Microbial Regulatory Mechanisms - American Chemical Society

4

Microbial Regulatory Mechanisms: Obstacles and Tools for the Biochemical Engineer

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H. E. UMBARGER Purdue University, Department of Biological Sciences, West Lafayette, IN 47907

The integration of metabolic activity of bacterial cells with the growth process can limit the extent to which a given activity can be made useful for industrial purposes. Since the mechanisms underlying this integration are genetically controlled, they can be often bypassed or compensated by judicious selection of mutants. The general physiological and molecular patterns of regulatory mechanisms that should be considered in the use of mutant methodology are briefly reviewed. Examples of the way such knowledge can be exploited to obtain valine-and isoleucine-producing strains are described. There i s today a widespread a p p r e c i a t i o n of the f a c t that metabolic pathways i n microorganisms are remarkably w e l l i n t e grated with the growth process. I t i s f u r t h e r remarkable that t h i s i n t e g r a t i o n i s p o s s i b l e under a broad range o f environmental conditions — c o n d i t i o n s that vary both chemically and p h y s i c a l l y . This i n t e g r a t i o n r e s u l t s from c o n t r o l s exerted i n two ways: one, the c o n t r o l of enzyme a c t i v i t y , and the other, the c o n t r o l of enzyme amount. ( S t r i c t l y speaking, t h i s l a t t e r category should a l s o i n c l u d e c o n t r o l of the amount o f other macromolecules, such as the ribosomal p r o t e i n s , ribosomal and t r a n s f e r RNA, and membrane components.) L e t me consider the p h y s i o l o g i c a l patterns found to a f f e c t enzyme a c t i v i t y , which I l i k e to c a l l the c o n t r o l of metabolite flow. (Throughout t h i s a r t i c l e , the words " c o n t r o l " or " r e g u l a t i o n " are meant to connote f a c t o r s a f f e c t i n g b i o l o g i c a l functions that can be modulated, i n contrast to a f a c t o r that has consequences on b i o l o g i c a l f u n c t i o n . An analogy with a domestic water supply would be an adjustable valve that can be manipulated, i n contrast to an o b s t r u c t i o n i n the supply l i n e . ) Broadly speaking, there are three b a s i c patterns a f f e c t i n g the c o n t r o l o f metabolite flow (Table I ) . The simplest i s endproduct i n h i b i t i o n i n which the endproduct o f a pathway i s an 0097-6156/ 83/0207-0071 $ 0 6 . 5 0 / 0 © 1983 American Chemical Society

Blanch et al.; Foundations of Biochemical Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Table I P h y s i o l o g i c a l Patterns o f C o n t r o l o f Metabolite Pattern En dp r ο duct inhibition

I n h i b i t i o n of threonine deaminase by i s o l e u c i n e

(1)

Qoli)

S t i m u l a t i o n o f l a c t a t e dehydro genase by f r u c t o s e - l , 6 - d i p h o s phate

Compensatory modulation

Reference

Example

(E.

Precursor activation

Flow

(Streptococcus

fecalis)

S t i m u l a t i o n o f carbamylphosphate synthetase by o r n i t h i n e (E.

(2)

(1)

coli)

i n h i b i t o r of the i n i t i a l enzyme o f the pathway. The i n h i b i t i o n of threonine deaminase i s a w e l l known example to which I r e f e r l a t e r i n t h i s a r t i c l e (1). There are v a r i a t i o n s on t h i s general theme i n which m u l t i p l e endproducts may act i n concert or synerg i s t i c a l l y upon a s i n g l e enzyme c a t a l y z i n g the f i r s t step i n a common pathway o r i n which a segment of a s i n g l e pathway i s con t r o l l e d by an intermediate i n h i b i t i n g an e a r l i e r step [e.g., the i n h i b i t i o n o f phosphofructokinase of Escherichia coli by phosphoenolpyruvate ( 2 ) ] . The reverse of endproduct i n h i b i t i o n i s precursor a c t i v a t i o n . Although i t i s more d i f f i c u l t to c i t e examples i n b i o s y n t h e t i c pathways, an example i s found i n the a c t i v a t i o n of c e r t a i n micro b i a l l a c t a t e dehydrogenases by fructose-l,6-diphosphate. Finally, there i s another p a t t e r n o f enzyme a c t i v a t i o n that I would c a l l compensatory modulation of a c t i v i t y . The example that i s p a r t i c u l a r l y w e l l s t u d i e d i s the a c t i v a t i o n of carbamyl phosphate synthe tase o f E. coli by o r n i t h i n e (3). This enzyme forms carbamyl phosphate needed f o r both pyrimidine n u c l e o t i d e b i o s y n t h e s i s and a r g i n i n e b i o s y n t h e s i s . The enzyme i s i n h i b i t e d by UMP, an i n d i cator of the g l o b a l pyrimidine p o o l . However, an ample supply o f carbamylphosphate f o r a r g i n i n e b i o s y n t h e s i s i s assured by o r n i thine s t i m u l a t i n g the enzyme and antagonizing the UMP-mediated i n h i b i t i o n . O r n i t h i n e , o f course, i s an intermediate that would appear only when the a r g i n i n e supply was low. This c l a s s i f i c a t i o n o f r e g u l a t o r y patterns implies nothing with respect to the mechanisms by which r e g u l a t i o n was achieved. The common mechanisms that have been recognized i n c o n t r o l l i n g carbon flow are c l a s s i f i e d i n Table I I . We can recognize mecha nisms that are s p e c i f i c i n that they are r e l a t e d to the s p e c i f i c r o l e the modulated enzyme plays and mechanisms that are general i n that they may a f f e c t many enzymes o r pathways that are not func t i o n a l l y r e l a t e d o r have l i t t l e to do with the p h y s i o l o g i c a l r o l e of the enzyme.


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Table I I Enzymic Mechanisms Underlying C o n t r o l of Metabolite Flow Example i n E. coli

Mechanism

Référer

S p e c i f i c mechanisms: Binding at catalytic site

I n h i b i t i o n of asparagine synthetase by asparagine

(4)

Binding at regulatory

I n h i b i t i o n of threonine deaminase by i s o l e u c i n e and i t s antagonism by valine

(

Enzyme modification

A d e n y l y l a t i o n of glutamine synthetase

(

Protein-protein interaction

S t i m u l a t i o n of α-subunit of tryptophan synthase by β-subunit

(

Energy charge

Retardation of aspartokinase I I I a c t i v i t y at low energy charge

(

Reducing potential

I n h i b i t i o n of pyruvate dehydrogenase a t low NAuVNADH r a t i o s

(

Substrate availability

None known

site

General mechanisms:


