Optimization of Fermentation Processes Through the Control of In Vivo

3 Optimization of Fermentation Processes Through the Control of In Vivo Inactivation of Microbial Biosynthetic Enzymes SPYRIDON N. AGATHOS and ARNOLD L. DEMAIN

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: January 18, 1983 | doi: 10.1021/bk-1983-0207.ch003

Massachusetts Institute of Technology, Department of Nutrition and Food Science, Fermentation Microbiology Laboratory, Cambridge, MA 02139 Prolonged production of antibiotics could be achieved by in vivo stabilization of biosynthetic enzymes. As a model system we have focused on the gramicidin S synthetase complex in Bacillus brevis. Our group previously reported an O -dependent loss of total biosynthetic activity preventable by anaerobiosis. We further found this inactivation to be independent of aerobic energy-yielding metabolism and of de novo protein synthesis through inhibitor studies. However, retardation of inactivation was achieved upon addition of utilizable carbon sources to aerated cell suspensions, the degree of stabilization being proportional to the ease of uptake and to the concentration of each C-source. Dithiothreitol contributed to retardation and partial reversal of the inactivation. These results suggest that the in vivo inactivation may be due to an energy deficiency at the end of exponential growth with a concomitant exposure of sulfhydryl groups on the synthetases to autoxidation. 2

Important commodity chemicals are c u r r e n t l y being produced by microorganisms i n a v a r i e t y of fermentation and bioconversion processes. The e f f i c i e n t production of these substances r e q u i r e s considerable enzyme l e v e l s c a t a l y z i n g the b i o s y n t h e s i s of metabol i t e s , as w e l l as good c a t a l y t i c a c t i v i t i e s and adequate operat i o n a l s t a b i l i t i e s . T h i s i s c e r t a i n l y true of both primary and secondary metabolites. U n t i l r e c e n t l y , most work aimed a t o p t i m i z i n g production of valuable secondary metabolites such as a n t i b i o t i c s has c o n s i s t e d of environmental and genetic approaches a f f e c t i n g almost e x c l u s i v e l y the l e v e l s and c a t a l y t i c a c t i v i t i e s of the relevant b i o s y n t h e t i c enzymes, rather than t h e i r o p e r a t i o n a l s t a b i l i t y . Environmental manipulations have included: (a) the a d d i t i o n o f appropriate precursors; (b) the d i s r u p t i o n of r e g u l a t o r y mechanisms

