1.0 Introduction
Penicillin G continues to remain an essential component of the medical toolkit, displaying unmatched activity against susceptible bacterial infections. To this day, it continues to be the focus of much research interest. Namely, this is due to its commercial and therapeutic importance, difficulty of cell growth, and consequence of engineering variables (Patnaik, 2001). That as a collective has created unique and diverse challenges throughout the production pipeline.
When attempting to produce cheaper and more effective penicillin, naturally, industry centres on strain development. But as research identifies, strain improvement and media development are intrinsically linked. That is to say, a strain cannot be chosen without a developed medium, and the optimum medium cannot be proposed without the finest strain (Singh et al., 2017). Considering the above description, industry has seen a renewed interest in the characterisation and design of a media composition that can accommodate strain development.
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When deciding on media design, it is necessary to first consider the most suitable fermentation settings and production medium components (carbon, nitrogen, mineral salts, etc). All of which must be recognised and optimised accordingly (Singh et al., 2017). For penicillin G fermentation, the media optimisation represents a significant cost and time factor in the bioprocess development. Increasing pressure on the demands of existing experimental procedures.
To gain a greater appreciation of media design and its part in penicillin G biosynthesis, 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 within the medium are characterised.
2.0 Penicillin is a secondary metabolite
Following the discovery of penicillin, researchers exploited microorganisms to produce secondary metabolites (Kumar et al., 2018). Unlike primary metabolites, they do not play a physiological role during exponential growth. Rather, they are formed during a subsequent growth stage called the idiophase. Distinct from the exponential phase; their production begins after the growth of a producing organism develops a nutrient imbalance (Kumar et al., 2018). A view held by Jarvis and Johnson; following analysis of batch culture data, concluded the precise rate of penicillin formation to be greatest when the organism growth rate was close to zero.
For large-scale production, industry has typically focussed on the filamentous microorganisms. With the filamentous fungal genus Penicillium, receiving the greatest interest. This interest lies in the penicillium's inherent capabilities to deliver a diverse range of metabolites. Among which, penicillin G represents the start of antibiotic production (Kumar et al., 2018).
For Penicillin G, Penicillium chrysogenum is the species of choice.
3.0 Penicillin biosynthesis is determined by its production medium
Formation of secondary metabolites involves the uptake of several intermediates (Figure 1), which are in effect the building blocks of the antibiotic. For penicillin G, this is of particular relevance. 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 certain amounts and types of nutrients be fed into the production medium. For as VanderMolen et al., (2013) describe, when variation occurs, the quality and biosynthesis of penicillin G is reduced.
Figure 1: The biosynthesis of penicillin G, with genes and their encoding enzymes defined (Peñalva, Rowlands and Turner, 1998).
Literature directs 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 biosynthesis pathway; that without, penicillin would not be produced. Though, perhaps even more important; certainly in terms of penicillins commercial value, is the formation of the penam nucleus (Revilla et al., 1986). 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.
Table 1: The major macronutrient elements, their physiological functions, growth requirements and common sources (Stanbury, Whitaker and Hall, 2017).
More recently, literature reports on the effects macronutrients; carbon, nitrogen and phosphate inflict on the quality and quantity of penicillin G.
4.0 Major macronutrients of penicillin G production medium
4.1 Carbon
For P. chrysogenum, penicillin G biosynthesis starts when glucose becomes exhausted from the production medium, and begins to consume a less readily utilised sugar (Soltero and Johnson. 1953). The supply of sugar is the carbon and thus energy source of P. chrysogenum. That no matter its type is instrumental in shaping the extent of biomass and titre of penicillin G (Marwick et al. 1999).
Figure 2: The effect of glucose at a number of different levels on penicillin biosynthesis. Adapted from Rokem, Lantz and Nielsen, (2007).
With the nutritional requirements of P. chrysogenum as drawn-out and diverse as the microorganism in question, the type of carbon source is greatly influential. Revilla et al. (1986) describe the biosynthesis of penicillin G to be regulated by glucose, sucrose; and to some extent galactose and maltose; but interestingly not lactose. This ultimately led to the initial widespread use of Lactose; for, without regulation, the catabolite repression of glucose was now avoided. A phenomenon known as the “glucose effect”, it prevents the overproduction of secondary metabolites (Martin and Aharonowitz, 1983).
Figure 3: The impact of different rates on penicillin production. The addition of sugar started after 24 hours (Soltero and Johnson. 1953).
Yet, sugar metabolism must also reflect the growth of the producing organism. For P. chrysogenum, this soon identified lactose to be 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. However, by running production as a batch culture; and feeding glucose slowly to the production medium, catabolite repression was avoided (Soltero and Johnson 1953).
Figure 4: Penicillin production from glucose supplied in a semi-continuous fashion. 0.5% of glucose was added every 12 hours commencing at 24 hours (Soltero and Johnson)
It is thought the high glucose concentrations were repressing the transcription of penicillin biosynthetic genes: pcbAB, pcbC and penDE (Martín et al., 1999).
