Some Origins of Biotechnology

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Oct 23, 2013 (3 years and 9 months ago)

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Some Origins of Biotechnology


Prof. R.K. Finn, School of Chemical Engineering, Olin Hall, Cornell University, Ithaca, N.Y.

14853
-
5201, USA


On the occasion of Professor A. Fiechter’s 65
th

birthday it is appropriate to reflect on
developments in biotechnology over the past fifty years. It was in 1940

41 that Florey,
Chain and their collaborators demonstrated the full therapeutic value of Fleming’s
p
enicillin [1]. Thereby was ushered in the

«
age of antibiotics
» and with it
modern
biotechnology

a fermentation industry capable of maintaining strict aseptic conditions on
a large scale in stirred, aerated tanks. This short discourse sets down in chronological order
some events and achievements of

modern biotechnology from its beginnings, with the
production of antibiotics in the early forties, to the advent of the
«
new biotechnology
»

with
genetically engineered cells in the mid
-
seventies
.


Earlier r
oots


From our vantage point fifty years later is it difficult to appreciate the radical implications of the
submerged culture of molds. The basis for this methodology was laid by Kluyver and his student
Perquin

in their 1933 paper from Delft on the use of shake

flasks for physiological studies [2].
Nevertheless there was grave doubt that sterile air and sterile seals on rotating shafts could be
maintained over week

long periods on an
industrial

scale

not just in the controlled environment
of the laboratory. Cons
equently surface culture continued in use during the first three years of
penicillin production in the United States; it was simply an extension of the method used for
citric acid production.

Prior to this time, the only experience in deep tanks had been
with fermentations to make
alcoholic beverages, baker’s yeast, or lactic acid. None of these required more than
pasteurization of the mash and steaming of the equipment. The sole exception was in the
manufacturing of acetone and butanol by solventogenic
Cl
ostridium

species, where phage
infections had nearly proved disastrous. That industry was able to recover however by
establishing strict asepsis; and since the fermentations did not require either aeration or agitation
this was relatively easy to accomplis
h.

Equally important, as a deterrent to developing a modern fermentation industry, was the
widespread view that
once their chemical structure was known, most natural products would be
more economically synthesized by organic chemists than by microorganism
s. Precedent had

been established not only with the popular sulfa drugs but also
with the various vitamins:
thiamine, pan
tothenate, ascorbic acid, even the relatively complex molecule of riboflavin.


The f
orties


The
manufactur
e

of pen
i
cillin

was followed

rapidly by that of streptomycin. Although the
Waksman patent was not issued until 1948 [3], production of streptomycin was already in full
swing from the Merck plant, built in Elkton, Virginia. Figures for 1947 to 1949 are shown in
Table 1.


Table 1:
Production of Penicillin and Streptomycin in kg from 1947 to 1949 in the United States

(Source:
US Dept of Commerce).


2


Antibiotics

1947

1948

1949

Penicillin

Streptomycin

24 856


9 676

57 513

37 709

80 078

83 699



Other antibiotics also became prominent during this period: chloromycetin, aureomycin from
Lederle Laboratories, bacitracin, neomycin, polymyxin. Tetracycline itself was not approved for
use until 1953.

An important development for establishing the validit
y of microbial technology was the
discovery of vitamin B
-
12 in 1948. It was not only of therapeutic value in treating pernicious
anemia, but turned out to be the long
-
sought animal protein factor (APF) and attained
widespread use in animal feeds. By 1952 t
he total sales (of 61 pounds, all produced by
fermentation) amou
nted to over $5.5 million [4].

Traditional fermentations waned during these years, especially as the petrochemical industry
developed. Thus the raw material for production of acetone/butanol w
as shifted from molasses to
grain, and feed
-
grade riboflavin from the fermentation residues became almost more valuable
than the solvents themselves.

During most

of

the forties, research and
development on the fermentations was conducted by
biologists. Ch
emical engineers were restricted to working on methods for recovery and
purification, despite growing recognition that submerged cultivation

of cells involved numerous
aspects of heat and mass transfer. In 1944
,

there appeared

a

seminal paper on the use of

sulfite
oxidation to characterize the aeration capacity of stirred tanks [
5] and during this period bench
-
scale fermentations were developed in industry and at the Northern Regional Research
Laboratories (NRRL), US Department of Agriculture, in Peoria, Il
linois.