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


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c o n t r o l l i n g the i n i t i a t i o n of a n t i b i o t i c s y n t h e s i s , such as r e p r e s s i o n and i n h i b i t i o n of t h e i r synthetases. For example, carbon c a t a b o l i t e r e g u l a t i o n i s r e l i e v e d through a d d i t i o n of a slowly u t i l i z e d carbon source at the beginning of a batch fermentation or through the slow a d d i t i o n of the otherwise r e a d i l y consumed carbon source i n various fed-batch type o p e r a t i o n s . S i m i l a r l y , n i t r o g e n and phosphate r e g u l a t i o n are bypassed e i t h e r through r e s t r i c t i o n of the r e s p e c t i v e N- or P- source or through a d d i t i o n of a slowly metabolized a l t e r n a t i v e source of those n u t r i e n t s . Feedback i n h i b i t i o n of a n t i b i o t i c synthetases has been minimized by approp r i a t e b i o r e a c t o r design, e.g., a d i a l y s i s c u l t u r e i n which the end product i s continuously removed; (c) f i n a l l y , the s p e c i f i c growth r a t e of the a n t i b i o t i c - p r o d u c i n g microorganism i s optimized during the idiophase (e.g., through l i m i t a t i o n of a key n u t r i e n t , pH c o n t r o l , etc.) s i n c e i n i t i a t i o n and maintenance of i d i o l i t e production i s favored at low growth r a t e s . Genetic s o l u t i o n s to the problems of the above mentioned regul a t o r y mechanisms have a l s o been a p p l i e d s u c c e s s f u l l y f o r the e f f i c i e n t production of secondary m e t a b o l i t e s . Mutation and s e l e c t i o n f o r s u p e r i o r producing mutants can account f o r much of the success of the modern a n t i b i o t i c i n d u s t r y . However, even with s u p e r i o r producer s t r a i n s and r a t i o n a l environmental s t r a t e g i e s f o r adequate expression and a c t i v i t y of the synthetases, these b i o s y n t h e t i c enzymes u s u a l l y decay q u i c k l y during the idiophase. Thus, the i n v i v o i n a c t i v a t i o n of b i o s y n t h e t i c enzymes i s widespread and appears to be a l i m i t i n g f a c t o r i n the prolonged production of a n t i b i o t i c s and r e l a t e d secondary m e t a b o l i t e s . Despite i t s importance, i n v i v o enzyme i n a c t i v a t i o n of secondary metabolism has not received any a t t e n t i o n as a d i s t i n c t b i o l o g i c a l phenomenon, although a fundamental understanding of the process(s) could hold the key to i t s successf u l circumvention as i s the case with other types of c e l l u l a r enzyme r e g u l a t i o n . We have reasoned that a novel approach to prolong the product i o n phase of secondary metabolites should be based on an attempt to prevent or r e t a r d the process of in v i v o i n a c t i v a t i o n of the enzymes (synthetases) c a t a l y z i n g t h e i r formation i n fermentations. Such an attempt would r e q u i r e an understanding of the chemical nature of the i n a c t i v a t i o n . T h i s knowledge could be subsequently t r a n s l a t e d i n t o process development and c o n t r o l i n a c t u a l ferment a t i o n s , which, f o r the most p a r t , are c a r r i e d out i n batch r e a c t o r s . If the object of the fermentation i s the recovery of the enzymes f o r f u r t h e r use i n a c e l l - f r e e system or the a c q u i s i t i o n of a c t i v e whole c e l l s f o r repeated use i n a f i x e d bed-type b i o r e a c t o r , the prevention or r e t a r d a t i o n of the i n a c t i v a t i o n process would ensure both an adequate margin of time for primary h a r v e s t i n g and a longer h a l f - l i f e of the a c t i v i t y f o r the b i o c a t a l y s t s i n the c e l l s . The purpose of t h i s communication i s to i l l u s t r a t e the p o t e n t i a l of the above-mentioned approach by f o c u s i n g on our

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


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i n v e s t i g a t i o n of the in v i v o i n a c t i v a t i o n of the enzyme (synthetase) complex r e s p o n s i b l e f o r the formation of g r a m i c i d i n S (GS), a c y c l i c peptide a n t i b i o t i c i n B a c i l l u s b r e v i s ATCC 9999. This i n v e s t i g a t i o n represents the f i r s t attempt, to our knowledge, t o understand and to c o n t r o l in v i v o i n a c t i v a t i o n of a synthetase i n v o l v e d i n the production of a secondary metabolite. The choice of GS s y n t h e s i s , as our model system, was i n f l u e n c e d by the r e l a t i v e l y l a r g e amount of i n f o r m a t i o n i n the l i t e r a t u r e about the enzymology of GS formation, the p a r t i c i p a t i o n of only two polyenzymes i n i t s b i o s y n t h e s i s and the existence of a c e l l - f r e e b i o s y n t h e t i c system. Also the p a t t e r n of appearance and disappearance of the enzyme complex (GS synthetase) i s t y p i c a l of a l a r g e number of a n t i b i o t i c fermentations. In a d d i t i o n , the producing organism, B a c i l l u s b r e v i s , i s a non-filamentous, r a p i d l y growing prokaryote and only a s i n g l e a n t i b i o t i c i s produced i n t h i s fermentation. I n v e s t i g a t o r s have noted that a f t e r the appearance of the two s o l u b l e GS synthetases at the end of exponential growth, they r a p i d l y disappear as the c e l l p o p u l a t i o n moves i n t o s t a t i o n a r y phase (12,L3) ( F i g - 1)· These changes have beeen observed using the assay which measures the t o t a l synthetase a c t i v i t y as w e l l as both L - o r n i t h i n e and L-phenylalanine-dependent ATP-PPi exchange r e a c t i o n s (assays f o r the i n d i v i d u a l heavy and l i g h t GS synthetases r e s p e c t i v e l y ) . F r i e b e l and Demain (.4,5.) found that i n a c t i v a t i o n of s o l u b l e GS synthetase i n v i v o i s oxygen-dependent and i r r e v e r s i b l e . They a l s o found that N gas prevented in v i v o i n a c t i v a t i o n of the enzyme complex under otherwise fermentation c o n d i t i o n s . However, under these c o n d i t i o n s no GS was produced, presumably due to the need of oxygen f o r ATP production i n t h i s aerobic organism. I t i s u s e f u l to review b r i e f l y some of the current motions on i n v i v o i n a c t i v a t i o n of m i c r o b i a l enzymes: 2