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The simplest of these mechanisms would be the a c t i o n of an i n h i b i t o r b i n d i n g at the a c t i v e s i t e . The i n h i b i t i o n of succinate dehydrogenase by oxaloacetate can be looked upon as such an exam p l e (10). It may be that t i g h t b i n d i n g of a product may a l s o play a r o l e i n r e g u l a t i n g the a c t i v i t y of an enzyme (11). The i n h i b i t i o n of asparagine synthetase by asparagine i n microorganisms provides a l i k e l y candidate f o r r e g u l a t i o n by t i g h t binding of product (4). Obviously more complex would be those enzymes on which a separate regulatory s i t e has been developed f o r b i n d i n g of e i t h e r a s t i m u l a t o r y or an i n h i b i t o r y l i g a n d that i s p h y s i o l o g i c a l l y r e l a t e d to the f u n c t i o n but not i t s e l f s t o i c h i o m e t r i c a l l y r e l a t e d to the c a t a l y t i c events. When such a l i g a n d i s an i n h i b i t o r , i t would be an example of what Monod and Jacob (12) had defined as an a l l o s t e r i c i n h i b i t o r , i n contrast to the previous example, which they defined as i s o s t e r i c . Many examples of t h i s k i n d of regulatory process have been described i n both c a t a b o l i c and b i o s y n t h e t i c pathways. However, i n r e f e r r i n g to binding of a r e g u l a tory l i g a n d to a d i s t i n c t r e g u l a t o r y s i t e , I would not wish to imply a common mechanism by which the i n h i b i t i o n or the s t i m u l a t i o n occurs. There probably are s e v e r a l mechanisms i n v o l v e d , and at the l e v e l of enzyme s t r u c t u r e there i s seldom complete agree ment among a l l i n v e s t i g a t o r s regarding the p r e c i s e d e t a i l s . At the present time, we can c i t e more examples of enzyme m o d i f i c a t i o n o c c u r r i n g as r e g u l a t o r y mechanisms i n animal metabo l i s m than we can i n m i c r o b i a l metabolism. In animal metabolism, phosphorylation by p r o t e i n kinases a f f e c t s many enzymes (13). There are examples of phosphorylation of b a c t e r i a l p r o t e i n s , but, with few exceptions, the r o l e or even the i d e n t i t y of the p r o t e i n i s unknown. One example that has been s t u d i e d i s the i s o c i t r a t e dehydrogenase of E. coli, which i s phosphorylated when the c e l l i s s h i f t e d from glucose to acetate as the carbon source and which thereby undergoes a very r a p i d decrease i n a c t i v i t y (14). The b e s t - s t u d i e d example of m o d i f i c a t i o n of an enzyme i s that of the a d e n y l y l a t i o n and deadenylylation of glutamine synthase of E. coli, which, i n e f f e c t , serves to i n h i b i t and to a c t i v a t e the enzyme (6). To some extent, the idea of p r o t e i n - p r o t e i n i n t e r a c t i o n s a f f e c t i n g the a c t i v i t y of b a c t e r i a l pathways may s t i l l be hypo thetical. In yeast, we can c i t e at l e a s t one c l e a r l y important example. That i s the i n h i b i t i o n of o r n i t h i n e transcarbamylase upon binding to arginase, an enzyme induced by adding a r g i n i n e to the medium (15). This p r o t e i n - p r o t e i n i n t e r a c t i o n thus prevents a f u t i l e c y c l e i n which o r n i t h i n e , formed from exogenous a r g i n i n e , would be converted back to a r g i n i n e . There are i n v i t r o models that can be c i t e d i n b a c t e r i a l metabolism that demonstrate the f e a s i b i l i t y of such a mechanism, i n which an a c t i v i t y of one pro t e i n i s markedly enhanced upon b i n d i n g to another. For example, the conversion of s e r i n e and i n d o l e to tryptophan i s slowly c a t a l y z e d by the α subunit of tryptophan synthase alone but i s


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stimulated some 30-fold upon b i n d i n g to 3-subunit dimers. Under the category o f general c o n t r o l s , I have l i s t e d energy charge and reducing p o t e n t i a l , which could be looked upon as spe c i a l cases of substrate a v a i l a b i l i t y . The energy charge e x p e r i ments of Atkinson (16) have demonstrated s e v e r a l examples of i n v i t r o response to energy charge, but i t i s s t i l l d i f f i c u l t to assess the i n v i t r o s i g n i f i c a n c e of such a response to energy charge, s i n c e c e l l s do have a remarkable c a p a c i t y f o r maintaining the energy charge w i t h i n rather narrow l i m i t s (17). Similarly, the extent to which a p y r i d i n e n u c l e o t i d e - l i n k e d r e a c t i o n proceeds can be modulated by the NAD/NADH (or NADP/NADPH) r a t i o (9). The question i s whether these r a t i o s can be so changed i n the growing c e l l to be e x p l o i t e d as a r e g u l a t o r y mechanism. One can a n t i c i p a t e that the f l u x through a pathway might be l i m i t e d not by some r a t e - l i m i t i n g step a t the beginning o r w i t h i n the pathway but by the r a t e a t which the i n i t i a l substrate f o r the pathway i s s u p p l i e d . To the extent that t h i s supply of sub s t r a t e i s modulated, such a mechanism might play a r e g u l a t o r y r o l e . That a l i m i t a t i o n i n supply can have important consequences on the extent that a pathway might f u n c t i o n w i l l become c l e a r i n the d e s c r i p t i o n of the development o f an i s o l e u c i n e - p r o d u c i n g strain later i n this a r t i c l e . However, i n the sense of the word " r e g u l a t i o n " as used here, t h i s example would not be considered a r e g u l a t o r y mechanism. Just as p h y s i o l o g i c a l patterns of c o n t r o l over metabolite flow can be recognized, so can p h y s i o l o g i c a l patterns of c o n t r o l over the amount of enzyme. Table I I I l i s t s a convenient c l a s s i f i cation. C l e a r l y , the amount o f enzyme a t any time i s a summation of the amount formed and the amount destroyed. Two s p e c i f i c patterns c o n t r o l l i n g formation might be recognized: repression by endproducts and i n d u c t i o n by substrate or a precursor. I t should be s t r e s s e d that the terms " r e p r e s s i o n " and " i n d u c t i o n " are o p e r a t i o n a l and should carry no connotations regarding the mechanisms. In a d d i t i o n , there are general mechanisms c o n t r o l l i n g l a r g e r groups o f enzymes or groups o f pathways. One general mechanism i s involved i n r e p r e s s i n g the formation of carbon- and energyy i e l d i n g pathways when there i s a good carbon and energy source ( t h i s i s b e t t e r known as c a t a b o l i t e repression) (27). An analagous c o n t r o l mechanism i s r e l a t e d to the n i t r o g e n supply, and many i n d u c i b l e c a t a b o l i c pathways y i e l d i n g ammonia are repressed when there i s already an adequate n i t r o g e n supply (21). Another important c o n t r o l mechanism i s that r e l a t e d to growth r a t e . At high growth rates i n a r i c h medium, the ρrotein-forming apparatus (ribosomes, tRNA, etc.) i s high, whereas the enzymes forming the small molecule b u i l d i n g blocks are repressed to an extent much greater than that accounted f o r by the presence o f the endproducts i n the medium (22). Recent s t u d i e s by Neidhardt and h i s colleagues (23) have revealed a newly recognized c l a s s of enzymes i n E. coli that are induced at e l e v a t e d temperatures.


