Media Composition And Its Role In The Production Of Penicillin
Penicillin G has continued to remain an essential component of the medical toolkit, exhibiting unrivalled activity against staphylococci, streptococci, and other susceptible bacterial infections. However even to this day, penicillin G production continues to be the focus of much research interest. There are many reasons for this; namely the commercial and therapeutic importance of penicillin, complexity of cell growth, and the impact of engineering variables. That as a collective has created unique and diverse challenges throughout the production pipeline.
To produce cheaper and more effective penicillins, industry has typically centred on the development of classical strain improvement or by optimising process parameters of the fermentation. But as extensive research has shown, both strain improvement and media composition are a “Catch-22” situation. You cannot choose a lead strain until you have the best medium and you cannot propose a finest medium until you have the lead strain. Considering the above information, there has been an increased effort by industry to further characterise and design a media composition to accommodate strain development.
For the design of production medium, the most suitable fermentation conditions (e.g., pH, temperature, agitation etc) and the appropriate medium components (carbon sources, nitrogen sources, mineral salts, trace elements etc) must all be identified and optimised accordingly. For penicillin G fermentation, the optimisation of media represents a significant cost and time factor in the bioprocess development. One that is undoubtedly putting increased pressure on the demands of existing experimental procedures.
To gain a greater understanding of media composition and its role in the production of penicillin G, a review of the literature was performed. The aim of this review was to demonstrate an appreciation for secondary metabolism, media design and the various ways nutrients may interfere with the biosynthesis of penicillin G.
2.0 Penicillin is a secondary metabolite
After the discovery of penicillin, researchers exploited microorganisms to produce secondary metabolites. Unlike primary metabolites, they do not play a physiological role during exponential phase of growth. Rather, they are produced during a subsequent stage of growth called the idiophase. Distinct from the exponential phase; their production begins when the growth of a producing organism becomes limited by the exhaustion of a key nutrient. A view held by Jarvis and Johnson; that from an analysis of batch culture data, concluded that the specific rate of penicillin production was at its highest when the organism growth rate was close to zero.
For large-scale production, industry has typically focussed on the filamentous microorganisms of fungi. With the filamentous fungal genus penicillium, receiving the greatest amount of interest. This interest lies in the penicillium inherent capabilities to produce a diverse range of secondary metabolites. Among which, penicillin represents the starting point of antibiotic production. With Penicillium chrysogenum, continuing to be the species of choice for industrial production of penicillin G.
3.0 Penicillin biosynthesis is determined by its growth medium
Formation of secondary metabolites involves the uptake of several intermediates acting as a source for the antibiotic. For penicillin G biosynthesis, this is particularly relevant. To ensure its formation, several metabolic pathways must first take place including: fatty acid metabolism, amino acid metabolism, carbohydrate metabolism and purine & pyrimidine metabolism. To initiate these pathways requires specific amounts and type of nutrients be supplied to the production medium. For as reference describes, when variation is present, the quality and production of penicillin G is impeded.
Literature has directed its attention to the transcriptional regulation of precursor amino acids: L-α-aminoadipic acid, L-cysteine and L-valine. Enabling the first step in the production pathway; that without, penicillin would not be formed. Though, perhaps even more important; certainly in terms of penicllins commercial value, is the formation of the penam nucleus. A molecule formed following the condensation of precursor amino acids. It houses the beta-lactam ring, a distinct chemical structure that confers their antibacterial properties.
More recently, literature reports on the role carbon, nitrogen and phosphate play in the quality and quantity of penicillin G.
4.0 Major macronutrients of penicillin medium
For P. chrysogenum, the biosynthesis of penicillin G starts when glucose becomes exhausted from the culture medium, and begins to consume a less readily utilised sugar reference. The supply of sugar is the carbon and thus energy source of P. chrysogenum. That no matter its form is instrumental in determining the extent of biomass and titre of penicillin G Marwick et al. (1999).
With the specific nutritional requirements of P. chrysogenum as complex and varied as the microorganism in question, the type of carbon source used also influences penicillin production. As reference describes, the biosynthesis of penicillin G is regulated by glucose and sucrose and to a lesser extent by other sugars (maltose, fructose and galactose); but interestingly not by lactose (Revilla et al. 1984, 1986). This led to the widespread use of lactose; for without regulation, the catabolite repression of glucose is avoided. A phenomenon known as the “glucose effect”, it prevents the overproduction of secondary metabolites.
However, sugar metabolism must also reflect the growth of the producing organism. For P. chrysogenum, lactose was soon identified as an ineffective energy source. In terms of commercial production, a lactose-enriched media would inhibit the growth of P. chrysogenum. So much so, that to employ lactose as its sole carbon source would lead to a decline in biomass and drop in penicillin G titre. Researchers found however, that the catabolite repression of glucose could be avoided in the form of a batch culture when glucose was supplied slowly. By slow feeding of glucose, titre levels of penicillin G surpassed lactose controls.
High glucose concentrations repress the transcription of penicillin biosynthetic genes; pcbAB, pcbC and penDE
Nitrogen and its concentration play a crucial role in penicillin G biosynthesis. As reference denotes, the beta-lactam ring of penicillin contains a nitrogen molecule. P. chrysogenum, uses both inorganic and organic sources of nitrogen. However, while specific amino acids can increase productivity, if unsuitable, can also decrease productivity (Marwick et al., 1999). Singh et al. (2009).
