Pharmaceutical Production of Antibiotics

Pharmaceutical Products and the Microorganisms That Make Them Uses microorganisms, typically grown on a large scale, to produce products or carry out chemical transformation Originated with fermentation processes Major organisms used are fungi Classic methods are used to select for high-yielding microbial variants

Properties of the Microorganisms Properties of a useful industrial microbe include Produces spores or can be easily inoculated Grows rapidly on a large scale in inexpensive medium Produces desired product quickly Should not be pathogenic Amenable to genetic manipulation

Industrial Products Produced by Microorganisms Microbial products of industrial interest include Microbial cells Enzymes Antibiotics, steroids, alkaloids Food additives Commodity chemicals Inexpensive chemicals produced in bulk Include ethanol, citric acid, and many others

Production and Scale Primary metabolite Secondary metabolite Produced during exponential growth Example: alcohol Secondary metabolite Produced during stationary phase

Production and Scale Secondary metabolites Not essential for growth Formation depends on growth conditions Produced as a group of related compounds Often significantly overproduced Often produced by spore-forming microbes during sporulation

Primary metabolite Secondary metabolite Alcohol Penicillin Cells Alcohol, sugar, or cell number Penicillin, sugar, or cell number Sugar Cells Sugar Figure 15.1 Contrast between production of primary and secondary metabolites. Penicillin Time Time

Production and Scale Secondary metabolites are often large organic molecules that require a large number of specific enzymatic steps for production Synthesis of tetracycline requires at least 72 separate enzymatic steps Starting materials arise from major biosynthetic pathways

Production and Scale Fermentor is where the microbiology process takes place Any large-scale reaction is referred to as a fermentation Most are aerobic processes Fermentors vary in size from 5 to 500,000 liters Aerobic and anaerobic fermentors Large-scale fermentors are almost always stainless steel Impellers and spargers supply oxygen

Fermenters are large vessels that monitor and react to changes allowing ideal conditions for bacterial growth to be maintained by; Supplying oxygen for respiration Stirring the suspension to maintain even temperature, oxygen and nutrient distribution Water cooling system to remove excess heat produced during respiration Monitoring pH etc to react to any changes

Figure 15.2 Fermentors. Motor Steam pH pH controller Acid–base reservoir and pump Sterile seal Viewing port Filter Exhaust Impeller (mixing) External cooling water out Cooling jacket External cooling water in Culture broth Figure 15.2 Fermentors. Sparger (high- pressure air for aeration) Steam in Sterile air Valve Harvest

Large scale microbe production Large scale production of microorganisms has many problems The bacteria need to be Kept at the right temperature Have nutrients supplied ie food, oxygen Have waste products removed ie waste and carbon dioxide

Antibiotics: Isolation, Yield, and Purification Compounds that kill or inhibit the growth of other microbes Typically secondary metabolites Most antibiotics in clinical use are produced by filamentous fungi or actinomycetes Still discovered by laboratory screening Microbes are obtained from nature in pure culture Assayed for products that inhibit growth of test bacteria

Antibiotics: Isolation, Yield, and Purification

The classical method for searching for antibiotics By random search in the soil The soil is a vast repository of microorganisms and it is to the soil that search is turned when antibiotics are being sought

The primary screening Several methods have been employed in primary screening The crowded plate The cross-streak method

The crowded plate A heavy aqueous suspension (1:10; 1:100) of soil is plated on agar. Organisms showing clear zones around themselves are isolated for further study. This method has the disadvantage that slow- growing antibiotic-producing organisms such as actinomycetes are usually over grown and are therefore hardly isolated.

Spread a soil dilution on a plate of selective medium Sterile glass spreader Incubation Colonies of Streptomyces species Overlay with an indicator organism Nonproducing organisms Incubate Zones of growth inhibition Producing organisms

The cross-streak method This method is used for testing individual isolates, especially actinomycetes which may be obtained from soil without any previous knowledge of their antibiotic- producing potential. The purified isolate is streaked across the upper third of plate containing a medium which supports its growth as well as that of the test organisms. A variety of media may be used for streaking the antibiotic producer. It is allowed to grow for up to seven days, in which time any antibiotic produced would have diffused a considerable distance from the streak. Test organisms are streaked at right angles to the original isolates and the extent of the inhibition of the various test organisms observed.

Isolation and screening of antibiotic producers. II. Testing Activity Spectrum Streak antibiotic producer across one side of plate Incubate to permit growth and antibiotic production Antibiotic diffuses into agar Streptomyces cell mass Cross-streak with test organisms Isolation and screening of antibiotic producers. Incubate to permit test organisms to grow Growth of test organism Inhibition zones where sensitive test organisms did not grow

Secondary screening Organisms showing suitably wide zones of clearing against selected target organisms are cultivated in broth culture in shake flasks using components of the solid medium in which the isolate grew best. Secondary screening is aimed at eliminating at an early stage any antibiotic which does not appear promising either by virtue of low activity, other undesirable properties or because it has been discovered previously

