Novel strain development for the mycoprotein fungus Fusarium venenatum

基于环境和健康的考虑，对肉类替代蛋白质来源的需求不断增长。真菌镰刀菌被用于生产真菌蛋白，这是一种高蛋白、低胆固醇的肉类替代品，以“Quorn”商标出售，在商业上非常成功。在20世纪60年代发现了一种特殊的菌株A3/5，被选择用于生产真菌蛋白，因为它在工业生长条件下产生高蛋白质地产品，毒素可以忽略不计。然而，也存在效率低下和问题。例如，在长时间生长后，真菌容易产生不适合Quorn生产的过多分支，某些维生素水平较低，并且仍然存在产生霉菌毒素的风险。也许令人惊讶的是，自20世纪70年代以来，相同的菌株A3/5一直在使用，没有菌株发展。因此，现在有商业和环境的兴趣，获得新的菌株与改进的特性，以生产真菌蛋白。与此同时，最近在真菌基因组学和基因操作方面取得了科学进展，这意味着真菌是一种有吸引力的生物技术开发模式生物，其研究具有重大基础科学和现实世界应用影响的潜力。实验方法、方法和技术我们计划使用领先的基因组学、分子生物学、生化和经典培养技术来探索F. venenatum的菌株改良。A3/5的基因组序列已经可用来促进调查。(1)黄颡鱼的性生物学。已知该物种只进行无性繁殖，这阻碍了菌株的改进。然而，我们最近开发了一种分子性别测试诊断方法，该方法已经鉴定出了有性生殖所需的不同MAT1-1和MAT1-2交配类型的F. venenatum分离株。我们计划通过qRT-PCR筛选分离株的性发育所需基因的表达，然后在有利于有性生殖的条件下选择交配分离株，使用一系列显微镜来监测发育。如果可以诱导性，那么具有理想特性（例如，更高的生长速度和维生素含量，不含霉菌毒素）的菌株可以杂交，后代可以确定具有有利性状的组合，这是令人兴奋的前景；性循环也可能产生全新的、有利的基因型。这种方法很有吸引力，因为有性繁殖是一种自然的，而不是转基因的菌株开发方法，可以利用菌株。在F. venenatum中发现性循环将是世界上第一次，并提供了对真菌性和无性行为的一般见解。(2)黄芪经典遗传学的操作。初步研究表明，在长时间发酵条件下，二倍体菌株比目前使用的单倍体菌株A3/5更稳定。我们将产生二倍体菌株，看看是否可以获得分支较少的更稳定的生产菌株，利用马洛食品公司开发的尖端显微镜和蛋白质组学方法来监测菌丝分支和蛋白质组成。我们还将发展拟性杂交，在单倍体菌株中产生新的性状组合。(3) venenatum的操作-基因组编辑。转基因和CRISPR基因组编辑工具将用于尝试和改变感兴趣的性状，例如真菌毒素合成和维生素生产。电子鼻气相色谱-质谱联用将用于识别不需要的风味挥发物，也可能被去除。虽然产生的菌株还不能用于商业用途，但它们将提供可能被上述（1+2）利用的关键见解。

There is ever growing demand for meat replacement protein sources on environmental and health grounds. The fungus Fusarium venenatum is used in the production of mycoprotein, a high-protein, low cholesterol meat substitute sold under the trade name 'Quorn', which has been very commercially successful. A particular strain A3/5 was identified in the 1960s, being chosen for mycoprotein production as it yields a high protein textured product with negligible toxins under industrial growth conditions. However, there are inefficiencies and problems. For example after prolonged growth the fungus is prone to excess branching unsuitable for Quorn production, has low levels of certain vitamins, and there remains a risk of mycotoxin production. Perhaps surprisingly the same strain A3/5 has been used since the 1970s with no strain development. Therefore there is now commercial and environmental interest in obtaining novel strains with improved characteristics for mycoprotein production. In parallel, scientific advances have recently been made in fungal genomics and genetic manipulation meaning that F. venenatum is an attractive model organism for biotechnological exploitation, with research having the potential for significant fundamental scientific and real world applied impact. Experimental Approaches, Methodology and Techniques We plan to use leading genomic, molecular biology, biochemical and classical culturing techniques to explore strain improvement in F. venenatum. A genome sequence of A3/5 is already available to facilitate investigations. (1) Sexual biology of F. venenatum. The species is only known to reproduce asexually, which has impeded strain improvement. However, we have recently developed a molecular sex-test diagnostic, which has identified F. venenatum isolates of different MAT1-1 and MAT1-2 mating type needed for sexual reproduction. We plan to screen isolates by qRT-PCR for expression of genes required for sexual development and then mate chosen isolates under conditions favourable for sexual reproduction, using a range of microscopy to monitor development. If sex can be induced, there is the exciting prospect that strains with desirable characteristics (e.g. higher growth rate and vitamin content, absence of mycotoxins) can be crossed and offspring identified with combinations of favourable traits; the sexual cycle may also generate completely novel, favourable genotypes. This approach is attractive as sexual breeding is a natural, rather than GM, method of strain development allowing exploitation of strains. The discovery of a sexual cycle in F. venenatum would be a world first and provide general insights into fungal sexuality and asexuality. (2) Manipulation of F. venenatum - classical genetics. Pilot studies suggest that diploid strains are more stable under prolonged fermenter growth than the haploid strain A3/5 in current use. We will generate diploid strains to see if more stable production strains with less branching can be obtained, drawing on cutting-edge microscopy and proteomic methods developed at Marlow Foods to monitor hyphal branching and protein composition. We will also develop parasexual crossing to generate novel combinations of traits in haploid strains. (3) Manipulation of F. venenatum - genome editing. GM and CRISPR genome-editing tools will be used to try and alter traits of interest e.g. mycotoxin synthesis and vitamin production. Electronic nose GC-MS will be used to identify undesirable flavour volatiles that also might be removed. Although arising strains could not yet be used commercially, they will provide key insights which might be exploited by (1+2) above.