INDUSTRIAL MANUFACTURING OF VITAMIN B2

INDUSTRIAL MANUFACTURING OF VITAMIN B₂

Introduction

Vitamin B2, also called Riboflavin, is an essential part of human and animal diets. Primarily, the vitamin serves as the precursor for flavoenzymes – flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) – which are central in redox reactions. Storhas and Metz (2013) explain that these enzymes are generally referred to as flavins, and they participate centrally in the metabolism of critical chemicals in the biological system, such as carbohydrates, fats, ketone bodies, and proteins. Other functions of riboflavin include participation in the metabolism of other vitamins like vitamins A, B3 and B6, recycling of glutathione and homocysteine metabolism.

The vitamin is categorized among essential nutrients. Essential nutrients are the ones that the body cannot naturally produce and, therefore, must be supplied in the diet. From the assertion, it is clear that the vitamin cannot be synthesized in the body, and an individual must, therefore, make efforts to supply it by taking diets rich in it (Storhas & Metz, 2013). For this reason, thus, the industrial manufacture of the vitamin becomes appropriate. Its production is done mainly for animal consumption and supplementation of the human diet.

Initial Discovery

Initially, the vitamin was discovered as a yellow pigment obtained from milk. Later, the yellow enzyme was also purified from aqueous yeast extract. The enzyme could be fractioned to two enzymatically inactive components: a protein and a yellow pigment, according to Storhas and Metz (2013). The studies continued to advance until the structure of the yellow prothetic group was finally elucidated and published. This yellow group was named riboflavin.

Occurrence

The vitamin naturally occurs in food sources of different types. According to Ward ( 2014), the primary natural source of the vitamin, as pointed in the previous paragraph, is milk. The vitamin was initially extracted from milk at the time of its discovery. However, the vitamin is also available in other foods like dairy products, eggs, and lean meat. Further spores include green leafy vegetables, fish, leguminous plants, cereals, and nuts. Because the vitamin is sensitive to light, it is vital that the storage of foods containing the vitamin be stored in dark environments to prevent the ultimate deterioration of the vitamin.

Recommended Dietary Allowance for Humans

The Recommended Dietary allowance for this vitamin generally varies depending on the age of the individual. For infants, the intake ranges between 0.3 and 0.4mg/day (Ward, 2014). The intake ranges for children are between 0.5 and 0.9mg/day. However, among adults, gender-based variations become essential. Males are expected to intake about 1.3mg/day while females are expected to intake about 1.1mg/day. Pregnancy increases the intake to 1.4mg/day, while lactation raises it to about 1.5mg/day.

Deficiencies and Complicating Factors

Riboflavin deficiencies in biological systems of humans and animals rarely occur independently. Most times, they occur together with other nutrient deficits. With Riboflavin deficiency, comes an increased risk for cardiovascular disease and impaired iron metabolism that always result in anemia. Globally, the overall prevalence of cardiovascular diseases and related complications and iron deficiency anemia is massive. Generally, as postulated by Pinto and Zempleni (2016), these two diseases are the main contributors to mortality and mobility associated with vitamin deficiencies. Ariboflavins are severe cases that result, usually, in developmental abnormalities and retarded growth. These are states of a total absence of riboflavin in the diet. On the contrary, however, because riboflavin is a water-soluble vitamin, excesses can easily be eliminated in urine.

Some diseases are known to exacerbate the effects of vitamin deficiencies—the main of these diseases are cancers. Generally, cancers of different types are always associated with increased demand for nutrients, and sometimes, a faster loss of essential nutrients (Pinto and Zempleni, 2016). Thus, the result is the amplified effects of deficiency. Other diseases that cause such are cardiac diseases and diabetes. In these cases, therefore, a patient is advised to increase their intake of vitamins and other nutrients to maintain the functionality of biological systems and improve quality of life.

Manufacturing Process

Two main approaches are commonly used in the industrial manufacture of vitamin B2. These include the use of microorganism fermentation and a from-scratch biosynthesis of the vitamin (Fischer and Bacher, 2011). Many microbes produce the vitamin naturally. This is a simple method because microorganisms only need to be cultured. However, only a few bacteria can produce significant amounts of the vitamin.

On the other hand, complete biosynthesis, at first, involved the addition of purines to cultures of some bacterium. Following the increased understanding of the process, a method of producing the vitamin in the laboratory under the catalysis of the Enzyme GTP cyclohydrolase was developed (Fischer and Bacher, 2011). In this paper, the diverse approaches to the manufacture of the vitamin are addressed in detail.

History of the Development of Process Techniques

The process of industrially producing riboflavin has undergone significant development over the years. The initial steps towards producing the vitamin started way back, at the time the vitamin was discovered. The first approach to the production of vitamin B₂ was the chemical biosynthesis of the vitamin, a six-steps process beginning with glucose (Ruvuelta et al., 2016). However, later, in the 1980s, a better, though a complicated, chemoenzymatic process of synthesis was developed. This process involved four steps, and glucose was still used as the primary raw material. Microbial fermentation and molecular engineering approaches, however, have fast-evolved to become mainstream methods of riboflavin production. This section discusses the main means of production and the development that has taken place over the years.