In Vivo Enzyme I n a c t i v a t i o n . Enzyme l e v e l s i n microorganisms are known to be c o n t r o l l e d through r e g u l a t i o n of the r a t e of t h e i r s y n t h e s i s ( i n d u c t i o n / r e p r e s s i o n ) whereas enzyme a c t i v i t i e s are c o n t r o l l e d by non-covalent b i n d i n g of v a r i o u s ligands ( a c t i v a t i o n / i n h i b i t i o n ) . However, through s t u d i e s that have focused almost e x c l u s i v e l y on the disappearance of enzymes of primary metabolism, i t i s now apparent that an a d d i t i o n a l type of enzyme r e g u l a t i o n i s o p e r a t i v e i n microorganisms, i . e . , the c o n t r o l of enzymatic a c t i v i t y by s e l e c t i v e i n a c t i v a t i o n . This i n a c t i v a t i o n of p a r t i c u l a r enzymes i s widespread among microorganisms, i s brought about by s e v e r a l mechanisms, and i s observed under s p e c i f i c p h y s i o l o g i c a l c o n d i t i o n s (2,LI). In v i v o i n a c t i v a t i o n i s defined as the i r r e v e r s i b l e l o s s of an enzyme's c a t a l y t i c a c t i v i t y i n the c e l l . This d e f i n i t i o n i s designed to c o n t r a s t i n a c t i v a t i o n from i n h i b i t i o n , which i s the r e v e r s i b l e l o s s of enzyme a c t i v i t y through u s u a l l y non-covalent b i n d i n g of an i n h i b i t o r that can be d i a l y z e d o r d i l u t e d away to r e s t o r e a c t i v i t y . I n v i v o enzyme i n a c t i v a t i o n can


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GROWTH

4

5

6

7

8

9

FERMENTATION TIKE (hours) Figure 1. Dynamics of appearance and disappearence of GS synthetases. Key: O, growth; GS; and A, total GS biosynthetic activity (Synthetase scale).


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be classified into modification inactivation, which involves usually a covalent modification of the protein molecule and dégradât ive inactivation, which involves cleavage of at least one peptide bond of the enzyme protein. The in vivo inactivation of microbial enzymes i s usually observed as the cells approach or enter stationary phase i n a batch reactor. It occurs mainly at a point i n which the microbe's nutritional/energetic state changes upon exhaustion or nutrients or a shift in carbon or nitrogen source. Possibly, i t i s a general regulatory mechanism aimed at phasing out a particular enzyme when a metabolic shift renders i t wasteful of metabolites and/or directly harmful to the c e l l .


Materials and Methods The culture used i n these experiments was Bacillus brevis ATCC 9999. The preparation of spores and inoculum and the determination of growth were as previously described (9). Protein was determined by the biuret method (6). In order to study the i n vivo disappearance of GS synthetase, we have used a system that exhibits inactivation kinetics i n short-term experiments. The system u t i l i z e s frozen and thawed c e l l s of IJ. brevis harvested after growth i n yeast extract-peptone (YP) medium in a 180-1 fermentor and frozen at -20°C immediately after harvesting. Small batches of cells were thawed just before performing i n vivo inactivation studies according to the methodology of Friebel and Demain (4). In brief, these cells are agitated under a i r i n buffer for various periods of time in the presence of appropriate compound vs. nitrogen-covered controls, crude cell-free extracts are subsequently prepared using lysozyme, and the extracts are assayed by the overall GS synthetase assay (incorporation of L-[ H]ornithine into GS). 3

Results and Discussion A typical kinetic pattern of the in vivo loss of GS synthetase in our frozen-thawed c e l l system i s shown i n Figure 2. The enzyme survives over 8 hours under nitrogen gas, but i s usually lost at the end of 2-3 hours under conditions of agitation at 250 rpm, 37°C i n a i r (simulated aerobic fermentation conditions). The time scale should not be taken as an absolute measure of the enzyme's survival, because the rate of the inactivation depends upon the previous growth history of the c e l l s . Therefore, comparisons are made only between experiments performed with c e l l s from the same batch of c e l l s . Lack of Inactivation by Energy-yielding Cellular Metabolism. We f i r s t examined whether the dependence of synthetase inactivation on oxygen reflects a requirement for aerobic energy-yielding metabolism. Some enzymes are inactivated i n vivo i n the presence of 0 due to the operation of energy-yielding circuits i n the c e l l , 2




