Production by Chemical Synthesis

Production by Chemical Synthesis

The idea was developed by Randolph Major who was the first director of Merck’s R&D Company which focused on synthesizing vitamins. The company produced vitamins in large quantities and sold them at low prices. The primary goal was to meet the increased demand for vitamin supplementation in health care setups.

For around 5 decades the commercial riboflavin was exclusively synthesized via chemical processes consisting of 6 to 8 chemical steps. Glucose was the used as the starting substrate. Later the process was revised to a four-step chemo enzymatic reaction sequence also starting with glucose. In the first step Glucose is converted to D-Ribose by fermentation using Bacillus tkt mutants. Afterwards D-Ribose is converted to Riboside after addition of xylidine. Riboside is then hydrogenated to produce Ribamine where crystallization is used to purify it. A reaction between Ribamine and Phenyl diazonium salt yields phenylazoribitylamine. This compound is later crystallized, dried and converted to vitamin B2 by cyclocondensation with barbituric acid. This process can yield up to 60 – 96% of purified product. However, many toxic agents are involved in the process which necessitate careful environmental control of the waste products eliminated. Tyhe figure below is an illustration of the process. Chemical structures are used in place of the complex names discribed in this paragraph..

Production by Microbial Fermentation

Simplistic methods were initially used in the microbial production of vitamin B2. These contyinued to advance over time to the complicated processes that are used today. The first commercial synthesis of Vitamin B2 using microbial fermentation was based on the anaerobic bacterium Clostridium acetobutylicum and 2 fungi Eremothecium ashbyii and Ashbya Gossypii.

The process, over time, became a luxurious investment that attracted many companies and investors. In 1965 several companies started production of riboflavin using anaerobic fermentation but could not match up with chemical process which led to their closure. Arnold Demain played a key role in the development of microbiological riboflavin while working at the Merck’s Microbiology Department. He commonly used Ashbya Gossypii fungus which would make up to 5g/L riboflavin after the fermentation process and this was low. With time, however, they came up with A. Gossypii strains which would yield more than 15g/l riboflavin.

Further developments continued to stream in. Later in 1988, Roche discovered the use of gram positive, non-toxigenic bacterium Bacillus subtilis in biological preparation of riboflavin. This was only after many years of genetic engineering research in collaboration with other researchers. The research, specifically led to the development of a strain of this bacterium which could efficiently convert glucose to riboflavin. Advancements from the Roche’s idea utilizing B. subtilis bio-process took place later which led to the establishment of a large production plant at Grenzach-Wyhlen, Germany in 2000. This plant could produce up to 3000 tons of riboflavin per year.

Metabolic Engineering of Riboflavin Production

Genetic engineering involves the production of genes coding for a particular substance by laboratory methods and introducing them into expression systems. Such genes that are produced by artificial methods are usually called recombinant genes. Many expression systems have been used in genetic engineering to produce different products in the laboratory. For example, bacteria, fungi, plant cells, and animal cell lines have all been used. Each proves to have several advantages over the other. However, each also has its limitations. On comparing fungal and bacterial systems, it is determined that fungal expression systems are generally better than bacterial ones. This is mainly based on the ease of purification, ability to express large proteins, and post-translational modifications done by these microorganisms. However, the first key step that determines success is the availability of a clear understanding of the characteristics of the substance being produced.

Classical methods of mutagenesis such as modern strategies of metabolic engineering have been applied in development of riboflavin. Chemical and radiational mutagenesis is a rapid and efficient method used at the initial stages of strain development for metabolite production. The process is highly effective when the antimetabolites specific for the target biosynthetic pathway are available. The microorganisms described here are the ones that have atracted the most reseaerch and developmnt. These two are the most commonly used.

Bacillus subtilis

This bacterium was used for a very long time in the production of the vitamin in question here. However, inasmuch as it was effective, many challenges were met in the process. In this, the initial improvement was by selecting mutants resistant to purines (8-azaguinine, methionine sulfoxide, decoyinine) or riboflavin analogs such as reseoflavin. This classical mutagenesis could yield no more improvements as it had reached its limitation. This led to employment of other gene targeted metabolic engineering strategies. The integration of multiple copies of the RIB operon in the genome and substitution of the native promoter with strong constitutive promoter to yield competitive B.subtilis strain was the main engineering approach selected. This, as was the goal, led to better and increased production.

Ashbya gossypii

A detailed molecular characterization of the riboflavin bio-synthetic genes (RIB gene) is usually necessary prior to the application of metabolic engineering strategies in A. gossypii. Simply, it is vital to characterize the metabolite of interest before commencing its production by molecular engineering. It is impossible to produce an unknown metabolite.

The process of producing a metabolite by artificial processes is complex. The same complex processes were used in the production of riboflavin. These molecular methods included recycling selectable markers, an insertional mutagenesis technique, efficient electrotransformation method, an assortment of constitutive and regulatory promoters of different strength and the sequencing of the genome.

With these complex methods, there was need to develop a highly competent strain of the microorganism used. A research conducted by BAST-based research groups and Metabolic Engineering group at the University of Salamanca, Spain, led to development of industrially competitive A.gossypii strains. Rational metabolic design led to effective production of large amounts of riboflavin focused on different processes.

GTP is one of the committed precursors in the bio-synthesis of riboflavin. Thus, the de-novo purine biosynthesis attracts some good attention. This purine biosynthesis is a long complex and tightly regulated pathway at the transcriptional and metabolic levels. There are 2 enzymes which have been showed to play a very important role in the biosynthesis of purines – PRPP amidotransferase and PRPP synthetase. These enzymes are subjected to feedback inhibition by their end products and are the major control steps of the purine biosynthesis pathway.

Accordingly, strains overexpressing inhibition – resistant forms of PRPP synthatase and PRPP amidotransferase – twofold and tenfold increase respectively in riboflavin production. Insertional mutagenesis allowed the isolation of several mutants with improved vitamin B12 production yields. These mutation have been showed to control the purine biosynthesis. Of note is the mutation on the transcription factor AgBAS1 which is has been reported to transcriptionally control purine biosynthesitic pathway.

Glycine also participates in the purine biosynthesis by stimulating riboflavin production in A.gossypii. Therefore, increasing intracellular levels of glycine have been showed to increase riboflavin production. To improve this precursor of purine synthesis, the gene encoding threonine aldolase was overexpressed resulting in significant increase in riboflavin production. Similarly it has also been postulated that disruption of the gene encoding one of two isoenzymes of serine hydroxymethyltransferase (AgSHM2 resulted in increased riboflavin production.

Expression of alanine; glyoxylate aminotransferase encoding gene (AGX1) was also showed to increase the glycine precursors with subsequent increase in riboflavin synthesis.