Project Description

2. Transformation of indigo dyeing process:

Indigo is produced by three methods: plant extraction, chemical synthesis, and microbial synthesis. India Indigo pigment has been extracted directly from the leaves of some leguminous plants for a long time in Egypt and China, mainly by direct dyeing technology. Knead the blue leaves with the fabric Or first knead out the blue juice and then soak it in the fabric, supplemented by plant ash to help dyeing. The action of plant ash can hydrolyze Isatis glycoside. , free indophenol.[9-11]Dip Knead Dyeing is actually the process of making indigo on the fiber. Dyeing by dipping and kneading is limited by the origin and harvest season of bluegrass It is extremely inconvenient to popularize and apply. In the long-term practice of the ancient people, The reduction dyeing technology was invented, which broke the time and space constraints of dyeing.[5-7] In the 1890s, a group of chemists led by Bayer opened the door to the chemical synthesis of indigo and realized the efficient and rapid synthesis of indigo[2]. In 1866, Bayer, a German chemist, obtained indole in the process of burning zinc powder and indophenol to synthesize the parent of indigo. In 1870, Bayer and his students treated isatin with phosphorus trichloride, and the product was reduced by zinc powder-hydrochloric acid to obtain indigo. The isatin used at that time came from bluegrass plants. Bayer mastered the method of preparing indigo carmine from phenylacetic acid and reduced it to indigo carmine with phosphorus trichloride, phosphorus and acetyl chloride. The chemical reaction equation is as follows:

This is an important milestone in the history of artificial organic synthesis of natural dyes, marking the beginning of artificial synthesis of complex organic compounds. The 1905 Nobel Prize in Chemistry was awarded to Bayer (Adolf von Baeyer) for his outstanding contributions to the synthesis of indigo and hydrogenation of aromatics.[3] In 1890, K. Heumann, a German chemist, invented the method of synthesizing indigo with aniline as the initial raw material, thus realizing the industrial production of indigo. Synthetic indigo produced by BASF in West Germany came out in 1897 and was not put into production in other countries until 1917. In 1901, the blue vat dye (C. I.) was synthesized by alkali fusion with amino quinone as raw material. Reduced Blue 4), which is the first quinone vat dye synthesized by human beings. Due to the toxicity of raw materials, catalysts and intermediate products in chemical synthesis, it causes serious harm to people and the environment. For this reason People began to explore green and sustainable methods for the synthesis of indigo.[8-12]

3. Research status of synthetic methods of biological dyes:

Directed evolution technology【13-14】】is the core technology of indigo biosynthesis, which can improve the synthesis rate, total reaction yield, even change the product and its composition, and also improve the activity and thermal stability of enzymes. Yes Site-directed mutagenesis can be used to change the substrate range of enzymes in wild strains that do not produce indigo. For example, HbpA from Pseudomonas azelaica HBP1 can not hydroxylate indole and its derivatives. However, its mutants D222V and V368a have the ability to hydroxylate indole, 4 (5) -hydroxyindole and 5-bromoindole to form indigo pigments. -4-monooxygenase is subjected to directed mutagenesis, and the mutant can change the regioselectivity of the substrate, the ratio of the primary product, and can synthesize otherProduct. Microorganisms and their enzymes are the undertakers of biosynthesis and transformation. Therefore, large-scale and deep-level development of microbial and enzyme resources is the premise and material basis of biocatalysis process. Indigo-synthesizing strains are mainly obtained by pure culture method Studies have shown that non-culturable microorganisms account for more than 99% of the total number of microorganisms, so relying solely on traditional culture technology can no longer meet the needs of the development of biological resources. Metagenomics technology developed in recent years has solved this bottleneck problem. By directly extracting the DNA of all microorganisms from environmental samples and constructing metagenomic libraries, the research strategies of genomics are used to study the genetic information and community functions of all microorganisms contained in environmental samples. For example Gene resources capable of synthesizing indigo and indirubin were screened from forest soil by culture-free method. A novel styrene monooxygenase[33]capable of synthesizing indigo was isolated from soil by van Hellemond using a function-driven screening method.

4. Research direction:

In recent years, with the improvement of people's awareness of safety and environmental protection, more and more attention has been paid to the synthesis of indigo by microbial method. Although some research results have been achieved, there are still some problems: 1) that yield of the indigo is low: the yield of the indigo is very low due to a plurality of factor such as limited expression level of a gene in a recombinant, low enzyme activity, stability and regeneration rate, toxicity of a substrate to a strain and the like, and industrial production is not practical; 2) low product purity: most strains will produce other indole oxidation products in the synthesis process, which on the one hand affects the product purity, and on the other hand, the existence of these substances and other intermediate substances will increase the cost of downstream purification and separation; 3) Expensive substrates: The commonly used synthetic substrates indole and tryptophan are expensive, so the whole process of bioconversion of cheaper raw materials (glucose, etc.) To indigo needs to be discussed in depth. Because Therefore, the development of new, stable and high-yielding strains, the optimization of metabolic regulation and fermentation processes, and the development of efficient and low-cost separation and extraction processes will become the mainstream development direction of indigo synthesis by using various omics, bioinformatics and systems biology technologies. Our team mainly chooses the method of biological dye synthesis to produce indigo glycoside, so as to achieve the goal of efficient production and less pollution. In order to reduce the cost of the strain, we chose Corynebacterium glutamicum to synthesize indigo glycosides in vivo through gene editing and other technologies. In order to improve the yield, we planned to increase the yield of indigoside with high concentration of ammonia as the supply of L-glutamine. In order to improve the purity of the product, we decided to actively explore effective methods to avoid the impact of oxidation products on the synthesis.