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BIOCHEMICAL ENGINEERING Table I I I P h y s i o l o g i c a l Patterns o f Regulation of P r o t e i n Amount T y p i c a l examples

Pattern

Reference

E f f e c t on Enzyme Formation Specific: Repression by endproducts

Repression o f arginine biosyn t h e t i c enzymes

(18)

Induction by substrates or precursors

Induction of lac operon

(19)

Related to carbon supply

Catabolite repression of lac i n d u c t i o n

(20)

Related to n i t r o g e n supply

Histidase induction i n glucose-containing, N ^ - f r e e media

(21)

General:

(K.

aerogenes)

Related to growth r a t e

Derepression of the protein synthesizing system at f a s t growth rates

(22)

Related to temperature

"Thermometer" p r o t e i n s , which increase o r decrease i n amount with growth temperature

(23)

The s t a b i l i z a t i o n of glutamine phosphoribosylpyrophosphate amidotransferase of

(24)

E f f e c t on Enzyme D e s t r u c t i o n Specific: S t a b i l i z a t i o n by l i g a n d s

B.

subtilis

by

substrates General: Starvation of c e l l s f o r carbon or n i t r o g e n

Increased p r o t e i n turn over with onset o f starvation

(25)

Serine protease i n h i b i I n h i b i t i o n of protease (26) activity t o r of ±. υ· B. subtilis *The examples c i t e d are found i n E. coli, except where i n d i c a t e d . Ό

*

S

^

^

»


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F i n a l l y , the degradation of an enzyme by p r o t e o l y s i s can be modulated i n s p e c i f i c ways through substrate o r product s t a b i l i z a t i o n (24). These would c o n s t i t u t e s p e c i f i c c o n t r o l s over enzyme destruction. I t i s a l s o known that, when c e l l s are starved f o r amino a c i d s , n i t r o g e n , o r energy, there i s increased turnover o f many c e l l p r o t e i n s (25). Such a c o n t r o l might be considered a general one. Some of the mechanisms underlying the p h y s i o l o g i c a l responses l i s t e d i n Table I I I are given i n Table IV. Enzyme d e s t r u c t i o n , which w i l l not be considered f u r t h e r , occurs o f course by proteol y t i c breakdown and, i n that i t can be impeded by ligands ( e i t h e r substrates o r i n h i b i t o r s ) that bind to the p r o t e i n , could be subj e c t to a s p e c i f i c c o n t r o l . P r o t e o l y s i s might be enhanced by f a c t o r s that a c t i v a t e proteases o r r e l e a s e protease i n h i b i t o r s . Such an enhancement might be considered a general c o n t r o l over p r o t e o l y t i c breakdown. The formation o f p r o t e i n s can be r e g u l a t e d at two l e v e l s , e i t h e r by a f f e c t i n g formation o f mRNA ( t r a n s c r i p t i o n ) or i t s u t i l i z a t i o n as template ( t r a n s l a t i o n ) . In turn, t r a n s c r i p t i o n can be c o n t r o l l e d i n two ways. One i s a c o n t r o l o f i n i t i a t i o n of t r a n s c r i p t i o n ( i . e . , whether the RNA polymerase forms an i n i t i a t i o n complex). The other i s a c o n t r o l over how f a r a polymerase t r a v e l s and whether i t proceeds i n t o a s t r u c t u r a l gene or not ( i . e . , whether a t t e n u a t i o n o f t r a n s c r i p t i o n o c c u r s ) . T r a n s l a t i o n a l c o n t r o l would more l i k e l y be achieved by c o n t r o l l i n g r i b o some b i n d i n g to the mRNA, although mechanisms a f f e c t i n g ribosome t r a v e l are conceivable. Among the molecular mechanisms c o n t r o l l i n g the i n i t i a t i o n of t r a n s c r i p t i o n i n b a c t e r i a that might be considered s p e c i f i c mechanisms o f c o n t r o l are those that i n v o l v e a negative c o n t r o l element, or the repressor of the o r i g i n a l model of Jacob and Monod (44). Two kinds o f c o n t r o l were envisioned i n that model. One i n v o l v e d a repressor p r o t e i n that was a c t i v e only i n the presence o f the endproduct of the pathway. The b e s t - s t u d i e d example i s the product of the trpR gene, which becomes a c t i v e only a f t e r b i n d i n g to tryptophan. The b i n d i n g o f the a c t i v a t e d repressor t o the operator s i t e s o f the trp operon, of the aroH gene, o r o f the trpR gene and the binding o f RNA polymerase to the corresponding promoters are mutually e x c l u s i v e (28). The inverse o f t h i s p a t t e r n i s the i n a c t i v a t i o n of the repressor p r o t e i n by the substrate^ (or a d e r i v a t i v e ) . The b e s t - s t u d i e d example o f t h i s p a t t e r n i s the i n a c t i v a t i o n o f the lac repressor by i t s b i n d i n g o f a l l o l a c t o s e , a d e r i v a t i v e o f l a c t o s e formed by the a c t i o n o f 8-galactosidase on l a c t o s e (29). Although i t was considered as one p o s s i b i l i t y by Jacob and Monod (44) i n t h e i r formulation o f a model f o r c o n t r o l o f gene expression, r e g u l a t i o n by p o s i t i v e c o n t r o l elements was considered a l e s s l i k e l y mechanism f o r c o n t r o l of gene expression. There have been s e v e r a l examples described i n which p o s i t i v e c o n t r o l


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BIOCHEMICAL ENGINEERING Table IV Molecular

Basis of Regulation

Mode o f C o n t r o l

of P r o t e i n Formation T y p i c a l Examples

Reference

Transcriptional Control C o n t r o l of T r a n s c r i p t i o n I n i t i a t i o n Specific: Negative C o n t r o l Elements A c t i v a t e d by endproducts

Repression operon by

trp

(28)

I n a c t i v a t e d by substrate or precursor

Induction of lac operon by a l l o l a c t o s e

(29)

A c t i v a t e d by s u b s t r a t e or precursor

Induction of acetohydroxy isomeroreductase by acetohydroxy acids

(30)

( I n a c t i v a t i o n by endproduct)

None known

of the trp

P o s i t i v e C o n t r o l Elements

General: P o s i t i v e C o n t r o l Elements A c t i v a t e d by cAMP

Dependence of lac operon i n d u c t i o n on cAMP-CRP i n t e r a c t i o n

(31)

A c t i v a t e d by temperature

Dependence of s e v e r a l heat-induced p r o t e i n s on the htp gene product

(32)

σ-Like p r o t e i n s

S h i f t o f RNA polymerase s p e c i f i c i t y by B a c i l l i during s p o r u l a t i o n