Figure 2. Typical kinetic pattern of in vivo inactivation of GS synthetase complex in frozen-thawed cells of B. brevis. Standard conditions for inactivation: agitation at 250 rpm, 37°C under air, vs. N -covered control. t


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such as glycosis, the c i t r i c acid cycle, electron transport, etc (11)· Especially i n Bacillus s u b t l l i s , i t has been demonstrated that inhibitors of such energy-yielding pathways prevent the i n vivo inactivation of aspartase transcarbamylase, a process requiring oxygen and entry into the stationary phase (15). In our case, inhibitors of glycolysis (NaF), the c i t r i c acid cycle (malonate) and oxidative phosphorylation (DNP), i n concentrations known to inhibit their respective metabolic c i r c u i t s , were not effective i n preventing enzyme inactivation (Table I)·. Therefore, i t appears as i f the inactivation i s not due to energy-yielding metabolic c e l l a c t i v i t y .


Table I. Effect of Energy Metabolism Inhibitors on in vivo Stability of GS Synthetase 0

a

Atmosphere

Additive

Activity

Nitrogen Air Air Air Air Nitrogen Nitrogen Nitrogen

None None 50 mM NaF 50 mM Malonate 0.2 mM DNP 50 mM NaF 50 mM Malonate 0.2 mM DNP

100 4 2 0 6 77 85 103

%

Incubation with shaking for 90 min at 250 rpm, 37°C.

Independence of Inactivation from Protein Synthesis. A similar experiment was carried out to determine whether the inactivation was dependent upon protein synthesis. Conceivably the process of in vivo synthetase inactivation could be enzymatic, involving the mediation of an enzyme that i s derepressed at some point during late exponential growth i n actively growing cultures of 1J. brevis, e.g., a protease with an unusual requirement for oxygen or an oxygenase or an unknown "inactivase". Nevertheless, when a protein synthesis inhibitor (chloramphenicol) was included i n the aerated c e l l suspensions of our system i n a concentration known to inhibit de novo protein synthesis i n IJ. brevis, no prevention or slowdown of the inactivation process was noted, thus suggesting that the process i s not protein-synthesis dependent. Carbon Source-mediated Retardation of Inactivation. As mentioned previously, a shift in C-metabolism i s one of the physiological conditions most commonly associated with in vivo inactivation of


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m i c r o b i a l enzymes. I t might be assumed that as B. b r e v i s moves i n t o the s t a t i o n a r y phase, the o p e r a t i o n of the ITS synthetase f o r continued formation of the a n t i b i o t i c from i t s c o n s t i t u e n t amino a c i d s becomes wasteful of p r o t e i n p r e c u r s o r s . Thus enzyme i n a c t i v a t i o n may spare b u i l d i n g blocks and energy and may be brought about by s i g n a l s of p r o g r e s s i v e d e p l e t i o n of carbon and energy source and/or the i n t r a c e l l u l a r ATP p o o l . To determine whether carbon sources have any e f f e c t on i n a c t i v a t i o n of GS synthetase, v a r i o u s u t i l i z a b l e ( g l y c e r o l , f r u c t o s e , i n o s i t o l ) and n o n u t i l i z able (glucose, s o r b i t o l ) carbon sources were added to f r o z e n thawed c e l l suspensions a g i t a t e d under a i r . As shown i n Table I I , s e v e r a l C-sources were able to p a r t i a l l y s t a b i l i z e the enzyme. Table I I . E f f e c t of Carbon Sources on i n v i v o S t a b i l i t y of GS Synthetases

Atmosphere

Additive

Nitrogen Air Air Air Air Air Air

None None 1% G l y c e r o l 1% Fructose 1% Glucose 1% I n o s i t o l 1% S o r b i t o l

a

O v e r a l l synthetase specific activity "g GS 1 Lmg-hr J

Γ

7.8 0.5 7.0 5.4 3.6 3.1 0.4

I n c u b a t i o n with shaking f o r 90 min at 250 rpm,

Activity (%)

100 6 90 69 46 40 5 37°C.

