The Impact of Chemistry on Biotechnology - ACS Publications

Chapter 2

Impact of Biotechnology on the Chemical Industry J o n a t h a n J. M a c Q u i t t y

Downloaded by SUNY STONY BROOK on February 9, 2015 | http://pubs.acs.org Publication Date: January 7, 1988 | doi: 10.1021/bk-1988-0362.ch002

Genencor,

Inc., 180 K i m b a l l W a y , South S a n Francisco,

CA

94080

Recent rapid developments i n some of the newer technologies such as protein engineering, high level protein secretion systems, and microbial pathway engineering, have reduced or eliminated many of the technical obstacles to the application of biotechnology to the production of specialty chemicals. Indeed, some of these developments are already being translated into commercial applications. It i s predicted that the impact of biotechnology on the chemical industry w i l l increase and extend well into the next century. There has been a tendency i n the popular press and i n the biotech industry i t s e l f to c l a s s i f y many d i f f e r e n t technologies as "biotechnology". C l e a r l y , t h i s i s an o v e r s i m p l i f i c a t i o n . From a technical standpoint there are a wide v a r i e t y of techniques involved and moreover, from a commercial point of view, there are d i s t i n c t differences i n the way these technologies should be applied. This paper attempts to c l a s s i f y the technologies involved with p a r t i c u l a r reference to the production of s p e c i a l t y chemicals and to review the technical and commercial l i m i t a t i o n s that might exist i n applying these technologies. In the f i r s t section, the production of chemical products by a v a r i e t y of b i o l o g i c a l approaches i s examined. These approaches are characterized i n terms of chronological phases or "waves" of development. The waves have rather d i f f e r e n t commercial c h a r a c t e r i s t i c s and require d i f f e r e n t business strategies i f the technologies involved are to be applied s u c c e s s f u l l y . In the second section, the technical b a r r i e r s or l i m i t a t i o n s that exist i n applying these b i o l o g i c a l approaches t o the chemical industry are reviewed p a r t i c u l a r l y as applied to the production of s p e c i a l t y chemicals. The thesis put forward i s that most, i f not a l l , of these b a r r i e r s can now be overcome because of a number of recent and very impressive technical advances.

0097-6156/88/0362-0011$06.00/0 © 1988 American Chemical Society

In The Impact of Chemistry on Biotechnology; Phillips, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

T H E IMPACT OF CHEMISTRY ON BIOTECHNOLOGY

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In the t h i r d section, some commercial b a r r i e r s that e x i s t today and l i m i t the f u l l impact of biotechnology on the chemical industry are reviewed. Several approaches are suggested f o r minimizing these b a r r i e r s and some examples are given where such l i m i t a t i o n s have been removed r e s u l t i n g i n improvements i n the cost or performance of s p e c i f i c products.


The Four "Waves" of Biotechnology There has been an evolution i n b i o l o g i c a l approaches to chemical problems, and these can be separated somewhat crudely into d i f f e r e n t chronological phases or "waves" (Table I ) . These waves have d i f f e r e n t technical bases and, more important, require d i f f e r e n t commercial strategies i n order to be u t i l i z e d successfully. Table I . The Production of Chemicals Using B i o l o g i c a l Approaches

1st Wave

1940s

C l a s s i c a l Fermentation

e.g. p e n i c i l l i n , c i t r i c ac i d

2nd Wave

1970s

rDNA Technology

e.g. i n s u l i n , hGH

3rd Wave

1980s

Protein Engineering

e.g. modified s u b t i l i s i n s

4th Wave

1980s

Pathway Engineering

e.g. new pathway to ascorbic acid

The F i r s t Wave; C l a s s i c a l Fermentation. The f i r s t wave started i n the mid-forties with the scaleup and commercial production of primary and secondary metabolites, such as c i t r i c acid and p e n i c i l l i n and has now evolved into a w e l l developed fermentation industry (1-2). Production i s achieved using fermentation, both surface and submerged, with submerged fermentation now the more important. The b i o l o g i c a l techniques involved i n t h i s f i r s t wave include: — — —

i s o l a t i n g a microorganism which produces the chemical of interest by means of screening/selection procedures. improving production y i e l d s by means of random mutagenesis of the microorganism. increasing y i e l d s s t i l l f u r t h e r by optimization of culture media and fermentation conditions.