(33)

[ppGpp-mediated regulation]

I n h i b i t i o n of RNA polymerase b i n d i n g to rrn promoters. Enhancement of polymerase pausing i n rrn opérons

(34)

[Nitrogen-mediated regulation]

(35)

The glnG-dependent a c t i v a - (36) t i o n of genes i n v o l v e d i n catabolism of n i t r o g e n c o n t a i n i n g compounds


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Mechanisms

Table IV, cont'd. Control of Transcription

Termination

General: p-Dependent

termination

trpt* terminator of trp operon t

p-Independent

termination

R 1

terminator

a t end

of λ

(37) (38)

Termination o f t r a n s c r i p t i o n at the end of leader sequence f o r amino a c i d b i o s y n t h e t i c operon

(39)

Polymerase pause s i t e i n rmB operon

(AO)

A n t i t e r m i n a t i o n by s p e c i f i c proteins

λ Ν gene-dependent t r a n s c r i p t i o n beyond the tp2 terminator

(41)

A n t i t e r m i n a t i o n by ribosome h a l t at s p e c i f i c codon

Attenuation o f ilv, trp, his, and other amino a c i d b i o s y n t h e t i c codons

(39)

Binding o f small r i b o somal p r o t e i n s a t r i b o some b i n d i n g s i t e f o r the rbsL (str) operon

(42)

Transient terminâtionpolymerase pausing Specific:

Control of T r a n s l a t i o n SpecificMasking of ribosomal b i n d i n g s i t e s by p r o t e i n binding By p e r t u r b a t i o n of secondary s t r u c t u r e of message

The masking o f erm (43) (erythromycin r e s i s t a n c e ) ribosome b i n d i n g s i t e by retarded t r a n s l a t i o n o f the erm leader t r a n s c r i p t

General: (Amino a c i d supply)

None known

(Inhibition)

None known

Mechanisms enclosed i n ( ) may be h y p o t h e t i c a l , s i n c e no examples have been reported. Mechanisms enclosed i n [ ] remain unexplained, i . e . , p r e c i s e mechanism i s unknown.


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provides the primary c o n t r o l over expression of a gene. One ex ample i s the p o s i t i v e c o n t r o l element ( u p s i l o n , the product of the ilvY gene) required f o r the i n d u c t i o n of acetohydroxy a c i d isomeroreductase, the i n d u c i b l e enzyme i n the i s o l e u c i n e and va l i n e b i o s y n t h e t i c pathway of E. coti (30). ( I t should be pointed out that some regulatory elements can play both a p o s i t i v e and a negative c o n t r o l r o l e . An example i s the araC gene product r e q u i r e d f o r i n d u c t i o n of the a r a b i n o s e - u t i l i z i n g pathway (45). In the absence of arabinose, i t acts as a negative c o n t r o l element or repressor, and i n i t s presence i t acts as a p o s i t i v e c o n t r o l element.) Again, the inverse of t h i s p a t t e r n , i n a c t i v a t i o n of a p o s i t i v e c o n t r o l element by the endproduct of a pathway, might provide a mechanism f o r r e p r e s s i o n . However, t h i s w r i t e r i s aware of no example of such a mechanism. Several of the general c o n t r o l s over enzyme formation appear* to be achieved by a f f e c t i n g t r a n s c r i p t i o n i n i t i a t i o n . There i s no reason to assume that these general c o n t r o l s could not be me diated v i a p o s i t i v e or negative c o n t r o l elements of broad s p e c i f i c i t y , and i t may be that many are. A w e l l - s t u d i e d example of a p o s i t i v e c o n t r o l element a f f e c t i n g many c a t a b o l i c systems i n bac t e r i a i s the i n t e r a c t i o n between cAMP and i t s binding p r o t e i n (CAP), which allows binding of CAP to the DNA near the promoters of c e r t a i n c a t a b o l i c enzymes, a f t e r which RNA polymerase can more r e a d i l y bind (31). The heat-induced p r o t e i n s r e c e n t l y demon s t r a t e d i n E. coli by Neidhardt's group may be considered another example (23). The i n d u c t i o n of these enzymes appears to be depen dent upon a p o s i t i v e c o n t r o l element s p e c i f i e d by the htp gene (32). Another kind of c o n t r o l has been found to be important i n the B a c i l l i and might be important i n other organisms as w e l l . This i s a c o n t r o l of l a r g e numbers of genes by a l t e r i n g the s p e c i f i c i t y of RNA polymerase by the binding of d i f f e r e n t sigma (σ) f a c t o r s (33). Thus, during the s p o r u l a t i o n phase, the 55-kd σ f a c t o r that i s predominantly bound to RNA polymerase i s replaced by a 29-kd σ f a c t o r which allows some of the s p o r e - s p e c i f i c genes to be expressed. To date, at l e a s t four d i f f e r e n t σ f a c t o r s have been i d e n t i f i e d i n B. siibtiUs. Two other general regulatory mechanisms have been s t u d i e d that appear to a f f e c t t r a n s c r i p t i o n i n i t i a t i o n . Neither, however, are w e l l enough understood to decide whether they might c o n s t i t u t e unique mechanisms or are s p e c i a l cases of p o s i t i v e c o n t r o l e l e ments that exert a g e n e r a l i z e d c o n t r o l r o l e . One i s the ppGppmediated r e g u l a t i o n that impedes s t a b l e (ribosomal and t r a n s f e r ) RNA formation and, at l e a s t i n v i t r o , stimulates t r a n s c r i p t i o n of many b i o s y n t h e t i c and c a t a b o l i c opérons. While ppGpp does prevent RNA polymerase binding to s t a b l e RNA promoters, the pronounced e f f e c t of ppGpp on s t a b l e RNA formation cannot s o l e l y be explained on t h i s b a s i s alone (see below) (34,35). The other i s the c o n t r o l that i s an e f f e c t of n i t r o g e n l i m i t a t i o n and leads to the expression of genes involved i n r e l e a s i n g NH3 from n i t r o g e n -


4.