The best s t a b i l i z a t i o n was obtained with g l y c e r o l , followed by f r u c t o s e , glucose, and i n o s i t o l i n decreasing order of e f f e c t . S o r b i t o l was i n c l u d e d i n t h i s experiment s i n c e i t i s not a source of carbon f o r growth but i s known to s t a b i l i z e at high concentra t i o n s ("20%) some c e l l - f r e e enzymes. In our case i t was i n a c t i v e . The r e s u l t s suggest that in v i v o synthetase s t a b i l i z a t i o n by c a r bon sources i s l i n k e d with the u t i l i z a t i o n of these compounds by the c e l l s under the c o n d i t i o n s of a e r a t i o n used here. Studies by A s a t a n i and Kurahashi ÇI) have revealed that there e x i s t systems of a c t i v e uptake f o r both g l y c e r o l and f r u c t o s e i n Β· b r e v i s . The uptake of g l y c e r o l i s c o n s i d e r a b l y f a s t e r than that of f r u c t o s e and they are both c a t a b o l i z e d through the g l y c o l y t i c (EMP) path way. The same workers have reported that glucose, while not able to support growth as sole C-source f o r B^. b r e v i s , can d i f f u s e



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p a s s i v e l y and very slowly i n t o the c e l l s and, once i n s i d e , i t can be c a t a b o l i z e d through the EMP pathway. In our frozen-thawed c e l l s we are e v i d e n t l y experiencing increased p e r m e a b i l i t y of the c e l l membrane to glucose as a r e s u l t of the freeze-thaw treatment. I n o s i t o l has been found by Vandamme and Demain (14) to be a poor source of carbon f o r t h i s organism. Our r e s u l t s i n d i c a t e that there i s a d i r e c t r e l a t i o n s h i p between magnitude of the s t a b i l i z a t i o n e f f e c t brought about by the C-sources i n question and t h e i r u t i l i z a t i o n . This c o r r e l a t i o n favors the idea of a metabolic b a s i s f o r the observed s t a b i l i z a t i o n in v i v o . I t i s p o s s i b l e that the c e l l s of B^. b e v i s are starved f o r a C- (and energy) source at the point of the in v i v o i n a c t i v a t i o n and that such n u t r i e n t s must be added to r e t a r d the i n a c t i v a t i o n process o c c u r r i n g under a i r . An experiment was c a r r i e d out i n which 1% g l y c e r o l was added to a c e l l suspension a f t e r i t had been subjected to i n a c t i v a t i n g con d i t i o n s ( i . e . , shaking i n a i r a 37°C). Table I I I shows that the i n a c t i v a t e d synthetase i n these c e l l s f a i l e d to be r e a c t i v a t e d by g l y c e r o l , demonstrating that g l y c e r o l i s e f f e c t i v e i n r e t a r d i n g synthetase i n a c t i v a t i o n i n a i r but cannot restore a c t i v i t y a f t e r i t has been l o s t . Table I I I . F a i l u r e of Carbon Source to R e - a c t i v a t e i n a c t i v a t e d GS Synthetases

Atmosphere

Additive

Nitrogen Air Air

None None None, i n i t i a l l y ; 1% g l y c e r o l added after inactivation

a

O v e r a l l Synthetase specific activity Ug GS L mg'hr J

Γ

Τ

10.3 0.4 0.4

I n c u b a t i o n with shaking f o r 90 min at 250 rpm,

37°C.

Given the immediate i m p l i c a t i o n s of a simple step, such an a d d i t i o n or p u l s i n g of carbon source f o r prolonged p r e s e r v a t i o n of the synthetase a c t i v i t y i n whole c e l l s , f u r t h e r experiments were conducted over s e v e r a l hours, to examine the time course of the enzyme a c t i v i t y i n the presence of s e v e r a l l e v e l s of g l y c e r o l . Figure 3 shows that p r o g r e s s i v e l y higher l e v e l s of carbon source from 1 to 4% are able to p r o p o r t i o n a t e l y r e t a r d the i n a c t i v a t i o n