Production by Chemical Synthesis

Randolph Major, who was the first director of Merck’s R&D Company, which focused on synthesizing vitamins (Revuelta et al., 2016), developed the idea. 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 fifty years, riboflavin was exclusively produced by a chemical biosynthetic process involving 6 to 8 steps. In this process, glucose was used as the raw material. Later, however, Revuelta et al. (2016) explain that the process was revised to a more straightforward four-step chemoenzymatic reaction that still started with glucose. In the first step, Glucose is converted to D-Ribose by fermentation using Bacillus mutants. Afterward, D-Ribose is converted to Riboside after the addition of xylidine. Riboside is then hydrogenated to produce Ribamine, where crystallization is used to purify it. Ribamine reacts with Phenyl diazonium salt to produce phenylazoribitylamine. It is this compound that is then crystallized and dried before it is transformed to riboflavin – the target of the reaction – by cyclo-condensation with barbituric acid. This process can yield up to 60 – 96% of the purified product (Revuelta et al., 2016). However, many toxic agents are involved in the process, which necessitates careful environmental control of the waste products eliminated. The figure below is an illustration of the process. Chemical structures are used in place of the complicated names described in this paragraph.

Source: Hiltunen et al. (2012)

Production by Microbial Fermentation

Simple methods were initially used in the microbial production of vitamin B₂. These continued to advance over time to the complicated processes that are used today (Revuelta et al., 2016). The first commercial synthesis of Vitamin B₂ using microbial fermentation was by Clostridium acetobutylicum. Also, two fungi – Eremothecium ashbyii and Ashbya gossypii – can be used.

The process, over time, became an expensive investment that attracted many companies and investors. In 1965, several companies started production of riboflavin using anaerobic fermentation but could not match up with a chemical process that led to their closure. Arnold Demain played a crucial role in the development of microbiological riboflavin while working at 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 (Revuelta et al., 2016). 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 the biological preparation of riboflavin (Revuelta et al., 2016). This was only after intensive research and biotechnological studies that were done by many researchers. The research specifically led to the development of a strain of this bacterium, which could efficiently convert glucose to riboflavin. Advancements from Roche’s idea utilizing B. subtilis bio-process took place later, which led to the establishment of a massive riboflavin factory in Germany in the year 2000 as Revuelta et al. (2016) clarifies. This plant could produce up to 3000 tons of riboflavin per year.

Source: Jha (2020)

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 means are usually called recombinant genes, as stated by Ledesma-Amaro, Serrano-Amatriain, Jiménez, and Revuelta (2015). 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, the ability to express abundant proteins, and post-translational modifications done by these microorganisms.  However, the first vital 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 the development of riboflavin. Mutation induced by chemicals and radiation is an essential step in the process of modifying cells to enhance the production of specific metabolites. On this, Revuelta et al. (2016) state that the process of mutagenesis is more efficient when anti-metabolites that are specific to the pathway of biosynthesis are present. The microorganisms described here are the ones that have attracted the most research and development. 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 active, many challenges were met in the process. In this, the initial improvement was by selecting purine (8-arginine, methionine sulfoxide, decoyinine) resistant strains or riboflavin analogs such as riboflavin (Revuelta et al., 2016). This classical mutagenesis could yield no more improvements as it had reached its limit. This led to the employment of other gene targeted metabolic engineering strategies. However, Hao et al. (2013) assert that integrating into the genome many copies of the RIB operon and substitution of the native promoter with the strong constitutive promoter to yield competitive B.subtilis strain was the first engineering approach selected. This, as was the goal, led to better and increased production.

Ashbya gossypii

It is essential to have enough information. Thus, intensive characterization of the vitamin, as well as its biosynthetic DNA sequences (RIB gene), is usually necessary before the biotechnological approach can be employed in A. gossypii (Revuelta et al., 2016). Naturally, 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 methods is complex. The same complex processes were used in the production of riboflavin. According to Ledesma-Amaro, Serrano-Amatriain, Jiménez, and Revuelta (2015), these molecular methods included selectable recycling markers, an insertional mutagenesis technique, efficient electrotransformation method, a variety of promoters, both regulatory and constitutive promoters, of varying ability and the sequencing of the genome.

With these sophisticated methods, there was a need to develop a highly competent strain of the microorganism used. Research conducted by BAST-based research groups and a group of researchers from the University of Salamanca in Spain, resulted in the development of highly competitive industrial strains of A.gossypii as stated by Gu and Li (2016). Rational metabolic design led to the effective production of large amounts of riboflavin focused on different processes.

The synthesis of riboflavin involves many metabolites. GTP is one of the significant precursors employed in the process. For this reason, the purine biosynthesis pathway attracts major atention.. However, Rubuelta et al. (2016), while discussing the purine biosynthesis, state that the process is complex and highly regulated. It is a two enzyme process. They (Ruvuelta et al.) prove that enzymes – PRPP amidotransferase and PRPP synthetase – play central roles in the process. Also, based on the fact that the enzymes are regulated by a feedback mechanism that emanates from the products of the reactions they catalyze, the two enzymes are important to control steps in the process.

