DEPARTMENT OF BIOTECHNOLOGY

FEDERAL UNIVERSITY DUTSE

Course; industrial biotechnology

Assignments

Question: discuss on biosynthesis of streptomycin and tetracycline.

ABDULRAZAK SALE YUSUF

FSC/BTG/18/2010
INTRODUCTION AND HISTORY

STREPTOMYCIN is an antibiotic agent produced by certain strains of Streptomyces griscus . It was found
as the result of a search for an agent that would be active against gram-negative bacteria yet "nontoxic
to the body and would therefore offer possibilities as a chemotherapeutic agent. It was first obtained as
a crude concentrate in 1944 by Selman A. Waksman and Albert Schatz at the New Jersey Agricultural
Experiment Station, Rutgers University. The concentrate isolated by Dr. Waksman showed sufficient
therapeutic value to stimulate considerable effort by Merck & Co., Inc., toward the establishment of its
chemical composition, the development of methods for its production, and the study of its medical
performance. Encouraged by the success of these combined evaluations, Merck started plans for large
scale production in July 1945.

Streptomycin is an antibiotic medication used to treat a number of bacterial infections, including

Mechanism of action

Streptomycin has two mechanism of action depending on what conformation (isomer) is at in the
system in which it will work. Isomer A functions as a protein synthesis inhibitor. It binds to the small 16S
rRNA of the 30S subunit of the bacterial ribosome irreversibly, interfering with the binding of formyl-
methionyl-tRNA to the 30S subunit. This leads to codon misreading, eventual inhibition of protein
synthesis and ultimately death of microbial cells through mechanisms that are still not understood.
Speculation on this mechanism indicates that the binding of the molecule to the 30S subunit interferes
with 30S subunit association with the mRNA strand. This results in an unstable ribosomal-mRNA
complex, leading to a frameshift mutation and defective protein synthesis; leading to cell death. As
human and bacterial both have ribosomes, streptomycin has significant side effects in human cells. At
low concentrations, however, streptomycin only inhibits growth of the bacteria by inducing prokaryotic
ribosomes to misread mRNA.
Streptomycin isomer B is a peptidoglycan synthesis inhibitor much like lysozyme. It binds to the
glycosidic linkages and breaks them through a SN2 mechanism. This leads to bacterial cell walls' integrity
being compromised, ultimately resulting in death of microbial cells.

Streptomycin is an antibiotic that inhibits both Gram-positive and Gram-negative bacteria, and is
therefore a useful broad-spectrum antibiotic.

Biosynthesis of
Streptomycin:

Streptomycin is directly
derived from glucose. Though the
enzymes involved in the synthesis
of N- methyl glucosamine are
not yet known, it is expected that
about 28 enzymes take part in the
conversion of glucose into
streptomycin as precised
in Fig. 6.11.
Fermentation Process of Streptomycin:

Industrially streptomycin is produced by submerged culture method, whose flow sheet is given in Fig.
6.12.
When Woodruff and Mc Daniel (1954) suggested medium consisting of soyabean meal (1%), glucose 1%
and sodium chloride (0.5%), Hocken hull (1963) recommended the medium consisting of glucose (2.5%),
soyabean meal (4.0%), distillers dry soluble (0.5%) and sodium chloride (0.25%) and pH 7.3-7.5 for
production of streptomycin by S. griseus.

(i) The Inoculum Production:

Spores of S. griseus maintained as soil stocks or lyophilized in a carrier such as sterile skimmed milk, is
employed as stock culture. The spores from these stock cultures are then transferred to a sporulation
medium to provide enough sporulated growth to initiate liquid culture build-up of mycelial inoculum in
flasks or inoculum tanks. After sufficient mycelial growth, it is fed to production fermenter.

(ii) Preparation of the Medium:

A production medium contains carbon source and nitrogen source. Glucose is one of the best carbon
sources which helps in the greater yield of streptomycin, because it provides basic carbon skeleton for
the streptomycin production. Apart from glucose, fructose, maltose, lactose, galactose, mannitol, xylose
and starch can also be used as carbon source. Polysaccharides and oligosaccharides generally give low
yields.