The fifties

In the year 1950
,

a number of significant papers
appeared

which set the tone for process
engineering during the ensuing decade. These included the work on aeration and agitation by
Gaden

at Columbia University [6] along with similar studies from the team at Merck & Co.,
under the guidance of Professor Wilhelm of Princeton [7]. Students led by Professor Marvin
Johnson at Wisconsin and by Professors Halvorson and Piret at Minnesota began to

publish their
studies on controlled fermentations. In that same year the chemostat was born (Mon
od and
Novick & Szilard) [8].

I
n fact, the theme of continuous fermentation was important throughout the decade, led by
groups in Scandinavia, Czechoslovakia,

and a
t Porton in England [9]. In 1951,

the First
International Symposium on Chemical Microbiology was held in
Rome and at the time an
international research center was inaugurated there, under the direction of Professor E.B. Chain.
A «First Symposium on C
ontinuous Cultivation of Cells» was held in Prague 1958. The VIIth
International Congress for Microbiology held in Stockholm in August of that same year included
symposia not only on continuous culture but also on «Recombination mechanisms in bacteria»

and

«Role of protein in the nucleic acid synthesis and role of nucleic acid in protein synthesis».
The American Society for Microbiology began publication of Applied Microbiology in 1953
(now Applied and Environmental Microbiology).

This was also the time whe
n the Institute of Biochemistry in Uppsala, Sweden, led by Tiselius,
was germinating many of the techniques still being used for separation of proteins and other
macromolecules. These included preparative electrophoresis, variations on column
chromatograph
y, and partitioning between immiscible aqueous phases. Co
-
workers included
3


leaders
such
as

Porath, Albertsson
,

and Flodin.

Meanwhile, an exciting new development came from the pharmaceutical

industry when the
Upjohn C
ompany announced simplification in the

synthesis of adrenal cortex hormones. In 1952
,

a
hydroxy group was introduced at the 11
-
alpha position of progesterone in yields of 85% [10].
In 1953 independent work by the Squibb and Ciba groups showed that microorganisms can
introduce double bonds into

the steroid nucleus at specific sites. The word
bioconversion
began
to take on added significance.


The sixties


The First International Fermentation Symposium held in Rome during May 9

14, 1960 was the
culmination of two decades of rapid development. It

was a memorable occasion, attended by 130
scientists and engineers from around the world plus 50 from Italy itself. Many of the topics
discussed then are still under investigation


aeration, shear, continuous culture. Oxygen
electrodes were described as
well as pH measurements. A special
panel

discussion was devoted
to genetics.

Many other institutions were founded also in this decade when modern biotechnology came into
full flower. In the United States, the Division of Microbial Chemistry became a separ
ate Division
of the American Chemical Society in 1963. The first textbook on bioprocess engineering, by
Aiba, Humphrey and Millis, appeared in 1965. The Journal of Biochemical and Microbiological
Technology and Engineering (now Biotechnology and Bioenginee
ring) had been established a
little earlier, in 1959.

This was, above all, the decade of microbial enzymes. The commercial availability of
glucoamylase in 1958 made possible the completely enzymatic conversion of corn starch to
glucose during the sixties.
Microbial
p
roteases were fir
st used in detergents during this period.
Although there were early scattered reports of covalently immobilized enzymes, Manecke in
Germany and Katchalski in Israel, along with Crook and others in England developed techniques
wh
ereby catalytic activity was largely retained. Dunnill and Lilly established a center for enzyme
study at University College in London.
Some expected an even greater impact of enzyme
engineering than did indeed occur (just as, a decade earlier, the assessm
ent of continuous
fermentation was sometimes too sanguine). In any case, during this period considerable support
for enzyme research was provided by the National Science Foundation

in the United States. By
the end of the decade, immobilized enzymes were us
ed for resolution of optical isomers of acyl
amino acids and by 1970 immobilized glucose isomerase was used to produce high fructose corn
syrup. Finally, an important conference on «Enzyme Engineering» was held at Henniker, New
Hampshire, USA in 1971. The
study of immobilized cells, either encapsulated in gels or retained
in hollow fibers or other membrane devices, also began at about this time.

The discovery of Kinoshita et al. of bacteria tha
t excreted large amount of glu
t
am
ic

acid [11] led
to the rapid exploitation of commercial methods for producing natural amino acids. Monosodium
glutamate, widely used a flavor enhancer, was manufactured from molasses by fermentation
more economically than in the prior art, namely extraction

and hydrolysis of precursors derived
from agricultural residues.