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81

containing compounds (36). The glnG gene i s somehow i n v o l v e d , but whether that product i s a p o s i t i v e c o n t r o l element a c t i n g i n a way analogous to that by which cAMP bound to CAP acts on carbon and energy producing pathways i s not c l e a r . Table IV a l s o l i s t s some o f the f a c t o r s that c o n t r o l t r a n s c r i p t i o n termination. T r a n s c r i p t i o n termination plays two r o l e s . One, of course, i s the termination that occurs a short distance beyond the end of a given s t r u c t u r a l gene or beyond the end of the operon. The other i s a termination that serves to attenuate the t r a n s c r i p t or to terminate t r a n s c r i p t i o n at a s i t e upstream of that at which the maximal-sized t r a n s c r i p t i s terminated (39). I t i s the modulation of the l a t t e r kind o f t r a n s c r i p t i o n termination that provides an important mechanism of t r a n s c r i p t i o n a l c o n t r o l . T r a n s c r i p t i o n termination i s s i g n a l l e d by e i t h e r p-dependent or p-independent sequences i n the DNA (and RNA). The l a t t e r are r e a d i l y recognized as regions o f dyad symmetry i n the DNA which y i e l d stem and loop s t r u c t u r e s i n the RNA followed by a sequence of s e v e r a l u r i d i n e residues i n which t r a n s c r i p t i o n i s terminated (46). p-Dependent termination s i t e s seem to be c h a r a c t e r i z e d by t r a n s c r i p t s with more weakly base-paired stem and loop s t r u c t u r e s near the termination s i t e i n the t r a n s c r i p t s and with l e s s p r e c i s e p o i n t s of termination than those found i n p-independent terminat i o n s i t e s (47,48). C l o s e l y r e l a t e d to the s i t e s of p-dependent and p-independent termination s i t e s are s i t e s at which pausing o f RNA polymerase occurs. Pausing i s a l s o apparently c h a r a c t e r i s t i c of termination s i t e s , but the " r u l e s " f o r polymerase pausing are even l e s s c l e a r than those f o r termination (40,49). Polymerase pausing and t r a n s c r i p t i o n termination can thus a f f e c t the extent to which the DNA downstream of the pausing or termination s i t e are t r a n s c r i b e d independently of the t r a n s c r i p t i o n i n i t i a t i o n event. That pausing or termination can be modul a t e d allows a c o n t r o l of the process. I t i s an enhancement by ppGpp o f polymerase pausing at a s i t e e a r l y i n ribosomal RNA opérons that may account f o r the s t r i k i n g e f f e c t that t h i s nucleot i d e has on the s y n t h e s i s of s t a b l e RNA over and above that which occurs a t the polymerase b i n d i n g step (40). The prolonged pausing of polymerase progress at a s p e c i f i c p o i n t has been termed " t u r n s t i l e " attenuation. S i m i l a r l y , when termination at a s i t e w i t h i n an operon or a t the end o f a leader region i s a f f e c t e d by a s p e c i f i c r e g u l a t o r y s i g n a l , an e f f i c i e n t c o n t r o l can be achieved. One of the most s t u d i e d c o n t r o l s over a p-dependent termination i s that which occurs at the XtRl s i t e and i s prevented by the λ Ν gene product (41). L i k e the a c t i v i t y of ρ i t s e l f , the a c t i v i t y of the Ν gene product i s dependent on the elongation subunit of RNA polymerase s p e c i f i e d by the E. coli, nusA gene. I t might be a n t i c i p a t e d that a t t e n u a t i o n of t r a n s c r i p t i o n by a s p e c i f i c a n t i t e r m i n a t i n g p r o t e i n i s a general p a t t e r n that w i l l be found to occur frequently i n m i c r o b i a l systems.


82


Another c o n t r o l over termination that has been e x t e n s i v e l y s t u d i e d i s the attenuation of t r a n s c r i p t i o n at the end of the leader regions of s e v e r a l amino a c i d b i o s y n t h e t i c opérons (or genes) i n E. coli and r e l a t e d organisms (39). The primary i n d i c a t i o n that such a mechanism i s involved i n the c o n t r o l of an amino a c i d b i o s y n t h e t i c pathway i s an endproduct r e p r e s s i o n that i s dependent on the amino a c i d being t r a n s f e r r e d to i t s cognate tRNA at an ample r a t e . Thus, derepression of the h i s t i d i n e biosynthet i c pathway can be achieved by any mechanism that reduces the i n t r a c e l l u l a r l e v e l of h i s t i d y l tRNA, f o r example, by l i m i t i n g the supply of h i s t i d i n e i t s e l f or by l i m i t i n g the a c t i v i t y of the h i s t i d y l tRNA synthetase (51). Thus f a r , the amino a c i d b i o s y n t h e t i c genes c o n t r o l l e d i n t h i s way are c h a r a c t e r i z e d by the presence of a leader region between the promoter and the s t r u c t u r a l gene (52,53). The leader i s t r a n s c r i b e d at a frequency dependent upon promoter strength and polymerase a v a i l a b i l i t y ( i n the case of the trp operon, t r a n s c r i p t i o n i n i t i a t i o n i t s e l f i s c o n t r o l l e d by the trp repressor). The leader t r a n s c r i p t c a r r i e s the information f o r a short peptide that i s unusually r i c h i n the amino a c i d ( s ) f o r which the pathway i s concerned. Whether RNA polymerase stops at the end of the leader or proceeds i n t o the s t r u c t u r a l gene i s dependent upon whether a ribosome can t r a n s l a t e the leader t r a n s c r i p t to the stop codon (amino a c i d excess) or i s s t a l l e d at a codon f o r the amino a c i d for which the pathway i s concerned (amino a c i d l i m i t a t i o n ) . The mechanism underlying termination i s that competing secondary s t r u c t u r e s of the leader t r a n s c r i p t are s e l e c t e d by the extent of ribosomal t r a v e l and that one of these a l t e r n a t i v e secondary s t r u c t u r e s contains a t y p i c a l ρ (rho)-independent termination s i g nal. The general features have been studied best i n the amino a c i d b i o s y n t h e t i c opérons of s e v e r a l Enterobacteriaceae (39). Although the nature of the b a c t e r i a l c e l l makes p o s s i b l e spec i f i c c o n t r o l over t r a n s c r i p t i o n i n ways that cannot be achieved i n eukaryotic systems, c o n t r o l of gene expression at the l e v e l of t r a n s l a t i o n i s a l s o important i n b a c t e r i a . Control of t r a n s l a t i o n i s achieved p r i m a r i l y by the prevention of ribosomal binding. Such a c o n t r o l might be achieved by masking the binding s i t e e i t h e r by s p e c i f i c b i n d i n g by p r o t e i n s or by the secondary s t r u c ture of the message i t s e l f . The b e s t - s t u d i e d examples are the ribosomal p r o t e i n s (54). Although the genes f o r a l l ribosomal p r o t e i n s have not yet been examined, the general pattern that seems to be emerging i s that one of the ribosomal p r o t e i n s s p e c i f i e d by a m u l t i c i s t r o n i c , ribosomal p r o t e i n operon prevents message t r a n s l a t i o n by binding at the t r a n s l a t i o n a l s t a r t s i t e on the message. The b a s i s for the s p e c i f i c binding i n the case of r i b o somal p r o t e i n s S4 and S7 i s that the RNA i n the v i c i n i t y of the ribosomal s t a r t s i t e has a s t r u c t u r e s t r o n g l y homologous to that of the region on 16S RNA that binds to S4 (or to S7) during r i b o some assembly (42). The c o n t r o l over the erythromycin r e s i s t a n c e gene, which


4.