From a commercial standpoint, the f i r s t wave i s l i m i t e d to those chemicals produced i n nature. I t i s also l i m i t e d by i t s t r i a l and error approach: i t can take years or even decades t o achieve a s i g n i f i c a n t improvement i n product y i e l d s . For example,


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Impact of Biotechnology on the Chemical Industry

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i t took 20 years applying these techniques to improve the fermentation y i e l d of p e n i c i l l i n from an i n i t i a l l e v e l of a few milligrams per l i t e r to 7 grams per l i t e r (1). The techniques involved here are often referred to as " c l a s s i c a l " techniques. However, with modern improvements i n the a b i l i t y to screen and assay f o r desired p r o p e r t i e s , t h i s approach i s now more powerful and more targeted than when o r i g i n a l l y applied. C l a s s i c a l mutagenesis and s e l e c t i o n s t i l l remains a highly v i a b l e approach to the development of b i o l o g i c a l production processes. The Second Wave: rDNA Technology. The second wave started i n the mid-seventies with the pioneering work of Cohen and Boyer which established techniques f o r recombinant technology i n simple procaryotes such as E. c o l i (3). This was followed by intensive e f f o r t s to demonstrate the expression of simple proteins from these procaryotes (A). The techniques involved here include: — —

— —

i s o l a t i n g the gene coding f o r the p r o t e i n of i n t e r e s t from a natural source. cloning the gene i n t o appropriate vectors f o r expression including necessary upstream and downstream regulatory elements. transforming a desired production microorganism with these vectors. expressing the gene using these regulatory elements so as to produce high y i e l d s of the p r o t e i n of i n t e r e s t .

The implications of t h i s second wave of rDNA technology are to open up a large range of proteins for consideration as s p e c i a l t y chemicals. Previously, these chemicals might have existed only i n minute quantities i n nature and thus might not have been available f o r commercial use. Another difference from the c l a s s i c a l fermentation techniques described above i s that the timeframe f o r y i e l d improvement can be made much f a s t e r . The a p p l i c a t i o n of these techniques began with the cloning and expression of human genes such as i n s u l i n (5) but has since been extended to other mammalian genes such as rennin (6), microbial genes such as glucoamylase (7), and even plant genes such as thaumatin (8). The Third Wave: Protein Engineering. The t h i r d wave, protein engineering, started i n the early eighties as a spinoff of rDNA technology. However, i t i s rather d i f f e r e n t from the second wave since i t i s concerned with producing new ( i . e . , man-made) proteins which have been modified or improved i n some way over t h e i r natural counterparts. The techniques involved i n p r o t e i n engineering are also more complicated than those described previously. They involve: — —

introducing changes i n t o s p e c i f i c l o c a t i o n s or regions of a gene i n order to produce a new gene. expressing the new protein from such a gene using the approach as described above.


T H E IMPACT OF CHEMISTRY ON B I O T E C H N O L O G Y

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—

characterizing the structure of t h i s new p r o t e i n by crystallographic or other techniques, determining the functional parameters f o r the new protein. s e l e c t i n g new locations or regions to modify as a r e s u l t of t h i s structure:function comparison.


— —

This i s shown schematically i n Figure I . As can be seen, such an approach i s n e c e s s a r i l y an i t e r a t i v e approach, and might appear to be time consuming as a r e s u l t . Fortunately there are some ingenious short cuts which have been developed recently that can keep these i t e r a t i o n s to a minimum. For example, using "cassette" mutagenesis i t i s possible to produce, i n a s i n g l e experiment, some 19 proteins d i f f e r i n g only by a s i n g l e amino acid at a designated s i t e i n the protein (9). Hybrid enzymes can also be produced where parts of two or more "parent" genes are combined to form novel hybrids (10) . These and other mutagenesis techniques allow d i f f e r e n t proteins to be produced i n large numbers, with controlled d i v e r s i t y from the o r i g i n a l natural p r o t e i n , and i n f a i r l y rapid fashion. I t i s also possible to accelerate the characterization of these new proteins both s t r u c t u r a l l y and f u n c t i o n a l l y . For example, by using area detectors, the c o l l e c t i o n of x-ray crystallographic data can be accelerated s i g n i f i c a n t l y . Indeed, i f the structure of a parent protein i s w e l l resolved, the structures of related proteins can now be analyzed i n 2-3 weeks using Fourier difference techniques (Bott, R., Genentech, So. San Francisco, CA, unpublished data). S i m i l a r l y , new assay techniques have been developed f o r screening functional parameters, and new computer software can be employed to speed up the process of analyzing these data. For example, new computational methods have been developed f o r the spectrophotometry determination of enzyme k i n e t i c s . This has enabled important k i n e t i c parameters (e.g. k » ) be determined i n a matter of minutes compared to the several days required by previous techniques (11). From a commercial point of view i t i s worth noting that since these novel proteins are man-made, e x c e l l e n t patent protection should be obtainable. The composition-of-matter type claims which can be made f o r these new p r o t e i n s , could p o t e n t i a l l y provide a much stronger proprietary base than the process claims a v a i l a b l e from rDNA technology on i t s own. K

c a t

t o

m

The Fourth Wave: Pathway Engineering. The fourth wave, pathway engineering, has r e a l l y only j u s t begun. This wave involves modification of various metabolic pathways using recombinant techniques so as to enhance production of a p a r t i c u l a r metabolite or indeed to include pathways not indigenous to the organism. Major steps i n t h i s fourth wave include: — —

i d e n t i f y i n g the enzymes involved i n the metabolic pathway of interest both from w i t h i n the production host and from other natural sources. obtaining the genes coding f o r these enzymes and modifying them, i f necessary, using the techniques described above.