Overexpression of the genes coding for the product of interest is vital as well. Towards this, Sanchez and Demain (2008) argue that the excess expression of inhibitory enzymes like PRPP synthase and PRPP aminotransferase commonly occurs. With insertional mutagenesis, however, it was possible to isolate still strains that exhibit increased production of the vitamin. These mutations have been showed to control the purine biosynthesis. Of note is the mutation on AgBAS1, the transcription factor that controls the purine biosynthetic pathway.

The participation of glycine in the purine biosynthetic pathway is also a noteworthy step. Glycine stimulates the production of riboflavin 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 sequence for threonine aldolase is intentionally over-expressed through engineering techniques, leading to a significant increase in the synthesis of riboflavin (Revuelta et al., 2016). Similarly, it has also been dedicated that disrupting the genes that encode the two isozymes of serine hydroxymethyltransferase can cause an increase in the production of vitamin B2. Further, glycine precursors can be increased by expressing alanine with a subsequent rise in riboflavin synthesis.

Life Cycle and Sustainability Assessment

Over the years, the processes employed in the production of vitamin B₂ have continued to change. These changes have primarily been fueled by advancements in technology and discoveries that seem to be more superior and more effective. For example, by the year 2018, the approach to the production of riboflavin was only microbial fermentation using metabolically engineered microbes (Acevedo-Rocha et al., 2019). A comprehensive bank of strains of B. subtilis is available, as well as a great deal of data on the bacterium.

As has been explained above, the bacterial and fungal processes begin with GTP and ribose phosphates, which are abundant in cells. Also, the biosynthetic enzymes used are highly efficient in their work. However, on the contrary, riboflavin metabolism enzymes are extremely slow (Acevedo-Rocha et al., 2019). This calls for strong and stable overexpression of the encoding genes and stabilization of mRNA. This stabilization is done by modified flavin mononucleotide riboswitches, which have continued to become essential components of modern production systems.

Still, nevertheless, many unresolved issues are present. Firstly, the superordinate regulator protein that was discovered recently in B. subtilis affects the working of the FMN riboswitches and therefore pauses a threat to the production of riboflavin in this bacterium (Acevedo-Rocha et al., 2019). Secondly, the phosphates of the riboflavin pathway have not been enumerated yet. If these are not identified soon, it may prove difficult to sustain the process in the future. Thirdly, flavins are highly reactive metabolites. Thus, Sepúlveda Cisternas, Salazar, and García-Angulo (2018) enumerate that flavins found within the cell, as well as flavoproteins, can be a rich source of reactive oxygen species and free radicals that may cause damage to cells. This has not been clearly understood, and no remedies have been developed so far (Acevedo-Rocha et al., 2019). A way to reduce the possibility of damage by reactive oxygen species and free radicals could, most likely, reintroduce the use of flavin binding proteins that keep flavins bound and prevent them from reacting freely in the medium.

Other sustainability issues include the fact that the hydrophobic dimethyl benzene portion of the isoalloxazine ring system of riboflavin supports the diffusion of vitamin B₂ across the cell membrane in the absence of a transport system (Acevedo-Rocha et al., 2019). Here, it appears clear that the bacterial cell does not have an intrinsic ability to export flavins. The introduction of the riboflavin transporter system by modification engineering dramatically increases the production of the vitamin. However, the transporter from Streptomyces devaonensis negatively affects FMN riboswitches and other flavoenzymes.

A life cycle assessment of the entire process reveals that the process is efficient and meets the requirements for sustainability. Initially, a process developed over many years ago was used. In the process, the vitamin was synthesized from scratch by the use of glucose as a raw material and complex bio-synthetic pathways (OECD, 2001). However, the process has evolved from the former method to a more advanced and environmentally friendly approach that utilized bacteria and fungi to produce vitamins.

Companies producing riboflavin commercially have developed a culture of carrying out internal life cycle assessments of their processes to determine if the processes can be sustained or not. A perfect example of this is the Roche Company. With the most reliable measure of sustainability being the use of renewable raw materials and sources of energy, the company, together with many other companies involved in the production of vitamins, has dramatically ensured the use of renewable sources (OECD, 2001). For instance, the production of the vitamin of interest, in this case, is done primarily by the use of glucose as a raw material. Glucose is a widely available carbohydrate metabolite. It is stated that 90% of the materials used are renewable (OECD, 2001). This confirms that the process is sustainable. The biological process, when compared with the chemical process that was initially used, however, is more sustainable and better set for the modern-day world.

Conclusion

Vitamin B2 is an essential nutrient that needs to be supplied in the diet of humans and animals if growth is to occur normally. Because of the commonality of vitamin B2 deficiencies, the vitamin is commercially produced in industrial processes to supplement natural supplies. This is attained through two main processes, that is, microbial fermentation and chemical bio-synthesis. Of these, fermentation by genetically engineered microbes is the most commonly used. The fore sections of this paper describe the two processes in depth and also discuss the life cycle and sustainability assessments associated with the process.

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