UMBARGER

Microbial

Regulatory

Mechanisms

83

s p e c i f i e s a ribosomal methylase, i s a l s o achieved by i n t e r f e r i n g with t r a n s l a t i o n of the message (43). Masking of the ribosome masking s i t e i s not by a p r o t e i n binding to i t but by a secondary s t r u c t u r e o c c u r r i n g i n the message whenever a t r a n s l a t i n g ribosome i s able to t r a n s l a t e the peptide s p e c i f i e d by the leader sequence (as could occur only i f already modified ribosomes were f u n c t i o n ing i n the presence of erythromycin or i f normal ribosomes were f u n c t i o n i n g i n the absence of erythromycin). The ribosome binding s i t e of the methylase message would be opened when the t r a n s l a t i n g ribosome s t a l l e d i n the e a r l y part of the leader (as could occur i f unmodified, s e n s i t i v e ribosomes were f u n c t i o n i n g i n the presence of erythromycin). This mechanism, a f f e c t i n g a c c e s s i b i l i t y of a ribosome b i n d i n g s i t e i s thus reminiscent of the c o n t r o l of formation of a t r a n s c r i p t i o n termination s i t e by the t r a n s l a t i o n of the leader sequence of s e v e r a l amino a c i d b i o s y n t h e t i c genes described above. These examples are a small sample of many that could be c i t e d that make i t q u i t e c l e a r that m i c r o b i a l metabolism i s r a t h e r r i g i d l y c o n t r o l l e d f o r the primary f u n c t i o n of the microbe: to grow and reproduce i t s e l f as e f f i c i e n t l y as p o s s i b l e . These are what I would term obstacles to the biochemical engineer who may wish to e x p l o i t a p a r t i c u l a r enzyme r e a c t i o n or a p a r t i c u l a r pathway to a f u n c t i o n that may w e l l be detrimental to the primary f u n c t i o n of the organism. However, i n every case, the c o n t r o l mechanisms that c o n s t i t u t e these o b s t a c l e s are g e n e t i c a l l y cont r o l l e d and, t h e r e f o r e , are subject to mutation. To the extent that mutations can be s e l e c t e d that are advantageous to the engineer without being l e t h a l to the organism, these c o n t r o l mechanisms can be considered " t o o l s to be manipulated and c a r e f u l l y honed. It i s , however, sometimes d i f f i c u l t to e x p l o i t the capacity of an organism to perform a u s e f u l biochemical transformation. Often Nature has s e l e c t e d organisms i n which the s t r i c t u r e s found i n most organisms are modified, and the organism occupies a unique niche. Such organisms, i f discovered, can serve u s e f u l f u n c t i o n s . Just such organisms are those that are c u r r e n t l y being used i n the Japanese amino a c i d fermentation i n d u s t r y . In most cases, the organisms have been i s o l a t e d and i d e n t i f i e d by e m p i r i c a l research and screening procedures. In some cases, the b a s i s f o r the nonnormal formation of amino a c i d s can be explained post facto, but seldom does i t appear that these organisms were developed by a preconceived, r a t i o n a l approach. I would t h e r e f o r e l i k e to c i t e and analyze what I think provides an exception. The e x c e p t i o n a l case i s one i n which the i n v e s t i g a t o r s j u d i c i o u s l y combined e m p i r i c a l and r a t i o n a l approaches to prepare an organism capable of producing l a r g e amounts of the amino a c i d i s o l e u c i n e . The p r o j e c t was performed by i n v e s t i g a t i o n at the Tanabe Seiyeku Company i n Osaka, Japan, and i n v o l v e d the organism Serratia marcescens (55). Before review of t h i s work, i t i s necessary to consider b r i e f l y s e v e r a l aspects of the pathways by which 11



84

i s o l e u c i n e and the c l o s e l y r e l a t e d amino a c i d , v a l i n e , are formed. Figure 1 shows these pathways. An important feature i s that the four steps l e a d i n g to v a l i n e are c a t a l y z e d by enzymes that are a l s o needed f o r i s o l e u c i n e formation. In a d d i t i o n , a f i f t h enzyme, threonine deaminase, i s r e q u i r e d i n most p l a n t s and micro organisms f o r the formation of α-ketobutyrate. Several d e t a i l s of the pathways are given i n the legend. Also shown i n Figure 1 i s the arrangement of the s t r u c t u r a l genes found i n E. coli. I t should be pointed out that a l l of the genes, except those f o r the two v a l i n e - i n s e n s i t i v e acetohydroxy a c i d synthases, are contained i n the ilv gene c l u s t e r at the 84minute region of the E. coli chromosome. I t might t h e r e f o r e be a n t i c i p a t e d that an E. coli s t r a i n c a r r y i n g a plasmid with the ilv gene c l u s t e r would be an e x c e l l e n t overproducer of v a l i n e . (If the chromosomal ilv fragment was derived from the K-12 s t r a i n of E. coli i t would have to contain an ilvO mutation so that ilvG would be expressed. See Figure 1.) Such a plasmid would be even more e f f e c t i v e i f the i n s e r t e d DNA c a r r i e d an ilvA lesion, i . e . , was threonine deaminase negative. Overproduction of v a l i n e would be expected, s i n c e the v a l i n e pathway derives i t s carbon e x c l u s i v e l y from a key intermediate i n the c e n t r a l metabolic route, pyruvate. Flow i n t o the pathway would then never be r e s t r i c t e d as long as ample carbon source was present. An analogous c o n d i t i o n f o r i s o l e u c i n e overproduction would not be expected to a r i s e i f the corresponding plasmid c o n t a i n i n g an ilvA s t r u c t u r a l gene mutation l e a d i n g to a feedback negative threonine deaminase were to be employed. As can be seen i n Figure 2, carbon flow i n t o the i s o l e u c i n e pathway i s dependent not only on the supply of pyruvate but a l s o on threonine, an endproduct i t s e l f , and the s y n t h e s i s of which i s a l s o s t r i n g e n t l y r e g u l a t e d . It i s doubtful whether threonine could be formed f a s t enough i n an otherwise normal c e l l to e x p l o i t f u l l y the high l e v e l expres s i o n of the ilv genes c a r r i e d on a plasmid as d e s c r i b e d here. The procedures followed by Komatsubara and h i s coworkers i l l u s t r a t e s w e l l how t h i s k i n d of o b s t a c l e can be circumvented. The sequence of manipulations employed by Komatsubara et a l . (55) i s summarized i n Figure 3. The simplest step was to i s o l a t e a d e r i v a t i v e o f the parent S. maroeeoens s t r a i n , 8000, that was derepressed f o r the ilvGEDA operon and c a r r i e d a l e s i o n i n the ilvA gene that allowed the formation of an i s o l e u c i n e - i n s e n s i t i v e (feedback negative) threonine deaminase ( T D ) . This kind of i s o l a t i o n had e a r l i e r been accomplished by the s e q u e n t i a l s e l e c t i o n of an aminobutyrate-resistant s t r a i n (derepressed) followed by s e l e c t i o n of an i s o l e u c i n e hydroxamate-resistant d e r i v a t i v e (feed back r e s i s t a n t ) (58). The i n v e s t i g a t o r s were more fortunate i n the s e l e c t i o n of s t r a i n GIHVL-r6426, which arose as an i s o l e u c i n e hydroxamate-resistant s t r a i n a f t e r mutagenesis by n i t r o s o g u a n i dine, a mutagen known f o r causing c l o s e l y l i n k e d mutations i n the same c e l l . The p r e c i s e nature of the mutation a l l o w i n g derepres s i o n was not e s t a b l i s h e d , but, i n analogy with what has been y

r



Ο

L-Threonjne

3

AHSU

3

3

Ο

3

CH

I

AHSm

—ι—-pi

H

a - Aceto - a hydroxybutyrate

2

CH

CH -C-C-COO"

