National Food Institute, Technical University of Denmark, Lyngby, Denmark
National Food Institute, Technical University of Denmark, Lyngby, Denmark.
mBio. 2017 May 30;8(3):e00526-17. doi: 10.1128/mBio.00526-17.
Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify variants secreting vitamin B (riboflavin). Initially, we used a roseoflavin-resistant mutant of strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter) was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes. The food industry prefers to use classical approaches, e.g., random mutagenesis followed by screening, to improve microorganisms used in food production, as the use of recombinant DNA technologies is still not widely accepted. Although modern automated screening platforms are widely accessible, screening remains as a bottleneck in strain development, especially when a mild mutagenesis approach is applied to reduce the chance of accumulating unintended mutations, which may cause unwanted phenotypic changes. Here, we incorporate a droplet-based high-throughput screening method into the strain development process and readily capture variants with more efficient vitamin secretion from low-error-rate mutagenesis libraries. This study shows that useful mutants showing strong phenotypes but without extensive mutations can be identified with efficient screening technologies. It is therefore possible to avoid accumulating detrimental mutations while enriching beneficial ones through iterative mutagenesis screening. Due to the low mutation rates, the genetic determinants are also readily identified.
高效的筛选技术旨在减少在诱变群体中鉴定具有感兴趣表型的稀有突变体所需的时间和成本。在这项研究中，我们结合了一种温和的诱变策略和基于微流控液滴技术的高通量筛选，以鉴定分泌维生素 B（核黄素）的变体。最初，我们使用对玫瑰黄素具有抗性的突变体 菌株 MG1363、JC017，其分泌低水平的核黄素。通过使用荧光激活液滴分选，从诱变文库中很容易分离出比 JC017 分泌核黄素效率更高的几种突变体。筛选非常高效，分离出的候选突变体每百万碱基对（Mbp）的突变仅为 1.6 个。遗传特征表明，核黄素的产生是由抑制嘌呤生物合成的突变引发的，这令人惊讶，因为嘌呤核苷酸 GTP 是核黄素的前体。突变体中的嘌呤饥饿诱导了核黄素生物合成簇的过表达。当在生长培养基中补充嘌呤以缓解嘌呤饥饿时，结果是核黄素生物合成簇的立即下调以及核黄素产量的降低。最后，通过在牛奶发酵中应用新的分离株，与使用野生型菌株和原始玫瑰黄素抗性突变体 JC017 相比，牛奶中的核黄素含量（0.99mg/L）提高到 2.81mg/L，分别为 0.66mg/L 和 1.51mg/L。结果表明，当将经典诱变与基于液滴的微流控筛选技术结合使用以获得具有有用属性的微生物时，经典诱变技术是多么强大。食品工业更喜欢使用经典方法，例如随机诱变，然后进行筛选，以改善用于食品生产的微生物，因为重组 DNA 技术的使用尚未得到广泛接受。尽管现代自动化筛选平台广泛可用，但筛选仍然是菌株开发的瓶颈，特别是当应用温和的诱变方法来降低积累意外突变的机会时，这可能会导致不需要的表型变化。在这里，我们将基于液滴的高通量筛选方法纳入菌株开发过程中，并从低错误率诱变文库中轻松捕获具有更高维生素分泌效率的变体。这项研究表明，可以使用高效的筛选技术鉴定具有强表型但突变较少的有用突变体。因此，可以通过迭代诱变筛选来避免积累有害突变，同时富集有益突变。由于突变率较低，因此也可以很容易地确定遗传决定因素。