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process. In the same experiment, the d i s s o l v e d oxygen (D.O.) conc e n t r a t i o n s were a l s o monitored i n t e r m i t t e n t l y i n the v e s s e l s cont a i n i n g v a r i o u s g l y c e r o l l e v e l s . I t can be seen ( F i g . 3) that there e x i s t s a c o r r e l a t i o n between enzyme a c t i v i t y , l e v e l s of Csource and D.O. concentration. The r e t e n t i o n of enzyme a c t i v i t y i s g e n e r a l l y compatible with "lower" D.O. l e v e l s . T h i s could mean that the lower D.O. l e v e l s are maintained, at l e a s t p a r t l y , through increased r e s p i r a t o r y a c t i v i t y i n the e a r l i e r domains of such time-course experiments, and t h a t , i n the presence of C-source such as g l y c e r o l , r e s p i r a t o r y a c t i v i t y d e p l e t i n g the D.O. i n the c e l l suspension i s maintained f o r a longer period of time thus preventing premature 0 -mediated i n a c t i v a t i o n . I t i s a l s o p o s s i b l e that the catabolism of the C-source i s required to continue f u r n i s h i n g both ATP (a co-substrate and p o s s i b l e p o s i t i v e a l l o s t e r i c e f f e c t o r of the enzyme's s t r u c t u r a l i n t e g r i t y ) as w e l l as reduced p y r i d i n e n u c l e o t i d e s , NADH/NAD(P)H (whose l i k e l y r o l e i n r e t a r d i n g i n a c t i v a t i o n i s discussed below). On the other hand, c o r r e l a t i o n of D.O. with remaining l e v e l of enzyme a c t i v i t y i s a s i g n i f i c a n t r e s u l t not only because i t immediately suggests ways of engineering i n t e r v e n t i o n (e.g., through a c o n t r o l l e d regime of D.O. during production phase) i n a batch a n t i b i o t i c fermentation to achieve optimal GS production, but a l s o because i t might suggest a general model of regulatory a c t i o n of oxygen upon enzymes i n aerobic microorganisms producing secondary metabolites. A case i n point i s the secondary metabolic process of cyanogènesis (producion of HCN) by Pseudomonas species described by C a s t r i c and h i s colleagues £ 2 , 3 2 which i s a l s o subject to oxygen-mediated i n a c t i v a t i o n . I t i s p o s s i b l e that oxygen becomes harmful to b i o s y n t h e t i c enzymes of secondary metabolism i n o b l i g a t e aerobes at the point of C- and energy source d e p l e t i o n p a r t l y by v i r t u e of the f a c t that D.O. l e v e l s are kept low only during aerobic C-source catabolism. A p o s s i b l e mechanistic scheme that would l i n k d i r e c t l y the C-source catabolism with the molecular events c o n s t i t u t i n g the i n a c t i v a t i o n of the sytnthetase v i a oxygen, could be formulated as f o l l o w s : the a c t u a l target of the i n a c t i v a t i o n could w e l l be l a b i l e s u l f h y d r y l (SH-) a c t i v e groups on the enzyme s u r f a c e . According to Laland and Zimmer (8) i t seems that at l e a s t seven SH- groups are d i r e c t l y required f o r a c t i v i t y and p o s s i b l y some more are involved i n ensuring the s t r u c t u r a l i n t e g r i t y of the enzyme macromolecule. I t i s then p l a u s i b l e to assume that while such SH- groups are s u s c e p t i b l e to a u t o x i d a t i o n by D.O. the presence of an a c t i v e l y metabolized C-source could lead to a low i n t r a c e l l u l a r redox p o t e n t i a l which i s conducive to maintaining the i n t r a c e l l u l a r p r o t e i n c y s t e i n e residues i n the SH- ( i . e . , reduced) s t a t e . Conceivably NAD(P)H produced under such cond i t i o n s may be coupled to regeneration of SH- groups on the enzyme v i a t h i o l interchange ( n u c l e o p h i l i c s u b s t i t u t i o n ) with g l u t a t h i o n e (reduced) or lipoamide or t h i o r e d o x i n . For example, g l u t a t h i o n e , the most abundant i n t r a c e l l u l a r t h i o l i n most types of c e l l s may interchange with the enzyme as shown i n Figure 4.


2



AGATHOS A N DDEMAIN

0

Optimizing

1

2 time

Fermentation

3 4 [ hr ]

5

Figure 3. Time course of specific activity of G S synthetase under air in the presence of various levels of utilizable carbon source (glycerol). Shown also are the corre sponding levels of dissolved oxygen (DO). Standard conditions for inactivation: agitation at 250 rpm, 3TC. Glycerol levels: M, 4%; A, 2%; Δ, 1%; O, 0%.

