Penicillin: Strain Improvement and Media Development

1.0 Introduction

Penicillin G continues to remain an essential component of the medical toolkit, exhibiting unrivalled 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, the difficulty of cell growth, and the consequence of engineering variables. That as a collective has created unique and diverse challenges throughout the production pipeline.

When attempting to produce cheaper and more effective penicillin, naturally, the industry centres on strain development. But as research continues to show, 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. Considering the above description, the industry has seen a renewed interest to further characterise and design a media composition that accommodates strain development.

When designing the production medium, the most suitable fermentation settings (e.g., temperature, pH, agitation etc) and the suitable medium components (carbon sources, nitrogen sources, mineral salts, trace elements etc) must all be recognised and optimised accordingly. 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 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 within the medium are characterised.

2.0 Penicillin is a secondary metabolite

Following the discovery of penicillin, researchers exploited microorganisms to produce secondary metabolites. 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. 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, the industry has typically focussed on the filamentous microorganisms, particularly fungi. 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. For Penicillin G, Penicillium chrysogenum is the species of choice.

3.0 Penicillin biosynthesis is determined by its growth medium

The formation of secondary metabolites involves the uptake of several intermediates, that is 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. As the reference describes, when variation occurs, the quality and biosynthesis of penicillin G are impeded.

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 penicillin's 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 effects macronutrients; carbon, nitrogen and phosphate inflict on the quality and quantity of penicillin G.

4.0 Major macronutrients of penicillin 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 reference. The supply of sugar cab said to be the carbon and thus energy source of P. chrysogenum. That no matter its nature is instrumental in shaping the extent of biomass and titre of penicillin G Marwick et al.

With the nutritional requirements of P. chrysogenum as drawn-out and diverse as the microorganism in question, the type of carbon source used is greatly influential. Reference describes the biosynthesis of penicillin G to be regulated by glucose and sucrose; and to a degree lesser galactose, maltose and fructose; but interestingly not lactose. Lactose soon became largely popular throughout the industry; for without regulation, the catabolite repression of glucose was now avoided. A phenomenon known as the “glucose effect”, it prevented the overproduction of secondary metabolites.

Yet, 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 employing lactose as its sole carbon source would lead to a decline in biomass and a 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. The slow feeding of glucose led to penicillin G titre levels surpassing lactose controls. High glucose concentrations repress transcription of penicillin biosynthetic genes; pcbAB, pcbC and penDE.

4.2 Nitrogen

Nitrogen and its quantity 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 and inorganic sources of nitrogen. However, while specific amino acids can increase productivity, if unsuitable, can also decrease productivity.

Investigation into the effect amino acids as a source of nitrogen, imposed on the production of penicillin started in 1919, with reference reporting the greatest penicillin when supplied with this. However, differing results were detailed by reference, as media fed with this contained higher penicillin tites. Nonetheless, more recent results support reference, whereby the nature of nitrogen used can limit penicillin G biosynthesis. For example, ammonia ions have been found to increase cell growth, whereby slowly assimilated amino acids provoke penicillin biosynthesis reference.

4.3 Other notable nutrients found within media

4.3.1 Sulphur

As penicillin contains a sulphur molecule its 5-membered ring, 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 the production of specific penicillins, the appropriate side chain precursor is supplied. For penicillin G, this is Phenylacetic acid. 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.

4.3.3 Phosphorous

Phosphate is the decisive growth-limiting nutrient in most secondary metabolite fermentations.

However, when supplied to the production medium in unfavourable quantities, can decrease product synthesis. An observation first reported by reference, identifying when inorganic phosphate concentration was supplied in too high of quantity, the production of penicillin G subsequently declined. A result also confirmed by reference, suggests phosphate causes an indirect increase on the catabolite repression of glucose.

It 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 and shows distinct catabolite repression by glucose, well also being regulated by ammonium ion concentration. Furthermore, in terms of penicillin G biosynthesis, both a steady supply of sulphur and 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.

5.1 Room for improvement

Seemingly absent, at least in terms of commercial production, is the specific quantity of nutrients necessary for 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.

Yet, with raw materials/medium components a significant portion of the overall product cost, is their room for improvement in media design?

5.1.1 Current methods of media optimisation

In the run-up to the 1970s, media optimisation involved classical methods including OVAT. Methods that were expensive, time-intensive, and involved plenty of laborious experiments. This led to the inaccuracy of results. However, more recently media optimisation has been replaced by modern statistical techniques including; Response surface methodology (RSM) and Artificial neural networks that rely on mathematical models.

However, these techniques are not yet optimised. Whereby, regardless of the 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. 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.

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.

5.1.2 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 the 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 study 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.