MEDIEVAL BLUE GENES: Reducing Industrial Indigo Dye Pollution of the Environment

青蓝是世界上最古老的蓝色染料来源。自公元前6000年开始使用以来，在20世纪之前，它与植物的叶子可持续地隔离开来。它现在几乎全部来自化石石油，是世界上最重要的合成化学品之一。因此，每年合成50,000吨。其中95%用于对每年生产的40多亿件牛仔服装进行染色。它是牛仔布行业的中流砥柱。不幸的是，合成青蓝在其制造和作为染料的开发过程中都会产生大量的环境和可持续性问题。它的合成依赖于有毒的石化产品苯，并涉及许多其他危险化学品。在染色过程中，每染3公斤蓝，就会还原出过量的连二亚硫酸钠--2公斤。产生的硫酸盐和亚硫酸盐副产物具有腐蚀性，很难从废水中去除。这导致了肆无忌惮的染料厂老板将废染料倾倒到河流中，并造成了令人震惊的生态影响，例如中国的蓝色或黑色河流和印度的蓝色狗。在中世纪，布料在大桶中染色，在大桶中加入板蓝叶(Isatis TincVictoria)，自然发酵过程导致其生物还原。恢复以植物为基础的自然进程将产生真正的环境效益，特别是如果其有效性和再现性得到改善的话。然而，合理的改变是不可能的，因为细菌还原青蓝的机制尚不清楚。从中世纪食谱中科学地复制了一个大桶，使人们能够鉴定出负责的细菌是板蓝梭菌。要提高它的性能，重要的是要了解它是如何减少青色的。最有效的策略是使用基因工具分离不再还原青蓝的突变细菌，然后使用DNA测序来识别受影响的基因/蛋白质。然而，尽管基因工具是可用的，但到目前为止还没有将它们转移到板蓝根中的方法的报道。我们现在第一次证明了DNA转移到这种细菌中。因此，现在产生突变是可能的。同时，我们已经确定了细菌染色体的整个序列蓝图，这是鉴定任何突变的性质的先决条件。通过在染色体中随机插入一小段被称为转座子的DNA，扰乱它所插入的基因，就可以产生这些基因。如果该基因及其编码的蛋白质参与了还原青蓝的过程，那么细菌菌落将不再能够溶解青蓝并变成蓝色。通过筛选数千个随机突变体，所有参与蓝宝石还原的基因/蛋白都将被识别出来。合理改变影响因素，提高生物在染色过程中的生产能力。在确定这些因素之前，无法说明将要作出的改变的性质。除了它们的改进之外，一个可能的战略将是导致它们的过剩生产。或者，可能干扰识别过程的因素可以通过突变来灭活。在对棉纱和毛纱样品进行染色试验中，将比较经过合理修饰的菌株在还原靛蓝中的性能，并将其与亲本菌株进行比较，这些试验基本上重现了实验室中的中世纪大桶。染色样品的色牢度也将进行洗涤牢度、日晒牢度和摩擦牢度的评估。为了确保成功，我们将直接与工业染料和着色师接触，并加深对染色工艺和工业限制和需求的了解。该项目的长期成果将是开发一种经济和可持续的生物牛仔染色工艺。

Indigo is the oldest source of blue dye in the world. In use since 6000 BC, prior to the 20th century, it was sustainably isolated from the leaves of plants. It is now almost entirely derived from fossil oil and is one of the world's most important synthetic chemicals. Thus, >50,000 tons are annually synthesised. Of this, 95% is used to dye the over 4 billion denim garments that are produced every year. It is the mainstay of the denim industry. Unfortunately, synthetic indigo creates substantial environmental and sustainability issues during both its manufacture and its exploitation as a dye. Its synthesis is reliant on the toxic petrochemical benzene and involves many other hazardous chemicals. During the dyeing process itself, the insoluble blue indigo is reduced by an excess of sodium dithionite - 2 kg for every 3 kg of indigo. The sulfate and sulfite by-products generated are corrosive and problematic to remove from wastewater. This has led to the dumping of spent dye materials into rivers by unscrupulous dye mill owners and alarming ecological impacts, e.g., rivers of blue or black in China and blue dogs in India.In medieval times, cloth was dyed in a vat in which indigo was provided by adding leaves of woad (Isatis tinctoria) and a natural fermentation process brought about its biological reduction. A return to the natural plant-based process would have real environmental benefits particularly if its effectiveness and reproducibility were improved. Rational changes are, however, not possible because the mechanism by which the bacteria reduce indigo is unknown. Scientific recreation of a woad vat from a medieval recipe has enabled the identification of the bacterium responsible as Clostridium isatidis. To improve its performance it is important to understand how it reduces indigo. The most effective strategy is to use gene tools to isolate mutant bacteria that no longer reduce indigo, and then use DNA sequencing to identify which gene/protein is affected. However, although gene tools are available, until now no means of transferring them into C. isatidis had been reported. We have now demonstrated, for the first time, DNA transfer into this bacterium. It is, therefore, now possible to generate mutants. In parallel, we have determined the entire sequence blueprint of the bacterium's chromosome, a prerequisite for identifying the nature of any mutants made. These will be made by randomly inserting a small piece of DNA, called a transposon, into the chromosome, disrupting the gene into which it has inserted. If that gene, and the protein it encodes, is involved in the indigo reduction process, then bacterial colonies will no longer be able to solubilise indigo and turn blue. By screening thousands of random mutants, all of the genes/ proteins involved in indigo reduction will be identified. The factors involved will be rationally altered to improve the productivity of the organism in dyeing process. Until these factors are identified the nature of the changes to be made cannot be stated. Aside from their modification, a likely strategy will be to bring about their overproduction. Alternatively, factors that may be interfering with the identified process could be inactivated, through mutation. The performance of rationally modified strains in indigo reduction will be compared to the parental strain in dyeing trials of cotton and wool yarn samples that essentially recreate a medieval vat in the laboratory. Colour fastness of dyed samples will also be assessed for wash, light and rubbing fastness. To ensure success we will directly engage with industrial dyers and colourists and develop a greater understanding of the dyeing processes and industrial constraints and needs. The longer-term output of the project will be the development of an economic and sustainable process for a biological denim dyeing.