In the late fifties and early sixties, water soluble polysaccharides from bacteria were under
investigation at the NRRL in Peoria, especially xanthan gum from common soil bacteria. This
pro
duct was commercialized by the Kelco Company in San Diego, California during the period
1960


63 and has become a popular food additive.



4


The seventies

The search for unconventional sources of protein especially for animals, but also possibly for
human
s, is not a new venture. Nevertheless, in the late sixties and early seventies there was
intensive effort to convert petroleum fractions to edible protein by algae, yeasts, molds or
bacteria. At the time of the first international conference at the Massach
usetts Institute of
Technology (MIT) in 1967 the term single
-
cell protein or SCP was coined [12]. Most of the
projects at that time were still in the experimental stage, but by the time of the second MIT
conference in 1973 various large
-
scale plants around

the world were being completed. The rise
in oil prices during the mid
-
seventies combined with deep public concern over nutritional safety
of the products quenched such early enthusiasm in most countries. Interest in using methanol as a
feedstock for SCP p
roduction continued through the seve
nties but economic factors have
mitigated
against large scale production of animal feeds in most Western countries.

Spurred

on
though by such possibilities a number of sizeable research a
nd pilot plans around the world
w
ere
established during the seventies. Among the most notable were the GBF (Gesellschaft für
Biotechnologische Forschung mbH) at Braunschweig
-
Stöckheim in Germany and of course
Professor A. Fiechter’s Institute for Biotechnology at the ETH Zürich.

In 1973 i
t was first demonstrated that hybrid plasmids, constructed
in vitro

by restriction
endonucl
ease cutting, annealing and li
gation
, could be
functionally

expressed in
Escherichia coli

[13]. The next year eucaryotic DNA was successfully cloned, and by the end
of the decade
synthetic human insulin was produced in
Escherichia coli.

As if to underscore
revolutionary

events, hybridoma cells were developed in 1975; these in turn led to the production of
monoclonal antibodies. Surely the «new biotechnology» represent
s a qualitatively new era
involving radical changes for medicine and agriculture. The most recent development has
extended the genetic engineering approach to the economical manufacture of calf rennet


chymosin


from both
Escherichia coli

(Pfizer) and fr
om
yeast

(Gist
-
Brocades).


Literature

[1]


Chain
,
E., et al
.: Lancet 2, 226


228 (1940);
Abraham, E.P
.,
et al.:

Lancet 2, 177


189
(1941).

[2]
Kluyver, A.J., Perquin, L.C.H.:

Biochem. Z. 266, 68


81 (1933).

[3]
Waksman, S.A., Schatz, A
.: U.S. Patent 2,

449, 86
6 (Sept. 21, 1948).

[4]
Hester, A.S. Ward, G.E
.: Ind. Eng. Chem.
46,

237


243 (1954).

[5]
Cooper, C.M. Fernstrom, G.A. Miller, S.A
.: Ind. Eng. Chem.
36
, 504


509 (1944).

[6]
Hixson, A.W., Gaden, E. L. Jr.:

Ind. Eng. Chem.
42
, 1970


1801 (1950).

[7]
Bartholomew, W. H., et al
.: Ind. Eng. Chem.
42
, 1801


1818; 1827


1830 (1950).

[8]
Monod, J
.: Ann. Inst. Pasteur
79
, 390


410 (1950;
Novick, A., Szilard, L.
: Proc. Natl. Acad.
Sci. USA
36
, 708


719 (1950).

[9]
Herbert, D., Elsworth, R., Telling,

R.C
.: J. Gen. Microbiol.
14
,
601


622 (1956).

[10]
Peterson, D.H., Murray, H.C.:

J. Am. Chem. Soc.
74
, 1871


1872 (1952).

[11]
Kinoshita, S. Nakayama, K., Shimono, M
.: J. Gen. Appl. Microbiol.
3
, 193


205 (1957).

[12]
Mateles, R.I., Tannenbaum, S.R
. (eds
): Single
-
Cell Protein; MIT Press, Cambridge, Mass.,
USA (1968).

[13]
Cohen, S.N.,
et al.:

Proc. Natl. Acad. Sc
i., USA
70
, 3240


3244 (1973).


Reprinted from SWISS BIOTECH 7 (1989) No. 4, 15
-
17.