3

a-Acetolactate

CH

CH -C-C-C00~

Ο OH

Β AHSI

I—; 1

IR

IR

3

Ο Ο

3

2

Y

A

D

OH

DH ι

Ο -CH-C-COO"

E

CH

2

3

ÇH

q

a

e

G G G Gp

a-Keio-βmethy I valerate

3

CH -CH-C-COO"

CH, a-Ketoisovalerate

3

CH

ι—:—ι—;—ι—; —ι ; ι—; —ι—I, t i *ι "i I I ι ι TD DH TrB AHSU

C

CH α,β-Dihydroxy/3-methylvolerote

ι

CH

CH -C-CH-COO" 3

3

CH α,β-Dihydroxyisovalerate

CH -C-CH-COO~

Η Η Ο Ο

TrB

TrC TrB 3

NH

3

3

2

TD (ilvA), threonine deaminase; AHS I (ilvB) and AHS III (ilvHI), valine-inhibited acetohydroxy acid jynthases; AHS II (ilvG), valine-insensitive acetohydroxy acid synthase; IR (ilvC), acetohydroxy acid isomeroreduc~' tase; DH (ilvD), dihydroxy acid dehydrase; TrB (ilvE), transaminase B; TrC, transaminase C. In E. coli strain K-12, ilvG exhibits no activity owing to a polar lesion in the ilvO region of the gene. Certain frameshift mutations in ilvO result in ilvG expression and increased downstream expression. Control of the ilvGEDA operon occurs by attenuation at ilvGa of transcripts initiated at the promoter ilvGp. A putative 32-residue leader peptide, rich in isoleucirte, leucine, and valine is specified by ilvGe. The ilvY product, μ, is a positive control element needed for induction of ilvC by the substrates of its product, IR.

L- Isoleucine

CH

ι

CH

CH -CH-CH-COO" 3

ι

CH L- Valine

3

NH CH, -CH-CH-COO"

IR Figure 1. Biosynthesis of isoleucine and valine. The enzymes catalyzing the indicated steps are abbreviated, and the corresponding structural genes (where known) are indicated in parentheses as follows.

CH -CH - CH-COO"

3

TD-*-NH

a-Ketobutyrote

CH -CH-C-COO

3

Pyruvote

3

CH -C-COO'

3

CH -C-COO Pyruvate

AHSU


!

h a t e

BJdapB

Dihydrodipicolinic acid

Aspartic wVAjr ' semialdehyde

L-Lysine

cJ/ysA

meso-diaminopimelate

LL-Diaminopimelate

Ε I dapE

N-succinyl-LLdiaminopimelate

DjcfapC or D

N-succinyl- '' L-Aspartate r> Aspartyl metLM "~f"* P P

4.

UMBARGER

Microbial

Regulatory

87

Mechanisms

THREONINE AND ISOLEUCINE PRODUCING STRAINS (Komatsubara et a l . ) AECrl74

N-ll

uth ilvA

KNr31

uth ilvA

uth ilvA

?(Met-leaky)

? (Met-leaky)

r

lysC (AK ) lysCo ?

?(Met-leaky) 0.3 g T h r / 1

r

lysC (AK ) lye Co thrB/C

7 g Thr/1

T-570

HNr21

T-693

uth ilvA

uth ilvA

r

r

r

thrA (AK HD )

uth ilvA

r

thrA (AK HD )

?(Itet-leaky)

ileS

11 g T h r / 1

r

lysC (AK ) lyeCo ?

8 g Thr/1

r

r

thrA (AK HD )

île S 25 g T h r / 1 HNr59

E-60

uth ilvA thrA (HD ) ileS r

5 g Thr/1

uth ilvA thrA (HD ) ileS thrB/C r

T-803 r

GIHVL-r6426 r

ilvA (TD ) ilvGa ? Mu-910

uth

3.5 g I l e / 1 8000

uth ilvA (TD ) ilvGa ? ?(Met-leaky) r

lysC (AK ) lysCo ? r

r

thrA (AK HD )

ileS 25 g I l e / 1

Figure 3. The development of an isoleucine-producing strain from S. marcescens strain 8000. Key: φ, phage-mediated transduction; M , nitrosoguanidine mutagenesis. See text for details.


