Directed evolution for optimization of industrially-relevant protists

鞭毛虫被认为是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健康有各种有益的影响[1]。DHA的摄入尤其被证明可以改善大脑和心脏功能，对视网膜和免疫系统的发育以及帮助预防心血管疾病都显示出积极的影响，因为它是膜流动性、细胞相互作用和细胞信号传递的关键因素。因此，在人和动物饲料中，特别是在水产养殖业中，甲藻正在成为一种可持续的替代品。它们巨大的生物技术潜力还在于它们能够产生其他生物活性化合物，如类胡萝卜素色素、角鲨烯、胞外多糖和胞外酶。该项目旨在通过定向进化过程提高这些原生生物的表型，开发这种生物技术潜力来生产具有工业意义的化合物。到目前为止，适应性实验室进化(ALE)已被证明在选定的进化压力下对细菌、酵母和微藻进行菌株优化是有效的[2]。这种方法的一些潜在生物技术应用包括提高生物量生产，增强菌株对工业加工中通常发生的压力的耐受性，诱导潜在途径的激活以提高产品耐受性和非本地化合物的生产，以及确定菌株适应的基本遗传基础[3]。与基因工程相反，定向进化策略允许在不同的基因和调控基因网络中同时发生多个有益的突变。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[3]。根据所选择的压力因素和实验环境，该项目本身预计会产生许多结果。分子生物学工具将用于对选定的菌株进行生化表征，并阐明各种代谢途径以及它们如何相互联系以产生所需的化合物。下一代测序技术和转录组分析将用于分析与脂肪酸生产有关的关键基因的表达。该项目将研究多不饱和脂肪酸代谢途径的多样性，并旨在开发一种能增加omega-3多不饱和脂肪酸产量的突变菌株。为了促进ALE实验，必须对筛选进行优化，以确定具有所需功能的变体。此外，将使用包括气相色谱和尼罗河红脂可视化结合光学显微镜在内的分析化学方法，从头到尾研究进化的thraustochytrid菌株的生长特征、脂肪酸的量化和化学成分分析。该项目与Mialgae Ltd.合作，该公司是一家初创公司，通过回收工业副产品来培养富含Omega-3的微藻，在专门为微藻最佳生长量身定制的发酵容器中进行研究。通过生产已证实对人类和动物健康有益的藻油，Mialgae有助于减少全球对鱼类作为人和动物消费的Omega-3多不饱和脂肪酸来源的过度依赖。由于该项目是与一个工业合作伙伴合作的，因此选定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.