Industrial Microbiology

HEALTHCARE PRODUCTS

Production of Penicillin in Industrial Bioreactors

The name penicillin is applied to a variety of compounds produced by various species of
Penicillium and also related to many semi-synthetic types of penicillin, produced by
converting one antibiotic, such as penicillin G, into another, such as ampicillin.

Aspergillus nidulans, another mould fungus, also produces penicillins. The complete
biosynthesis pathway for penicillin is extremely complex and unlikely to be achieved in the
laboratory.

Commercial production of penicillin

Penicillin G, one of the most active and widely used forms, is manufactured commercially
using Penicillium chrysogenum.

The process is carried out in stainless steel fermenters of l0000dm3 capacity. The fermenter
is steam sterilised and loaded with sterilised growth medium (corn steep liquor) containing
lactose, amino acids, mineral salts and other substances (carbon source: glucose/sugars;
nitrogen source NH4+ / NO3- /amino acids other mineral salts; phenylethanoic acid, a
metabolic intermediate, is also added, to increase the yield).

An inoculum of strongly growing hyphae is added. Both glucose and nitrate are added
periodically. The pH requires adjustment from time to time, to neutralise ammonia
produced by the fungus. Temperature is set at first to give the maximum growth rate and
then altered to favour penicillin synthesis. The fermenter is continuously stirred and sterile
air blown in. An external cooling jacket is used for temperature control. The diagram below
shows the type of fermenter used.

Penicillin is a secondary metabolite, produced in large quantities only towards the end of the
growth period of the fungus therefore it is essential for all of the mycelium to reach peak

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growth at the same time. This is why batch fermentation, rather than a continuous process,
is appropriate for penicillin manufacture.

After about 160-200 hours, the broth is filtered. Penicillin passes through in the filtrate
which is further processed to crystallise the product. Antibiotics such as penicillin are
usually produced in large cylindrical vats, constructed of stainless steel, containing a liquid
medium in which Penicillium chrysogenum is grown.

Before use, fermenters must be sterilised, usually with superheated steam. Usually these
fermenters are operated in a batch process. After a certain amount of time for fungal
growth, followed by gradual production of antibiotic, the contents are removed and
processed to extract the antibiotics, then the fermenter is cleaned, sterilised and the process
is repeated.

Penicillin extraction
After 6-8 days of batch culture, the liquid medium is pumped out, filtered and concentrated.
The basic antibiotic - benzyl penicillin - is precipitated as crystals when potassium
compounds are added.

This antibiotic may then be modified by the action of other micro-organisms or by chemical
means, before being mixed with inert substances and pressed into tablets or converted into
syrup or injectable form.

Although the molecular structure of penicillin is known, and it may be synthesised by

chemical methods, it is not economic to do so. The production process still relies on fungal
fermentation based on biological principles, although modern strains are much more
productive than the early strains. This has been achieved through screening programmes
involving isolates from different sources, and treatment to encourage mutations.

References
http://www.biotopics.co.uk/microbes/penici.html (181008)

Production of Human Growth Hormone

Human growth hormone (hGH), a protein hormone produced and secreted by the
somatotropic cells of the anterior pituitary, plays a key role in somatic growth through its
effects on the metabolism of proteins, carbohydrates, and lipids. The biological properties of
hGH are diverse and complex, with the molecule exhibiting anabolic activity, insulin-like and
diabetogenic activities, and lactogenic activity, as well as producing effects on water and salt
retention. First isolated from the pituitary gland, hGH is produced using recombinant DNA
technology and has been approved for use as a human pharmaceutical in the treatment of
growth failure owing to hGH deficiency and in the treatment of Turner's syndrome in Europe
and Japan. In addition, hGH is being investigated for use in treating burns, various wounds,
osteoporosis, cachexia, and other conditions. Human growth hormone is one of the largest
selling therapeutic proteins produced by recombinant DNA technology.

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with
different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors
were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in
the amino acid extensions of the hGH precursors led to three-fold differences in specific
expression rates. The specific expression rate of the hGH precursors was inversely
proportional to the ratios of the specific growth rates of plasmid containing and plasmid free
cells ( +/ -) and also to the genetic stability. To ensure a satisfactory genetic stability in
production fermentors, an hGH precursor with moderate expression efficiency was chosen.

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The medium composition and growth conditions were studied, resulting in the choice of
glucose fed batch fermentation process using a complex medium. The fermentation process
comprised a glucose-limited growth phase followed by a second phase with increased
glucose feed and exhaustion of phosphate from the medium.

