The process involves fermenting a substrate with a fungus that produces statins.

1. FERMENTATION OF STATINS
INSTITIUTE OF PHARMACEUTICAL SCIENCES
KURUKSHETRA UNIVERSITY, KURUKSHETRA
SUBMITTED TO SUBMITTED BY
DR. SUKHBIR LAL KHOKHAR SAGAR SHARMA(32)
ASSISTANT PROFESSOR M PHARM 1st YEAR

2. CONTENT
• Introduction
• Statins
• Discovery
• Fungal Fermentation & Statin Production
• Fermentation Techniques
• Solid Substrate Fermentation
• Liquid Submerged Fermentation
• Applications

3. INTRODUCTION
• Fungi are used industrially to obtain a variety of products, from low value
bulk chemicals to high value drugs like, immunosuppressants, antibiotics,
alkaloids and statins.
• Lovastatin and compactin are natural statins produced as secondary
metabolites by predominantly Aspergillus and Penicillium species,
following a polyketide pathway.
• Lovastatin was one of the first cholesterol-lowering drugs. Many statins are
now chemically synthesized but lovastatin is still required to produce
simvastatin.
• Apart from reducing blood cholesterol levels simvastatin causes pleotropic
effects and has potential to treat various kinds of disorders including
neurodegenerative disease and cancer.

4. STATINS
• Statins are drugs prescribed to reduce serum cholesterol levels.
• The first statin to be approved by the FDA, lovastatin, is produced as a
result of fermentation of Aspergillus terreus.
• Statins act by inhibiting HMG-CoA reductase (HMGCR) through
competitive inhibition.
• This blocks the activity of HMGCR, the rate limiting enzyme in the
synthesis of cholesterol.

5. • Natural statins, including lovastatin and mevastatin (commonly known
as compactin) are produced by direct fungal fermentation.
• Semi-synthetic statins, simvastatin and pravastatin are synthesised by
the stereoselective hydroxylation of natural statins.
• Chemically synthesised statins include atorvastatin, rosuvastatin,
fluvastatin and pitavastatin.

6. DISCOVERY
• Discovery of statins Compactin, discovered by Akira Endo in 1973 as
a structural analogue for the HMG-CoA substrate, was produced by
Penicillium citrinum.
• Lovastatin, previously known as mevinolin K and mevinolin, was
produced from cultures of Monascus ruber and Aspergillus terreus
respectively.
• It was the first statin to be approved by the FDA in 19874.
• Pravastatin, the semi-synthetic derivative of compactin was
commercialized in
• 1989. Simvastatin remains a commonly prescribed statin.

7. FUNGAL FERMENTATION AND STATIN
PRODUCTION
• Statins are produced as a secondary metabolite from a polyketide
pathway. This pathway is regulated by polyketide synthase genes such
as LovB , LovF and LovD, that are responsible for the transcription
regulation and production of these secondary metabolites
• Statins are produced as a secondary metabolite during stress of the
fungi. Acetyl Co-A acts as precursor molecule that plays an important
role in bridging the primary metabolism with the secondary
metabolism leading to production of various secondary metabolites
such as terpenes and polyketides including statins.

8. • Lovastatin is commercially produced by fermentation of Aspergillus
terreus and simvastatin is produced by further chemical treatment of
lovastatin usually involving direct alkylation.
• Compactin is not as effective in inhibiting HMGCR as lovastatin,
however, a semisynthetic derivative of compactin, pravastatin, is
highly effective in lowering blood cholesterol levels.
• Different strategies have been adopted for the efficient and economic
scale up of these metabolites, such as media optimization, using cheap
raw substrates, mutagenesis and bioreactor optimization.

9. • Unlike simvastatin where conversion of lovastatin to simvastatin is a
chemical reaction, the reaction for pravastatin synthesis is
biotransformation Lovastatin can be directly methylated or deacylated
for the synthesis of simvastatin
• This involves a single step fermentation process and then direct
chemical conversion. Pravastatin production involves the
hydroxylation of compactin produced by P. citrinum by the
biotransformation using the bacterium Streptomyces carbophilus.

10. • This organism produces cytochrome P450 enzyme that is responsible
for the hydroxylation of compactin. This dual step fermentation is
economically not feasible. Recent studies have reported the production
of pravastatin in a single fermentative step.
• Enzymes responsible for the hydroxylation are genetically
incorporated in the penicillin-producing fungus Penicillium
chrysogenum. This results in efficient production of pravastatin at
industrial scale

11. FERMENTATION TECHNIQUES
• Different fermentation techniques including Solid State Fermentation (SSF)
and Sub Merged Fermentation (SMF) can be used for statin production.
• Large scale commercial production utilizes submerged batch fermentation.
• There is a controlled aeration and agitation in a bioreactor during SMF,
which increases the oxygen mass transfer and constant distribution of
nutrients to fungal mycelia, resulting in increased production of statins.
• Some studies have also reported fed - batch fermentation that were carried
out in a bioreactor with a capacity of 1000 L. Repeated fed batch processes
can also improve the productivity of desired metabolites.