The investigation on the role of amino acids as a source of nitrogen in the production of penicillin began in 1919, with reference reporting a maximum penicillin concentration when this was supplied. However, differing results were recorded by reference, as media supplemented with this contained greater penicillin tites. Nevertheless, more recent results favour the work of reference, whereby the type of nitrogen source used limits the biosynthesis of penicillin. For as another study highlighted, when supplied with ammonia ions, the nitrogen source would favour cell growth, but alternatively would increase penicillin production if supplied with slowly assimilated amino acids.
4.3 Other notable nutrients found within media
Due to the nature of penicillin containing a sulphur molecule its 5-membered ring, the addition of sulphur is indispensable. Whereby a steady supply of sulphur is supplied to the production medium, as sulphuric acid.
P. chrysogenum ensures the production of specific penicillins when the appropriate side chain precursor is fed into the production medium. For penicillin G, this tends to be Phenyl acetic acid. In its absence, carbon and nitrogen may initiate the biosynthesis of different precursors (Elibol, 2004).
Phosphate is the critical growth-limiting nutrient in many secondary metabolite fermentations.
However if supplied to the medium in unfavourable concentrations it can also decrease the product synthesis. An observation first reported by reference, that found when inorganic phosphate concentration was supplied in too high of quantity, the production of penicillin G declined. A result also confirmed by reference, characterised by a later study, showing phosphate to indirectly increase the catabolite repression of glucose (F. Antequera & J.F. Martín, unpublished results).
It appears the limitation of phosphate and sulphate would lead to a nutrient imbalance. That in addition to carbon and nitrogen, its regulation and control is essential when designing production media.
5.0 Media design is an essential step for penicillin biosynthesis
Productivity of penicillin is closely related to nutrients present within the production medium. Whereby both the quantity and quality of nutrients available and the ability to assimilate successfully, are the major determinants of microbial nature and its metabolic activity.
Literature reports penicillin biosynthesis is affected by the concentration of phosphate, and shows a distinct catabolite repression by glucose, well also being regulated by ammonium ion concentration. Furthermore, in terms of the specific production of penicillin G, it appears both a steady supply of sulphur and a careful addition of precursor is required. That seemingly in their absence would prevent the 5 membered-ring and correct derivative of penicillin from being formed respectively.
6.1 Room for improvement
Seemingly absent, at least in terms of commercial production, is the actual amount of nutrients required for growth or penicillin production. Certainly from the authors discussed, it appears the observations that greater amounts of phosphorus, sulphur, and iron are required for penicillin production than for growth are more important. An opinion supported by the shapes of penicillin response curves.
Yet, with raw materials/medium components covering a significant portion of the product cost, could the media design be further optimised?
6.1.1 Current methods of media optimisation
Before 1970s, media optimisation was typically carried out using classical methods such as OVAT. Methods that was expensive, time consuming, and involved plenty of laborious experiments. This ultimately led to an inaccuracy of results tested. However more recently, these techniques have been replaced with modern statistical techniques such as Response surface methodology (RSM) and Artificial neural network that rely on mathematical models
However, these techniques are not yet optimised. Whereby, Irrespective of media chosen; involve large number of experiments, accounting for a great deal of labour cost. Furthermore, there are not many rigorous studies regarding the comparison of medium performances at different scales yet carried out in this line (Gupta and Rao, 2003). Whereby, no matter how promising they may be, are unable to provide full-scale models that accurately reflect the production environment.
Indeed, such techniques continue to rely on shake flask technology, with the misconception that the best medium obtained in the shake flask culture method will equate to the best media in the fermenter (Kennedy et al., 1994; O’Kennedy et al., 2003).
When testing such environments, analytical techniques currently involve; HPLC, different versions of GC or various types of vibrational spectroscopy for sample analysis. Techniques that although highly selective and reliable; in terms of application to process control, are hindered by the need for expensive instrumentation, single element analysis, and complex sample preparation.
6.1.2 Moving forward
Although many of the steps of penicillin biosynthesis are now characterized, many more remain to be identified, and targets for improvement are no longer obvious. As such, a radically new concept has been put forward to replace the current inadequate methods of analytical techniques. The concept will take inspiration from the precise testing of hospital equipment, specifically their chemistry analyser unit.
By using a state of the art bio-analyser, one specifically designed for the testing of patient blood samples. It is hypothesized that the multi-element analysis capabilities of the bio-analyser could be reassigned to analyse the medium composition of penicillin G. It is postulated by doing so; the degree of accuracy and testing of various nutrients, enabled by the bio-analyser, would produce the biochemical profile of production medium. One that would allow industry to identify if the current media design chosen was truly optimal from a financial and production point of view.
Furthermore, with the close follow-up of a fermentation process critical for detecting unfavourable deviations, employing the bio-analyser over traditional techniques represents the potential to save downtime, materials and resources. This is extremely important for penicillin G. As a recent studied identified, a number of nutrients are frequently added in substantial excess of that required.
For untargeted metabolites profiling, an important but often forgotten issue is the necessity of method validation. Thus, the prerequisite of our proposed approach was to validate the reliability, repeatability and sustainability of the developed bio-analyser methods to achieve the following aims:
- A greater scope of nutritional control over the course of an entire production cycle
- Understand significant relationships between different media components in greater detail
- Maximise the efficiency and minimise the production cost and waste by-products to compete effectively against the traditional methods.
- Deliver recommendations to media design in terms of their utility, application and feasibility to maximize the metabolite yield produced by the fermentation process
- Help create a more sustainable media