Chemostat experiments showed that the glucose concentration and the metabolic condition
of the cells - i.e. with or without formation of acetate - were not critical per se in order to
obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH
is catabolite repressed by glucose. Furthermore it was shown that the specific production
rate of MAE-hGH was independent of the specific growth rate and it was further
demonstrated that the decrease in expression efficiency in glucose batch fermentation was
a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was
enhanced by a salt effect caused by increased consumption of acid and base used to control
pH.

References
Production of recombinant human growth hormone in Escherichia coli: Expression of
different precursors and physiological effects of glucose, acetate, and salts. E. Bech Jensen, S.
Carlsen *Novo Nordisk a/s, Novo Alle, DK-2880 Bagsvaerd, Denmark. Published online at
http://www3.interscience.wiley.com/journal/107622572/abstract (181008)

Production of Recombinant Human Interferon

Interferons are natural proteins produced by the cells of the immune system of most
vertebrates in response to challenges by foreign agents such as viruses, parasites and tumor
cells. Interferons belong to the large class of glycoproteins known as cytokines. Interferons
are produced by a wide variety of cells in response to the presence of double-stranded RNA,
a key indicator of viral infection. Interferons assist the immune response by inhibiting viral
replication within host cells, activating natural killer cells and macrophages, increasing
antigen presentation to lymphocytes, and inducing the resistance of host cells to viral
infection. When the antigen is presented to matching T and B cells, those cells multiply and
strategically and specifically wipe out the foreign substance. That is why antigen
presentation is so important to the immune response.

There are three major classes of interferons that have been described for humans according
to the type of receptor through which they signal:
• Interferon type I: All type I IFNs bind to a specific cell surface receptor complex known as
the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I
interferons present in humans are IFN-α, IFN-β and IFN-ω.
• Interferon type II: Binds to IFNGR. In humans this is IFN-γ.
• Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called
CRF2-4) and IFNLR1 (also called CRF2-12)

Production of Interferon gamma

Method of producing human gamma interferon comprises:
• providing Chinese hamster ovary cells bearing a first DNA sequence which codes on
expression for human gamma interferon and includes at least one intron of the genomic
human gamma interferon gene, said first DNA sequence being operably linked to a
constitutive promoter functional in such cells, said promoter being a non-interferon
promoter, said cells also bearing a gene coding on expression for dihydrofolate
reductase, said gene likewise being operably linked to a constitutive promoter functional
in such cells;

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• cultivating the cells in a medium containing methotrexate at a level toxic to cells which
do not constitutively express dihydrofolate reductase, whereby the DHFR gene and the
gamma interferon gene are co-amplified, and
• cultivating such cells under conditions in which the cells constitutively express human
gamma interferon in recoverable quantities.

Production of Interferon alpha

Escherichia coli TG1 transformed with a temperature-regulated interferon-alpha expression
vector was grown to high cell density in defined medium containing glucose as the sole
carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to
achieve an exponential increase in biomass at growth rates which minimized acetate
production. Thermal induction of such high cell density cultures resulted in the production of
approximately 4 g interferon-alpha/l culture broth.

Production of Interferon beta

The production of interferon- by NB1-RGB fibroblast cells cultured on protein and peptide
membranes prepared from silk fibroin, motif peptides of silk fibroin [(AG)n] containing
arginine-glycine-aspartic acid (RGD) peptide, and PronectinTM was investigated. The cell
density on various protein and peptide membranes was approximately the same, although
the production of interferon- depended significantly on the membranes where the cells
were cultured. The highest production of interferon- was observed when the cells were
cultured on (AG)6RGD(AG)7 membranes prepared with hexafluoroacetone (HFA) as the
casting solvent.

Both the chemical composition and the secondary and higher order structure of the peptide
membranes are important for enhanced production of interferon- .

References
http://www.freepatentsonline.com/4889803.html (181008)

Application Microbial Biotechnology. 2000 Jun; 53(6):655-60. Published online at

http://www.ncbi.nlm.nih.gov/pubmed/10919322 (181008)

Production of interferon-β by fibroblast cells on membranes prepared with RGD-containing

peptides. Akon Higuchi, Yasunari Takanashi, Nobuya Tsuzuki, Tetsuo Asakura, Chong-Su Cho,
Toshihiro Akaike, Mariko Hara. Department of Applied Chemistry, Seikei University,
Musashino 180-8633, Japan. Published online at
http://www3.interscience.wiley.com/journal/104528154/abstract (181008)