S Enz'i + GSH S

SH Enz' -h GSSG ^SH

^ ^ GSH reductase

n a d p*

Stabilizer compound

nad ρ

—>

—>

—>

—>

H

Τ

Figure 4. Hypothetical scheme illustrating the action of a stabilizer compound (e.g., utilizable carbon source) on an enzyme containing O -sensitive SH— groups. GSH, reduced glutathione; GSSG, oxidized glutathione. t


64


I n a c t i v a t i o n Through Autoxidation o f S u l f h y d r y l Groups. To examine f u r t h e r the p o s s i b i l i t y that SH- groups are o x i d i z e d and i n a c t i v a t e d by 0 i n v i v o we checked the e f f e c t of an organic t h i o l (DTT) on the a c t i v i t y of the synthetase i n long-term e x p e r i ments. Because hydrogen peroxide and superoxide f r e e r a d i c a l s are generated during most o x i d a t i o n s of organic t h i o l s , such as DTT, i n a i r (10), such reagents cannot be used as a d d i t i v e s i n f o r c i b l y aerated c e l l suspensions, but only under N gas. Results from long-term incubations are shown i n Figure 5. The experiment was c a r r i e d out i n a way which only assumes a l a c k of forced 0 transf e r to a l l c e l l suspensions [both experimental (with DTT) and c o n t r o l s (without DTT)]. This was achieved through a p a r t i a l atmosphere of N gas i n rubber-capped v i a l s , which, however, permit the gradual entry of a i r l a t e r i n the i n c u b a t i o n . DTT at 15 mM preserved p r a c t i c a l l y the e n t i r e i n i t i a l a c t i v i t y of the enzyme during 16 hours of incubation, whereas by that time the c o n t r o l v e s s e l (no DTT) had l o s t a l l enzyme a c t i v i t y . I t appears as i f the slow r e t u r n to a e r o b i o s i s i n the c o n t r o l v e s s e l s (no DTT) lead to a u t o x i d a t i o n and i n a c t i v a t i o n f a s t e r than i n the v e s s e l s cont a i n i n g DTT. S t i l l another approach to determine the p o s s i b l e involvement of SH- groups as t a r g e t s of o x i d a t i v e i n a c t i v a t i o n of the enzyme was to incubate c e l l s , having already been exposed to various degrees of 0 ~dependent i n a c t i v a t i o n , under N i n the presence of d i t h i o t h r e i t o l (DTT). I f the hypothesis i s c o r r e c t then the synthetase a c t i v i t y should increase under these c o n d i t i o n s . I t i s of course assumed that the postulated SH- a u t o x i d a t i o n has led to s u l f u r d e r i v a t i v e s of the enzyme, which are amenable to r e d u c t i o n back to the t h i o l s t a t e (SH-) i n the presence of excess d i t h i o t h r e i t o l . An i n i t i a l experiment along these l i n e s i s shown i n Figure 6. I t can be noted that moderate r e a c t i v a t i o n was achieved even from s t a t e s that assume t o t a l l o s s of a c t i v i t y under air. In t h i s experiment a 45-minute a d d i t i o n a l incubation of the p r e v i o u s l y exposed c e l l suspensions was c a r r i e d out under N i n the presence of 15 mM DTT. The r e s u l t s of t h i s experiment s t r o n g l y i m p l i c a t e SH- groups on the synthetase as the a c t u a l t a r g e t s of the 0 ~dependent i n a c t i v a t i o n . 2

2

2


2

2

2

2

2

Conclusions I t seems that u t i l i z a b l e C-sources are able to r e t a r d i n v i v o i n a c t i v a t i o n of the synthetases of GS and i t a l s o appears as i f the i n a c t i v a t i o n i s brought about by a u t o x i d a t i o n of SH- groups on the enzyme complex. Moreover the l o s s of synthetase a c t i v i t y i s , at l e a s t p a r t i a l l y , r e v e r s i b l e through e x t e r n a l l y added d i t h i o threitol. These r e s u l t s throw considerable l i g h t on the process of i n v i v o i n a c t i v a t i o n of the b i o s y n t h e t i c enzyme complex c a t a l y z i n g the formation of GS i n B^. b r e v i s . We b e l i e v e that these f i n d i n g s not only suggest f r e s h ways of optimizing the GS ferment a t i o n by c o n t r o l l i n g the r a t e of synthetase i n a c t i v a t i o n , but


3.