Peptones, soya extracts, meat extract, the residue from alcohol distillation, ammonium salts, nitrates
and glycine may be used as nitrogen source. Magnesium, calcium, potassium, boron and molybdenum
may be used as mineral source along with sulphates, phosphates and chlorides.

Phenylacetic acid, L-naphthalene acetic acid may be added as growth stimulating compounds. It is better
to add proline into the medium which helps in high streptomycin production. Fats, oils and fatty acids
may also be used along with glucose. If necessary antioxidants such as sodium sulphate or starch or agar
may also be added into the medium. There is no need of precursor in the production of streptomycin.

(iii) Fermentation:

Sterilized liquid medium with all the above substances is fed to the production fermenter. Appropriate
volume of inoculum (4-5%) is introduced into it. The optimum fermentation temperature is in the range
of 25 to 30°C and the optimum pH range is between 7.0 and 8.0. High rate of streptomycin production,
however, occurs in the pH range of 7.6 to 8.0.

The process of fermentation is highly aerobic and lasts approximately for 5 to 7 days and passes through
3 phases:

(a) The First Phase:

It takes about 24 hours to 48 hours. Rapid growth and formation of abundant mycelium occurs during
this phase. The pH rises to 8.0 due to release of ammonia into medium, due to proteolytic activity of S.
griseus. Glucose is utilized slowly and little production of streptomycin is witnessed.

(b) The Second Phase:

It lasts for 2 days. Streptomycin production takes place at a rapid rate without increase in the mycelial
growth. The ammonia released in the first phase is utilized, which results in the decrease of pH to 7.6-
8.0. Glucose and oxygen are required in large quantity during this phase.

(c) Third Phase:

Cells undergo lysis, releasing ammonia and increase in the pH, which falls again after a period of
continuous streptomycin production. Requirement of oxygen decreases and the contents of the medium
including sugar get exhausted. Finally streptomycin production ceases. A yield of 1200 micrograms per
milliliter of streptomycin is obtained.

(iv) Harvest and Recovery:

After completion of fermentation the mycelium is separated from the broth by filtration. Streptomycin is
recovered by several methods.

Introduction

The tetracyclines are a family of polyketide produced by Streptomyces genus of. The different form of
tetracyclines are
tetracycline, chlortetracycline demeclocycline,

oxytetracycline and other most active types. These

antibiotics are broad-spectrum in nature. At present,

reports on the clinical use of tetracyclines have been

generally confined to respiratory tract infection, sinuses,

middle ear infection, urinary tract infections, intestinal

infection and also gonorrhoea (Chopra and Howe,

1978, Speer, et al 1992). Moreover many derivatives of

tetracycline such as doxycycline, lymecycline,

meclocycline, methacycline, minocycline and

rolitetracycline used in the treatment of chronic

prostatitis, chlamydia and gum disease. More recently,

tetracycline derivative (doxycycline) has been reported

to be effective for the treatment for pelvic inflammatory

disease (Gjonnaess et al, 1978). This makes

tetracycline an attractive group of antibiotics with

Traditionally, tetracycline has been produced by

submerged fermentation (SmF) and used in a one-way

process in solution. In recent years, however Solid

State Fermentation (SSF) processes have been

increasingly utilized for the production of various

antibiotics. SSF also holds tremendous potential for the

production of antibiotics (Robinson et al, 2001). It can

be of particular relevance in those processes where a

crude fermented product may be used as an antibiotic

source (Barrios-Gonzalez, et al 1988). The selection of

a particular strain, however, remains a tedious task,

particularly when commercially significant antibiotic

yields are required. Agro-industrial residues are

generally considered the best substrates for the SSF

processes. The use of SSF for the production of

advantages over submerged fermentation (Johns,

1992) and these have been widely discussed in the

literature (Balakrshana and Panday,1996, Barrios-

Gonzalez, et al 1993, Ohno, et al 1992)

Pineapple fruits are abundantly available in India.

In 2009 the cultivation area of pineapple fruits was

80,000 hectares and annual production was 1, 17,832

tons. The pineapple peel wastes contain high

concentration of biodegradable organic material and

suspended solids. As a result it has a high BOD and

withstanding at extreme pH conditions (Kroyer, 1991).