88


learned about the c o n t r o l of the ilvGEDA operon i n E. coli, i t is most l i k e l y a mutation a f f e c t i n g the attenuator (ilvGa), thereby a b o l i s h i n g the m u l t i v a l e n t c o n t r o l of the p a r e n t a l s t r a i n . That ilvGy the s t r u c t u r a l gene f o r the v a l i n e - i n s e n s i t i v e acetohydroxy a c i d synthase, was derepressed assured that acetohydroxy a c i d i s o meroreductase would be h i g h l y induced by s u b s t r a t e , even though i t s s t r u c t u r a l gene, ilvC i s not part of the derepressed operon. The r e s i s t a n t mutant was shown to excrete up to 3.5 gm of i s o l e u cine per l i t e r i n a minimal medium c o n t a i n i n g 150 gm of sucrose as carbon source. That t h i s l e v e l was l i m i t e d by a v a i l a b i l i t y of threonine was l a t e r demonstrated by the f a c t that t r a n s f e r of the two l i n k e d ilv mutations to a s t r a i n overproducing l a r g e q u a n t i t i e s of threonine r e s u l t e d i n a much b e t t e r i s o l e u c i n e producer. The steps used to develop the threonine producer were complex and are a l s o o u t l i n e d i n F i g u r e 3. The f i r s t of these steps was the i s o l a t i o n of n i t r o s o g u a n i d i n e mutant (Mu-910) unable to u t i l i z e threonine as a n i t r o g e n and carbon source (59). The mutation was i n the uth gene, which s p e c i f i e s threonine dehydrogenase. To obtain an organism even l e s s able to break down exogenous t h r e o nine, mutagenesis to y i e l d an i s o l e u c i n e - r e q u i r i n g s t r a i n l a c k i n g threonine deaminase was employed. When preformed threonine was added to f u l l y grown c u l t u r e s of the r e s u l t i n g s t r a i n , D-60, the r a p i d disappearance noted with the o r i g i n a l parent s t r a i n was no longer observed (59). S t r a i n D-60 was then used to s e l e c t mutants r e s i s t a n t to the threonine analog, 3-hydroxynorvaline, f o l l o w i n g n i t r o s o g u a n i d i n e mutagenesis (60,61). Three kinds of r e s i s t a n t s t r a i n were saved for subsequent use. One of these (HNr31) had an incomplete but undefined b l o c k i n methionine b i o s y n t h e s i s , which may have accounted f o r the s m a l l amount o f threonine that was accumulated from homoserine being funneled i n t o the threonine pathway. The other two mutants produced much g r e a t e r amounts of threonine. One, s t r a i n HNr21, appeared to contain a s i n g l e mutation i n the thrA gene, which s p e c i f i e s the b i c e p h a l i c enzyme, a s p a r t o kinase I-homoserine dehydrogenase I (Figure 2). Normally, both of these a c t i v i t i e s are i n h i b i t e d by threonine. The mutation i n s t r a i n HNr21 a b o l i s h e d the threonine s e n s i t i v i t y f o r both a c t i v i ties. This l o s s of feedback c o n t r o l r e s u l t e d i n an overproduction of threonine to an extent of 11 gm of threonine per l i t e r . The other s t r a i n , HNr59, a l s o had a thrA l e s i o n , but t h i s one appeared to a b o l i s h only the threonine s e n s i t i v i t y of the homoserine dehydrogenase. There was another mutation i n the same s t r a i n which appeared to be i n the ileS gene. I f so, i t undoubtedly caused the corresponding gene product, i s o l e u c y l tRNA synthetase, to have a reduced a f f i n i t y f o r i t s s u b s t r a t e . Such an enzyme would l e a d to reduced l e v e l s o f i s o l e u c y l tRNA and derepressed l e v e l s of the thr operon and the ilvGEDA operon. Since the mutant was an ilvA mutant, the concomitant derepression of the ilvGEDA operon was of no consequence. However, the iVoA mutation made i t necessary f o r the s t r a i n to be grown i n the presence of exogenous i s o l e u c i n e and may 9


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have reduced the e f f e c t i v e n e s s of the l o w - a f f i n i t y i s o l e u c y l tRNA synthetase i n causing a derepression of the threonine b i o s y n t h e t i c enzymes. In an attempt to enhance threonine production, steps were taken to r e p l a c e the thrA gene mutation a f f e c t i n g only homoserine dehydrogenase I a c t i v i t y i n s t r a i n HNr59 with that of s t r a i n HNr21, which a f f e c t e d both aspartokinase I and homoserine dehydrogenase I a c t i v i t i e s . This t r a n s f e r was accomplished by i n t r o ducing a thrB (or C) mutation i n HNr59, to y i e l d s t r a i n E-60 (62). This s t r a i n then served as r e c i p i e n t f o r phage-mediated transduct i o n with s t r a i n HNr21 as donor. The r e s u l t i n g s t r a i n , T-570, contained the doubly i n s e n s i t i v e aspartokinase I-homoserine dehydrogenase I a c t i v i t i e s of s t r a i n HNr21 but had not l o s t the c l o s e l y l i n k e d ileS l e s i o n of s t r a i n E-60 (or i t s parent, HNr59). Surp r i s i n g l y , t h i s s t r a i n d i d not produce q u i t e as much threonine as did s t r a i n HNr21. Attempts were t h e r e f o r e made to increase carbon flow i n t o the threonine pathway by enhancing the a c t i v i t y of the l y s i n e - s e n s i t i v e aspartokinase. This was accomplished by s e l e c t i n g n i t r o s o guanidine- induced mutants r e s i s t a n t to aminoethylcysteine (61). One of these s t r a i n s , AECrl74, was probably another doubly mutated s t r a i n with a l y s i n e - i n s e n s i t i v e aspartokinase that was a l s o derepressed. The derepression might have been e i t h e r an "up" promoter or an operator mutation i n the lysC gene (as i s suggested i n Figure 3). This s t r a i n accumulated 7 gm of threonine per l i t e r . Mutagenesis to threonine auxotrophy y i e l d e d s t r a i n N - l l , which allowed i n t r o d u c t i o n of the thrA gene and ileS genes of s t r a i n T-570 by phage-mediated t r a n s d u c t i o n (62). The r e s u l t a n t s t r a i n , T-693, produced up to 25 gm of threonine per l i t e r and provided e x a c t l y the k i n d of background that might feed carbon i n t o an i s o l e u c i n e pathway that had l o s t both feedback and r e p r e s s i o n c o n t r o l . This i n t r o d u c t i o n was p o s s i b l e because s t r a i n T-693 c a r r i e d the o r i g i n a l ilvA l e s i o n of s t r a i n D-60. Phage grown on s t r a i n G1HVL-6426 was t h e r e f o r e used to render s t r a i n T-693 i s o l e u c i n e independent. In so doing, both the feedback r e s i s t a n c e of threonine deaminase and the derepression of the ilvGEDA operon were introduced. The r e s u l t i n g s t r a i n , T-803, produced up to 25 gm of i s o l e u c i n e per l i t e r . The carbon flow i n t o the u n c o n t r o l l e d threonine pathway had thus been almost comp l e t e l y d i v e r t e d to i s o l e u c i n e production with only about 3 gm of threonine now being formed by s t r a i n T-803. This r a t h e r i n v o l v e d example serves perhaps as an i n t e r e s t i n g model f o r the way e m p i r i c a l l y generated mutations can be r a t i o n a l l y manipulated to y i e l d u s e f u l organisms. In the example c i t e d , the u s e f u l t o o l of using analogs to s e l e c t s t r a i n s with a l t e r e d c o n t r o l c i r c u i t s was i n v a l u a b l e . There were r e a l l y no unexpected mutations, yet there was no way to p r e d i c t which combination of l e s i o n s would have supplemented each other to have y i e l d e d such p r o l i f i c overproducers. I t may be that the maximally e f f e c t i v e combination was not obtained i n s t r a i n T-803, and i t i s l i k e l y


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that the i n v e s t i g a t o r s t r i e d many more combinations than those that were reported. I n v e s t i g a t o r s are undoubtedly now being tempted to e x p l o i t the use o f recombinant DNA technology to produce s t r a i n s with cert a i n biochemical a c t i v i t i e s enhanced. The lesson the model system described here has f o r such i n v e s t i g a t o r s i s t h a t , i n a d d i t i o n to r e q u i r i n g elevated enzyme a c t i v i t i e s , i t i s a l s o necessary to assure that the c e l l can provide the substrates o r the carbon r e quired f o r the enhanced pathway to f u n c t i o n . The other lesson i s that s t r a i n development w i l l remain l a r g e l y an e m p i r i c a l process but that empiricism w i l l be more r e a d i l y harnessed i f the i n v e s t i g a t o r has a thorough knowledge o f the physiology and biochemist r y o f the process.

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Microbial Regulatory Mechanisms - American Chemical Society

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