ornithine transcarbamylase , arginase , and asparaginase (aspartyl transferase ... Aminohydrolase activity of asparaginase was assayed conductimetrically.
Biotechnologg Letters Vol 7 No 9
673-678
(1985)
GROWTH REQUIREMENTS AND THE ESTABLISHMENT OF A CHEMICALLY DEFINED MINIMAL MEDIUM IN
ZYMOMONAS MOBILIS
Irene Galani I , Constantin Drainas 2 and Milton A. Typas I * 1.Department of Biochemistry, Mollecular and C e l l u l a r Biology, and Genetics, Athens U n i v e r s i t y , Panepistemiopolis, Athens T.K. 15701 , Greece . 2.Department of Chemistry , Division of Organic Chemistry and Biochemistry, University of 1oannina , 1oannina , Greece . SUMMARY A chemically defined minimal medium which f u l f i l s the growth requirements of d i f f e r e n t Zymomonas mobilis s t r a i n s has been established . The k i n e t i c s of ethanol production o$---the strainsATCC 10988 , CUt , CP4 and 11163 grown on the minimal medium at d i f f e r e n t glucose concentrations were measured . All s t r a i n s produced ethanol at rates s i m i l a r to those on complete medium . The minimal medium described is s u i t a b l e to study spontaneous metabolic d e f i c i ciencies and r e g u l a t i o n of enzyme a c t i v i t i e s in' Z.mobilis . INTRODUCTION The bacterium Zymomonas mobilis has a t t r a c t e d considerable a t t e n t i o n f o r i n d u s t r i a l ethanol production since i t was discovered that i t possesses a number of advantages over the t r a d i t i o n a l yeast process~ . These include higher ethanol p r o d u c t i v i t i e s and increased ethanol y i e l d s (Swings and De Ley, 1977 ; Rogers et a l , 1979 ; Karsch et a l , 1983) . Although the biology of Z.mobilis has been described (Swings and De Ley, 1977) and a large number of researchers work today with Z.mobilis , l i t t l e is r e a l l y known about the physiology and t h e formal genetics of the bacterium . Studies in these areas have been considerably delayed by the lack of a chemical]y d e f i n e d minimal medium . Belaich and Senez (1965) f i r s t reported a chemically defined minimal medium and discussed the possible vitamin requirements of Z.mobilis . However, the growth requirements of a number of s t r a i n s were not p r e c i s e l y studied . Later r e p o r t s suggested the a d d i t i o n of NH~CI to overcome vitamin requirements (Forrest , 1969), or the a d d i t i o n of amino acids with some vitamins and baSes (Van Pee et a l , 1974 ; Goodman et a l , 1982) or the addition of yeast e x t r a c t in the medium (Skotnicki et a l , 1981) . This i n f o r m a t i o n : b r D u g h t a b o u t a great controversy about the growth requirements of Z.mobilis and the need to establish a chemically defined minimal medium . The c o n t r i b u t i o n of the present work is the establishment of a chemically defined minimal medium and the basic growth and ethanol production of w i l d - t y p e and mutant s t r a i n s of Z.mobilis on t h i s medium . MATERIALS AND METHODS Ss : Z.mobilis s t r a i n s CP4 , 11163 (NCIB) , ATCC 10988 and i t s d e r i v a t i v e C01-wh~ch lacks a t - l e a s t one plasmid species with concomitant reduced ethanol production (Drainas et a l , 1984) were used . Media : A medium containing 2% glucose , I% yeast e x t r a c t , 0.1% KH2PO4, 0.1% ~NR~2SO4and 0.05% MgS04 (added a f t e r autoclaving) served as complete medium (CM) . Ihe chemically defined minimal medium (MM) used throughout t h i s work
673
contained : glucose 2% , (NH4)2S04 or NH4Cl 0.1% , KH2P04 0.1% , K2HP04 0.1%, MgSO4.7H20 0.05% , NaCI 0.05% and pantothenate 0.5 g.ml - ~ . S o l i d media were prepared w i t h the a d d i t i o n of 2% agar . The amount of glucose f o r growth rate e s t i m a t i o n s was 2% , 10% and 20% as shown in the t e x t o Both l i q u i d and s o l i d c u l t u r e s were incubated in the dark at 30~ ~ The l i q u i d c u l t u r e s were grown without agitation ~ w167 : L i q u i d media c o n t a i n i n g the a p p r o p r i a t e glucose c o n c e n t r a t i o n were inoculated w i t h fresh inocula of Z . m o b i l i s s t r a i n s to give I0 v c e l l s per ml . The growth rates were estimated by reading the o p t i c a l d e n s