The solid waste from pineapple canning process was

estimated about 40 - 50 % from fresh fruit as pineapple

peels and core (Buckle, 1989). With the goal of being

economically competitive, pineapple peel a sugary

Substrate and microorganism

Pineapple peel waste procured from a local food

processing unit in Dharwad, India which was used as a

solid substrate. It was dried at 60oC for 72 h to reduce

the moisture content to around 5%, and ground to the

mean particle size (mm) of 12 x 4, 12 x 2, 6 x 4, 2.8–

2.0; 2.0–1.4 and 1.4–1.0 were segregated and

designated as A to F respectively. Streptomyces

strains [S. aureofaciens NCIM (2417, 2614, 2615), S.

rimosus NCIM 2213 and S. viridifaciens NCIM 2506]

obtained from National Chemical Laboratory, Pune,

India. These strains used throughout the study.

Bacillus cereus NCIM 11778 was used in antimicrobial

assay as test organism.

Growth conditions
The culture was maintained on MGYP slants

having the composition (%): malt extract 0.3, glucose

1.0, yeast extract 0.3, peptone 0.5 and agar 2.0. The

pH of the medium was adjusted to 6.4 to 6.8 and

culture was incubated at 30oC for 48 h. Subculturing

was carried out once in 2 weeks and the culture was

stored at 4oC.

Inoculum preparation

A 5 ml of sterile water was added to the slant and

the spores were scraped and transferred into 250 ml

Erlenmeyer flask containing 50 ml of inoculum medium.

The composition of the inoculum medium was; 2.5%

soluble starch, 1.0% corn steep liquor, 0.5% (NH4)2SO4,

2% CaCO3, 1% NaCl, 1% K2HPO4, 1% MgSO4 7H2O

and pH 7.5. The flasks were incubated at 28oC in

shaker incubator (at 220 rpm) for 5 to 7 days. Spores

and resuspended in 25 ml sterile saline solution. This

spore suspension was used as inoculum.

Media preparation and fermentation

The basal solid medium contained pineapple peel

waste 100 g and additional inorganic salts (1% w/w),

inorganic nitrogen source (1% w/w), organic nitrogen

source (10% w/w) and carbon source (10% w/w) were

included. The contents were thoroughly mixed and

autoclaved at 121oC (15 psi) for 20 min. The sterilized

medium is mixed thoroughly with spores of

Streptomyces strains [S. aureofaciens NCIM (2417,

2614, 2615), S. rimosus NCIM 2213 and S.

viridifaciens NCIM 2506] separately and incubated

statically in flask (the thickness of medium was about 2

cm) at 35°C for 2-10 days by stirring once a day. All

values were reported as the % variation less than 5%.

Extraction of antibiotics

The tetracycline from the fermented material was

recovered by simple extraction method. For this, the

fermented substrate was mixed thoroughly with 500 ml

of distilled water and the contents are agitated for 1 h

at room temperature in a rotary shaker at 150 rpm. At

the end of extraction, the liquid was filtered off through

Whatman No.1 filter paper and the resulting clear

filtrate was used for tetracycline analysis (Agenes, et al

2005, Yang and Yuan, 1990).

Analytical methods

The disc diffusion bioassay method that utilizes

the antibacterial property of tetracycline to produce a

zone of inhibition against Bacillus cereus NCIM 11778

Pharmacopoeia of India, 2007) was used. The method

employed the use of filter paper discs containing 10 µl

of supernatant from fermentation broth of

Streptomyces strains and negative control. These discs

were dried and placed on the surface of agar plates

seeded with Bacillus cereus NCIM 11778 strain.

Positive control was consisted of disc with known

amount of tetracycline. These plates were incubated at

37oC for 24 h. Zones of inhibition were measured in

mm. All experiments were conducted in triplicate, and

the mean of the three is represented as milligram of

tetracycline produced per gram of substrates. Standard

tetracycline (Himedia, India) was used to construct the

calibration curve. The moisture content of the

pineapple peel waste was estimated by drying 100 g of