12. Natural statins, Merck's Mevacor and Zocor and Bristol Myer's Pravachol, are all produced
in fermentation by fungal microorganisms

13. PRODUCTION OF LOVASTATIN

14. SYNTHESIS OF SIMVASTATIN &
LOVASTATIN

15. PRODUCTION
Production of natural & semi-synthetic statins by fungi.

16. MICROORGANISMS AND INOCULUM
PREPARATION
• Terreus strains are isolated from soil.
• Isolates were grown on Czapek-Dox agar slants at 28°C until complete
sporulation.
• Conidiospores were harvested from slants with 5 ml of sterile solution of
0.85% NaCl, 0.2% Tween 80 and transferred into 250 ml Erlenmeyer flasks
containing 50 ml medium (g/l): 10 g glucose, 10 g oat meal, 10 g corn steep
liquor, 0.2 g polyethylene glycol, and 10 ml of trace elements - 100 mg
Na2B4O7 • 10H2O, 50 mg MnCI2, 50 mg NaMoO4 • 5H2O, and 250 mg
CuSO4 5H2O - per litre of solution.
• The flask with medium was inoculated with 3 x107 conidiospores, held on
rotary shaker at 160 rpm for 2 days at 28-30°C and then is used as
inoculum.

17. SOLID SUBSTRATE FERMENTATION
• Lovastatin production and optimization of fermentation parameters
has been of great interest since its discovery.
• Many efforts and trials have been performed to increase the titre.
• Initially, all production processes were carried in Submerged
Fermentations (SMF) by varying physio-nutritional parameters.
• The submerged processes have not but yielded constant results and
higher yield.

18. • Hence a shift towards to Solid State Fermentation (SSF) was gaining
popularity for multiple industrially important products such as
enzymes, pigments, antibiotics etc.
• SSF has been widely employed in industrial productions because of its
advantages such as better process control, maximum substrate
utilisation, lower chances of contamination, easy downstream
processing etc.
• Many bacteria and fungi have been utilised for production of
industrially important products by SSF.

19. SOLID STATE FERMENTATION
• Substrates such as wheat bran, oat bran, rice bran, maize bran and mix of
wheat and peanut bran were used in the solid state fermentation process.
Before fermentation, substrates were ground to the size of 20 mesh. SSF
was performed in 500 ml conical flasks, containing 50 g of solid substrate.
• The flasks were autoclaved for 40 min at 121°C, the substrate's moisture
content was measured and adjusted to a level 55-65% with nutrient solution
(%): glucose- 11, glycerol-16, MgSO4- 0.75, (NH4)2HSO4 – 2 to 3, KH2PO4
- 2, maltose - 5, pH -7,5.
• After moistening of substrate, 2 to 5 ml of inoculum (with spore
concentration of 107 - 108 ml-l) was added. The flasks were shaken evenly
and incubated at 28°C for 14 days. At the end of incubation SSF substrate
was dried at 100-105°C, ground using a porcelain pestle and mortar to a fine
powder and used to estimate the lovastatin content by HPLC analysis.

20. LIQUID SUBMERGED FERMENTATION
• Different glucose and lactose based lovastatin production media are used for SMF. 10 ml
of conidiospores were inoculated in 300 ml Erlenmeyer flasks, containing 100 ml of the
following media (g/l):
1. Glucose - 10, corn steep liquor - 5, tomato paste - 40, oatmeal - 10, pH 6.
2. Glucose - 30, glycerol - 70, peptone - 8, soybean meal - 30, pH 6,4.
3. Glucose - 45, Na glutamate – 12.5, KH2PO4 - 5, K2HPO4 - 5, FeSO4 • 7H2O, MnS04 •
4H2O – 0.1, ZnSO4 • 7H2O – 0.2, MgSO4 7H2O - 0,1, trace elements - 1 ml, pH 6,5.
4. Lactose - 20, yeast extract - 8, KH2PO4 - 1,51, MgSO4 • 7H2O - 1,51, NaCl - 0,4,
ZnSO4 7H2O- 1, Fe(NO3) • 9H2O- 2, biotin – 0.04 ml, trace elements - 1 ml, pH 6,0.
5. Lactose - 70, yeast extract - 8, defatted soybean meal – 0.5, polyethylene glycol 2000-
0.5, KCl - 1, K2HO4 - 1, pH 6,5

21. • Fermentation is carried out at 28°C in flasks held on a rotary platform
shaker at 160 rpm for 24 days.
• Lovastatin was extracted only from biomass after centrifugation of
whole cultural suspension at 6000 rpm for 20 min.
• 1g of mycelium was washed with 0.05M HCl and extracted with 20 ml
of acetonitrile in a rotary shaker at 160 rpm for 60 min.
• Extracts were dried with Na2O4, concentrated to 2 ml by vacuum
evaporation and used for lovastatin estimation.

22. APPLICATION OF STATINS
• The mevalonate pathway is not only responsible for the synthesis of
cholesterol but also for the synthesis of other non-sterol isoprenoids
that are involved in protein prenylation such as binding and regulation
of target proteins.
• Statins may decrease protein prenylation, a key step during a cell
growth and signaling pathway. Statins can be used in combination
with cancer drugs to treat cancer.

23. APPLICATION OF STATINS
• Statins also reduce hepatic cholesterols leading to reduced gallstone
formation and reduced platelet aggregation.
• Recent studies have reported the role of statins in cognitive
impairment after sepsis by reversing the microvascular dysfunction
and reducing neuroinflammation.
• Simvastatin has been found to reduce the incidence of
neurodegenerative disorders such as Alzheimer's disease and
Parkinson's disease.