Figure 5: The effect of glucose and alkaline pH on the transcription of pcbAB, pcbC, and penDE genes. Black arrows depict positive effects while negative effects are shown by dotted lines. White arrows indicate the direction of transcription. Intergenic sequences are shown in dotted boxes. Alkaline pH exhibits a small positive effect on the pcbAB, pcbC, and pentacene promoters, while glucose negatively affects both promoters (Martín et al., 1999).
4.2 Nitrogen
Nitrogen and its quantity play a crucial role in penicillin G biosynthesis. As Tudzynski (2014) denotes, the beta-lactam ring of penicillin contains a nitrogen molecule (Figure 1). For the organism in question, P. chrysogenum, it may use both inorganic and organic sources of nitrogen. However, while specific amino acids can increase productivity, if unsuitable, can also decrease productivity (Demain and Vaishnav, 2006),
Investigation into the effect amino acids as a source of nitrogen began in 1919. With Demain and Vaishnav, (2006) reporting a drop in penicillin production when high ammonia concentrations were supplied. Indeed, as supported by more recent results, the nature and concentration of nitrogen can both increase and limit penicillin G biosynthesis. For as literature describes, ammonia ions tend to favour cell growth; whereby slowly assimilated nitrogen sources, such as glutamate, will increase penicillin production (Tudzynski, 2014).
4.3 Other notable nutrients found within media
4.3.1 Sulphur
As penicillin contains a sulphur molecule its 5-membered ring (Figure 1), the addition of sulphur is indispensable. Whereby a steady supply is provided to the production medium, typically as sulphuric acid.
4.3.2 Precursor
To ensure production of specific penicillins, the appropriate side chain precursor is supplied (Higuchi et al., 1946). For penicillin G, this is Phenyl acetic acid (Figure 3). It is important to ensure the appropriate precursor is present. For if without, carbon and nitrogen may initiate the biosynthesis of different precursors and thus different penicillin formation (Elibol, 2004).
4.3.3 Phosphate
Phosphate is the decisive growth-limiting nutrient in most secondary metabolite fermentations (Li, Zhao and Yuan, 2005).
However if supplied to the production medium in unfavourable quantities, can also decrease product synthesis. An observation first reported by F. Antequera & J.F. Martín, (1999), identifying inorganic phosphate when supplied in too high of quantity, can decrease penicillin G biosynthesis. A result later confirmed by Martin, (2000) suggesting phosphate to be acting indirect on the catabolite repression of glucose (Figure 6).
It thus appears the limitation of phosphate and sulphate leads to a nutrient imbalance. That in addition to carbon and nitrogen, its regulation and control is fundamental in media design.
5.0 Media design is an essential step for penicillin biosynthesis
Penicillin productivity is closely related to existing nutrients found within the production medium. Whereby both the quantity and quality of nutrients present and the ability to assimilate effectively, are determinants of P. chrysogenum nature and metabolic activity.
Literature reports penicillin biosynthesis to be affected by phosphate concentration, showing a clear catabolite repression by glucose, well subsequently being regulated by ammonium ion concentration. Furthermore, in terms of penicillin G biosynthesis, both a steady supply of sulphur and a careful addition of precursor are required. That in their absence would prevent the 5 membered-ring and correct derivative of penicillin from being formed respectively.
6.0 Room for improvement
Seemingly absent, at least in terms of commercial production, is the specified quantity of nutrients to allow growth or penicillin biosynthesis. Certainly from the authors discussed, it appears the observations that more amounts of sulphur, phosphorus and iron are needed for penicillin biosynthesis than for growth are more influential. An opinion reinforced by the shapes of penicillin response curves (Figure 6).
Yet, with raw materials/medium components a significant portion of overall product cost, is their room for improvement in media design?
6.1 Current methods of media optimisation
In the run up to the 1970s, media optimisation involved classical methods including OVAT (Singh et al., 2017). Methods that were expensive, time intensive, and involved plenty of laborious experiments (Panda, Ali and Javed, 2007). This led to an inaccuracy of results. However, more recently media optimisation has been replaced by modern statistical techniques including; Response surface methodology (RSM) and Artificial neural network that rely on mathematical models.
However, these techniques are not yet optimised. Whereby, regardless of media chosen, involve countless experiments that account for a great deal of labour cost. Furthermore, there are a limited number of rigorous studies concerning the comparison of medium performances at dissimilar scales yet performed in this line (Gupta and Rao, 2003). As such, no matter how promising these techniques may be; due to the inadequacy of testing, are unable to provide models that closely 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).
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.
7.0 Moving forward
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 understanding of nutritional control over the course of an entire production cycle
- Increase the efficiency and reduce the production cost and waste by-products to contest effectively against the traditional methods.
- Provide recommendations to media design regarding of their application, value and feasibility to further develop penicillin yield
- Understand if the current media design can be made more sustainable, to support reproducibility?
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