Industrial Production of Penicillins Penicillins are -lactam antibiotics Natural and biosynthetic penicillins Semisynthetic penicillins Broad spectrum of activity Penicillin production is typical of a secondary metabolite Production only begins after near-exhaustion of carbon source High levels of glucose repress penicillin production

Penicillin Fermenters are used to produce large amounts of penicillin The penicillin mould requires a lot of oxygen The mould grows rapidly but does not produce penicillin until most of the nutrients are used up Enough food is provided to let the mould grow then supplies are limited to maximize penicillin production There is therefore a lag between stating the process and penicillin production

Magic Bullet Penicillin and other beta-lactam antibiotics (named for an unusual, highly reactive lactam ring) are very efficient and have few side effects (apart from allergic reactions in some people. As an additional advantage, the enzymes attacked by penicillin are found on the outside of the cytoplasmic membrane that surrounds the bacterial cell, so the drugs can attack directly without having to cross this strong barrier

Penicillium The name Penicillium comes from penicillus = brush, and this is based on the  brush-like appearance of the fruiting structures

Glucose feeding Nitrogen feeding 100 90 80 Penicillin 70 Biomass (g/liter), carbohydrate, ammonia, penicillin (g/liter  10) Kinetics of the penicillin fermentation with Penicillium chrysogenum. 60 50 40 Cells 30 20 Lactose 10 Ammonia 20 40 60 80 100 120 140 Fermentation time (h)

Special fermenter Conditions for the production of Penicillin Most penicillins form filamentous broths that are pseudoplastic in nature. This means they can be difficult to mix due to their high (and not constant) viscosity Penicillin is an aerobic organism; therefore the rate of oxygen supply is critical to the fermentation The optimum pH for penicillin growth is 6.5. Thus the reactor must maintain pH efficiently (this is frequently done by addition of NaOH). Strain Stability problems do exist and careful strain maintenance is required. Biomass doubling is about 6h

Composition of early media % corn steep liquor (cotton seeds, peanut, CULTURE MEDIUM   Composition of early media % corn steep liquor (cotton seeds, peanut, Linseed or soybean meals) 2-4 lactose, glucose or beet molasses 2-4 CaCO3 or phosphates (buffer) 0.5-1 precursor 0.1-0.5 Catabolite repression of the enzymes responsible for penicillin biosynthesis occurs in high concentrations of glucose. Use of precursors to increase penicillin yield.  benzyl penicillin (penicillin G) is desired, phenylacetic acid is added

Use of precursors:  

Production It requires a batch fermenter, process is normally used to prolong the stationary period and so increase production Downstream processing is relatively easy since penicillin is secreted into the medium (to kill other cells), so there is no need to break open the fungal cells the product needs to be very pure, since it being used as a therapeutic medical drug, so it is dissolved and then precipitated as a potassium salt to separate it from other substances in the medium

Penicillin G Penicillinase (E.coli) 6 - APA Side Chain Modification Amoxycillin AUGMENTIN b-lactamase resistant Clavulanic acid

6-Aminopenicillanic Acid (6-APA) 6-APA: Raw material for production of new semisynthetic penicillins (amoxycillin and ampicillin) Fewer side effects Diminished toxicity Greater selectivity against pathogens Broader antimicrobial range including G- -ve Improved pharmacological properties Gastric acid stability & oral absorbability Resistance to beta-lactamases

The resulting penicillin (called penicillin G) can be chemically and enzymatically modified to make a variety of penicillins with slightly different properties. These semi-synthetic penicillins include penicillin V, penicillin O, ampicillin and amoxycillin

Penicillin G Penicillin G is not stable in the presence of acid (it is therefore said to be acid-labile). Since our stomach has a lot of hydrochloric acid in it (the pH can be around 2.0), if we were to ingest penicillin G, the compound would be destroyed in our stomach before it could be absorbed into the bloodstream penicillin G must be taken by intramuscular injection - to get the compound in our bloodstream. Many of the semi-synthetic penicillins can be taken orally.

Use of Biotechnology in Antibiotic improvement

Metabolic engineering of bacteria Increasing biological production of small molecules Random screening for overproducing strains (genome shuffling) Rational engineering of pathways

Increasing production of antibiotics -traditional methods Obtain organism that produces a specific compound--Penicillium mold originally made micrograms per liter of culture Randomly mutagenize the organism and screen for increased production, repeat using top producing organism Outcome: grams of penicillum per liter of culture (1000-fold increase in production)

Advance Method An alternative to simple random mutagenesis: genome shuffling Gene shuffling is the process of recombining the starting pool of sequences to generate new gene- sequences that subsequently can be screened for particular desired characteristics. The shuffling advantage: simultaneous recombination of entire genomes (breeding) with multiple parents

Increasing production of a biological compound: rational design 1)Increase production of a naturally produced commercial compound Modify existing genes 2) Obtain a new organism that can convert an existing compound into a commercial compound Introduce new genes

Method for introducing DNA Transformation (spontaneous) Transformation (chemical, electroporation) Conjugation Method for replicating DNA Plasmid replicon Integration into chromosome (homologous recombination)