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molecular modeling

molecular modeling


15

X-ray data

x-ray crystallography

3-D "Blueprint" for new enzyme

genetic engineering

assay

Utility

development

Figure 1. The Application of Protein Engineering to Chemical Products.


T H E IMPACT OF CHEMISTRY ON B I O T E C H N O L O G Y

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—


—

cloning the genes coding f o r these new improved enzymes into the production host along with the appropriate regulatory elements, optimizing the e f f e c t i v e i n t e r a c t i o n between these regulatory elements and the c u l t u r e and fermentation media conditions i n order to achieve proper regulation of the key metabolic pathways.

The l a s t step alone i s obviously c r i t i c a l and only recently has the f e a s i b i l i t y of t h i s been demonstrated. There has, f o r example, been excellent work on modifying amino acid production using t h i s technique (12) , 2-Keto-gulonic a c i d , a precursor to Vitamin C, has also been produced by using t h i s approach (13), From a commercial standpoint, there are some c l e a r differences between pathway engineering and c l a s s i c a l fermentation. For one thing, since the Supreme Court has allowed engineered organisms to be patented, i t i s now possible to e s t a b l i s h a better proprietary p o s i t i o n . Secondly, pathway engineering allows a much more targeted approach to y i e l d improvement. The approach allows research to be conducted on a much sounder technical basis than the c l a s s i c a l t r i a l and error methodology. Increasing Impact of Biotechnology. With the development and growth of the four "waves" described above, the impact of biotechnology on the chemical industry has increased dramatically. A clear example of t h i s increasing impact i s i n the area of i n d u s t r i a l enzymes. This i s summarized somewhat f i g u r a t i v e l y i n Table I I . As can be seen, each wave of biotechnology has expanded the p o t e n t i a l a v a i l a b i l i t y of d i f f e r e n t enzymes. This should allow the development of a number of enzyme product l i n e s and expand the commercial a p p l i c a t i o n of enzymes to many new i n d u s t r i a l areas.

Table I I . The Accelerating Impact of Biotechnology Example:

A v a i l a b i l i t y of I n d u s t r i a l Enzymes

Approximate Number of Enzymes Available

Classical Fermentation

rDNA Technology

Protein Engineering

Pathway Engineering

15-20

5-10,000

20',300

oo

Technical L i m i t a t i o n s to the A p p l i c a t i o n of Biotechnology In the f i r s t section, four d i f f e r e n t waves of biotechnology were i d e n t i f i e d . In t h i s section some of the technical factors are described which l i m i t the a p p l i c a t i o n of these waves of


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biotechnology t o the chemical industry. In p a r t i c u l a r the t e c h n i c a l requirements f o r the production of s p e c i a l t y chemicals using these biotechnology approaches are reviewed. The thesis being proposed i s that many of the t e c h n i c a l requirements l i m i t i n g the a p p l i c a t i o n of biotechnology t o the chemical production have now been reduced or eliminated. Lower Cost Biochemical Processes. One of the common b a r r i e r s c i t e d against b i o l o g i c a l approaches to chemical production i s the high cost of the biochemical processes involved compared to chemical a l t e r n a t i v e s and the lack of methods a v a i l a b l e to reduce these costs. Biochemical processes f o r the production of chemicals can be divided into two major categories: 1.

D i r e c t fermentation from a carbon feedstock. The desired end product i s recovered from the fermentation broth a f t e r fermentation. The Fermentation has t y p i c a l l y been performed i n a batch or modified batch mode but continuous fermentation processes are now emerging·

2.

Bioconversion using a b i o c a t a l y s t . Here the b i o c a t a l y s t can be m i c r o b i a l c e l l s (both l i v i n g and dead) or enzymes and the b i o c a t a l y s t can be f r e e l y suspended or immobilized.

The fermentation processes are h e a v i l y dependent on carbon feedstock costs and w i l l be discussed below. Bioconversion processes, however, need not be expensive processes and indeed several inexpensive biaconversions have been commercialized a t very large scale. Some examples are summarized i n Table I I I . The thermolysin dependent production of aspartame i s now being scaled up i n Holland by a j o i n t venture between Toyo Soda and DSM (14). The p e n i c i l l i n acylase route has now almost t o t a l l y displaced the more expensive chemical a l t e r n a t i v e s (15). In the case of high fructose corn syrup (HFCS) production, the cost of the immobilized glucose isomerase i s now around 0.2

The Impact of Chemistry on Biotechnology - ACS Publications

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