AGATHOS A N DDEMAIN

Optimizing

65

Fermentation


%

0

4

8

12 16 time [ hr J

20

24

Figure 5. Long-term incubation of frozen-thawed cells of B . brevis under N in the presence and absence of an organic thiol (DTT). Standard conditions: agitation at 250 rpm, 37°C. Key: O, N present; Δ, N + 15 mM DTT present. t

t

a

% S.a.

Figure 6. Effect of DDT on specific activity of GS synthetase at various degrees of prior exposure to standard inactivating conditions (air, 250 rpm, 37°C). The subsequent incubation of the cell suspensions was under N in the presence of 15 mm DTT at 37°C for 45 min. Key: O, air only; Δ, air, then N, + 15 mM DTT. g



66


a l s o that such knowledge should be d i r e c t l y a p p l i c a b l e to the e f f i c i e n t production of c l o s e l y r e l a t e d a n t i b i o t o c s of commerical s i g n i f i c a n c e , such as b a c i t r a c i n . The methodology o u t l i n e d i n t h i s i n v e s t i g a t i o n and p o s s i b l y s p e c i f i c f i n d i n g s may a l s o be exendable to d i f f e r e n t processes c a l l i n g f o r i n v i v o s t a b i l i z a t i o n of b i o s y n t h e t i c enzymes, p a r t i c u l a r l y o f oxygen-labile enzymes i n a e r o b i c organisms. Furthermore a fundamental understanding o f the i n v i v o i n a c t i v a t i o n of such synthetases appears to hold the pro mise f o r a more r a t i o n a l i n t e r v e n t i o n on the part of the biochemi c a l engineer i n making e f f i c i e n t use o f the b i o s y n t h e t i c p o t e n t i a l of a wide v a r i e t y of i n d u s t r i a l l y s i g n i f i c a n t microorganisms. While the immediate b e n e f i t from such an understanding would be an extended production phase i n batch fermentations, t h i s knowledge should a l s o be c e n t r a l i n designing a l t e r n a t i v e w h o l e - c e l l pro cesses (e.g., immobilized c e l l r e a c t o r s ) , i n which keeping the enzymes " a l i v e " i n t h e i r n a t i v e i n t r a c e l l u l a r micro-environment could be the optimal approach i n the o p e r a t i o n a l t r a n s i t i o n from a batch t o a continuous process. Acknowledgements We thank the Greek M i n i s t r y of Coordination and Planning f o r a NATO Science Fellowship f o r S. N. Agathos.

Literature Cited 1. Asatani, M.; Kurahashi, K. J. Biochem. (Tokyo) 1977, 81 813-822. 2. Castric, P.Α.; Ebert, R.F., Castric K.F. Current Microbiol. 1979, 287-292. 3. Castric, P.Α.; Castric K.F.; Meganathan, R. "Hydrogen Cyanide Metabolism," Conn, E . E . , Knowles, C . J . , Vennesland, B., Wissing F . , Eds.; Academic Press: New York, in press. 4. Friebel, T . E . ; Demain, A.L. J. Bacteriol. 1977a, 130, 1010-1016. 5. Friebel, T . E . ; Demain, A.L. FEMS Microbiol. Lett. 1977b, 1, 215-218. 6. Gornall, S.G.; Bardawill, C . J . , David, M.M. J. Biol. Chem. 1949, 177, 751-766. 7. Holzer, H. Trends Biochem. Sci. 1976, 1, 178. 8. Laland, S.;, Zimmer, T. Essays Biochem. 1973, 9, 31. 9. Matteo, C.C.; Glade, M.; Tanaka, Α.; Piret, J.: Demain, A.L. Biotechnol. Bioeng. 1975 17, 129-142. 10. Misra, H.P. J. Biol. Chem. 1974, 249, 2151. 11. Switzer, R.L. Ann. Rev. Microbiol. 1977, 31, 135. 12. Tomino, S.; Yamada, M.; Itoh, H.; Kurahashl, K. Biochemistry 1967, 6, 2552. 13. Tzeng, C H . ; Thrasher, K.D.; Montgomery, J.P.; Hamilton, B.K.; Wang, D.I.C. Biotechnol. Bioeng., 1975, 17, 143.


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Fermentation

14. Vandamme, E . J . ; Demain, A.L. Antimicrob. Agents Chemother. 1976, 10, 265-273. 15. Waindle, L.M.; Switzer, R.L. J. Bacteriol. 1973, 114, 517. June 1, 1982


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Optimization of Fermentation Processes Through the Control of In Vivo

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