recognized as important producers of omega-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which have proven to have various beneficial effects on animal and human health [1]. The consumption of DHA especially is shown to improve brain and heart functions, as well as exhibiting positive effects on the development of both retinal and immune systems and helping in the prevention of cardiovascular diseases, by functioning as a key factor in membrane fluidity, cell interactions and cell signalling. Therefore, thraustochytrids are emerging as a sustainable alternative in human and animal feeds, especially in the aquaculture industry. Their vast biotechnological potential lies, as well, in their ability to produce other bioactive compounds such as carotenoid pigments, squalene, exopolysaccharides and extracellular enzymes.This project would aim to exploit this biotechnological potential to produce industrially relevant compounds by enhancing the phenotype of these protists through the process of directed evolution. Adaptive laboratory evolution (ALE) has so far proven to be effective in strain optimization of bacteria, yeast and microalgae under chosen evolutionary pressures [2]. Some of the potential biotechnological applications for this method include improving biomass production, enhancing tolerance of strains to stresses that generally occur in industrial processing, inducing activation of latent pathways to improve product tolerance and production of non-native compounds, as well as identifying essential genetic bases of strain adaptation [3]. In contrast to genetic engineering, directed evolution strategies allow multiple beneficial mutations to occur in various genes and regulatory gene networks at a time. Additionally, depending on the selection pressure, ALE can mediate many different evolutionary trajectories [3]. The project itself is expected to have numerous outcomes depending on the chosen stress factors and the environment of experiments. Molecular biology tools will be used to characterize chosen strains biochemically and to shed light on various metabolic pathways and how they interconnect to produce a desired compound. Next-generation sequencing technology and transcriptome analysis will be used to analyse the expression of key genes involved in fatty acid production. The project will examine the diversity of polyunsaturated fatty acid metabolism pathways and aim to develop a mutant strain with increased production of omega-3 PUFAs. To facilitate the ALE experiments, screening will have to be optimized to identify variants with desired function. Additionally, growth characteristics, quantification of fatty acids and chemical composition analysis of the evolved thraustochytrid strains will be studied throughout by using analytical chemical methods including gas chromatography and Nile red lipid visualization combined with light microscopy.The project is in collaboration with MiAlgae Ltd., a start-up company that cultivates Omega-3 rich microalgae by recycling industrial co-products, in specially-designed fermenting vessels tailored for optimal microalgal growth. By producing algal oil with proven human and animal health benefits, MiAlgae contribute to decreasing global over-reliance on fish as a source of Omega-3 PUFAs for human and animal consumption. As the project is in collaboration with an industrial partner, selected mutant strains could be industrially relevant.