- Statins are drugs that lower cholesterol by inhibiting the enzyme HMG-CoA reductase. Some early statins like lovastatin are produced naturally by fungi through fermentation. - Lovastatin is produced commercially by fermenting the fungus Aspergillus terreus. Simvastatin is then produced from lovastatin through a chemical reaction. - Solid state fermentation and submerged fermentation techniques can be used to produce statins at an industrial scale. Optimization of fermentation parameters and media composition aims to improve statin yields.

1. FERMENTATION OF
STATINS
Presented by,
D.Keerthana - M.pharm 1st yr,
Dept.of pharmaceuticalchemistry

2. 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.

3. 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 terreus1 .
• 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
cholesterol2 .

4. • 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.

5. 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 commercialised in
1989. Simvastatin remains a commonly prescribed statin.

6. 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.

7. • Lovastatin is commercially produced by fermentation of A. 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.

8. • 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.

9. • This organism produces cytochrome P450 enzyme that is responsible for the
hydroxylation of compactin14,15. 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

10. FERMENTATION TECHNIQUES
• Different fermentation techniques including solid state fermentation (SSF) and
submerged fermentation (SMF) can be used for statin production.
• Large scale commercial production utilises 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 reportedfed - 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. PRODUCTION OF LOVASTATIN

13. SYNTHESISOF SIMVASTATIN AND LOVASTATIN

14. PRODUCTION

15. MICROORGANISMS AND INOCULUM PREPARATION
Terreus strains are isolated from soil.
• Isolates were grown on Czapek-Dox agar slants at 28degree 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 MnCl2, 50 mg Na2MoO4 ·5H2O, and 250 mg CuSO4·5H2O - per liter of
solution.
• The flask with medium was inoculated with 3 x107 conidiospores, held on rotary
shaker at 160 rpm for 2 days at 28-30o C and then is used as inoculum.

16. SOLID SUBSTRATE FERMENTATION
• Lovastatin production and optimisation 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 physico-nutritional parameters.
• The submerged processes have not but yielded constant results and higher yield.

17. • 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.

18. 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°С, 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 – 2,3, KH2PO4 – 2, maltose - 5, рH -7,5.
• After moistening of substrate, 2,5 ml of inoculum (with spore concentration of 107 -
108 ml-1) was added. The flasks were shaken evenly and incubated at 28°С for 14
days. At the end of incubation SSF substrate was dried at 100-105°С, ground using a
porcelain pestle and mortar to a fine powder and used to estimate the lovastatin
content by HPLC analysis.

19. 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, рН 6.
2: Glucose – 30, glycerol - 70, peptone – 8, soybean meal – 30, рH 6,4.
3: Glucose – 45, Na glutamate - 12,5, KH2PO4 – 5, K2HPO4 – 5, FeSO4 ·7H2O,
MnSO4 ·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 мг, 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, K2HPO4 – 1, рH 6,5

20. • Fermentation is carried out at 28℃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 Na2SO4, concentrated to 2 ml by vacuum evaporation
and used for lovastatin estimation.

21. 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.

22. • 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.

23. REFERENCE
1. Istvan, E. (2003) Statin inhibition of HMG-CoA reductase: a 3-dimensional
view. Atheroscler. Suppl. 4, 3–8. doi:10.1016/S1567-5688(03)00003-5.
2. Subhan, M. et al. (2016) Exploitation of Aspergillus terreus for the production of
natural statins. J Fungi 2, 1–13. doi:10.3390/jof2020013
3. Tobert, J.A. (2003) Lovastatin and beyond: the history of the HMG-CoA
reductase inhibitors. Nat. Rev. Drug Discov. 2, 517–526. doi:10.1038/nrd1112
4. Lorenz, R.T. and Parks, L. (1990) Effects of lovastatin (mevinolin) on sterol
levels and on activity of azoles in Saccharomyces cerevisiae. Antimicrob. Agents
Chemother. 34, 1660–1665. doi:10.1128/AAC.34.9.1660

24. THANK YOU