i t y of the c u l t u r e s every I-2 hours in a Perkin Elmer 295 Photometer a t 600 n m . Growth on s o l i d media was scored only when c l e a r colonies were grown or c o n f l u e n t growth had occurred f o l l o w i n g r e p l i c a p l a t i n g , s t r e a k i n g or v i a b l e count measurements . : As described p r e v i o u s l y (Drainas et a l , 1984) . mmPem igm_e _ 11_ mm _m mm : Cells o f Z . m o b i l i s s t r a i n s were grown up to l a t e exponential phase , as described above , in 100 ml l i q u i d c u l t u r e s . The c e l l s were harvested by c e n t r i f u g a t i o n at 10,O00rpm f o r 10 min. The p e l l e t was washed once in 20mM T r i s - h y d r o x y - m e t h y l - a m i n o methane ( T r i s - H C I ) pH 8 , and resuspended in 5ml 20mM Tris-HCl ph 8 c o n t a i n i n g ImM d i t h i o t h r e i t o l . The suspension was c h i l l e d and sonicated f o r I min. in an MSE soniprep 150 at 26 microns . The preparation was allowed to cool in an ice bath and s o n i c a t i o n was repeated as above . Cell d e b r i s were removed by c e n t r i f u g a t i o n a t 37,000 g f o r 20 min. using an SS34 r o t o r in a Sorval RC5B c e n t r i f u g e at 4~ . The supernatant was c o l l e c t e d and used in enzyme assays as the c e l l - f r e e e x t r a c t . Enz~me_assa~w : The enzymes glutamine synthetase , NADP-glutamate dehydrogenase, o r n i t h i n e transcarbamylase , arginase , and asparaginase ( a s p a r t y l t r a n s f e r a s e a c t i v i t y ) were assayed as described by Pateman (1969) , Kinghorn and Pateman (1973) , Davis (1965) , Davis and Mora (1968) and Drainas et al (1977) r e s p e c t i v e l y . Aminohydrolase a c t i v i t y of asparaginase was assayed c o n d u c t i m e t r i c a l l y f o l l o w i n g the change of c o n d u c t i v i t y due to the production of ammonia and a s p a r t a t e from the h y d r o l y s i s o f asparagine (Drainas and Drainas , sent f o r publication) . RESULTS AND DISCUSSION The combinations of various i n o r g a n i c n i t r o g e n and sulphur sources and trace elements which were examined f o r the establishment of a chemically defined MM are shown in Table I . All combinations (128) contained 2% glucose as the carbon source . Complexity increased by adding s u c c e s s i v e l y a supplement to the previous medium . The simple media contained o n l y one i n g r e d i e n t ' The r e s u l t s show t h a t NH4CI or (NH~)~SO~ served as the best n i t r o g e n sources and MgSO, as the best sulphur source . All s t r a i n s f a i l e d to grow on NaNO~ or H~S as the sole n i t r o g e n and sulphur sources r e s p e c t i v e l y . The combinations 3+5§ and 4+5+9+10+12 initially showed i d e n t i c a l growth curves when compared to CM . However , samples from l a t e exponential phase o f s t r a i n s CUt and 10988 showed impaired growth when subcultured i n t o the same media ( f i g . I ) . This i n d i c a t e d a growth requirement of the organism . Amino acids and/or v i t a m i n s were added to the above media in a l l p o s s i b l e combinations (Table I ) . Following growth e s t i m a t i o n s f o r a successive s e r i e s o f at l e a s t 8 s u b c u l t u r i n g s in the same medium , s t r a i n s 10988 and CUt were found to r e q u i r e pantothenate , whereas s t r a i n s 11163 and CP4 were v i t a m i n independent . The optimum c o n c e n t r a t i o n of pantothenate was estimated and t h u s , a s t r i c t l y chemically defined MM f o r a l l s t r a i n s of Z . m o b i l i s was e s t a b l i s h e d . This MM contains the f o l l o w i n g ( g . l - I ) : glucose 20.0 ; (NH,}~SO~ or NH~CI 1.0 ; K~HP04 1.0 ; KH~PO~ 1.0 ; MgSO~7H~O 0.5 ; NaCI 0.5 ; pantothenate 0.5 x 10 -~
674
1 . Construction
TABLE
of a chemically
defined
.Inorganic Sulphur sources
Nitrogen sources
Phosphorous Trace sources elements 11. FeSO47H2D I. NAN03 5. MgSO47HzO 9.KaHPO. 2. NAN02 6.(NH.)aSO. IU.KH2P04 12. N a C I 13. MnS04 3. NH~CI 7. NaaSO3 14. MnC]2 4.(NH4)2SD 4 8. HaS 15. CuS04 16. CoSO, 17. ZnSO, 18. Na2MoO~
minimal medium for Z.mobilis
Organic Amino acids Vitamins (20 ug.ml - I ) (O.1-1pg.ml") gly,i]e,leu nicotinic acid cys,ser,thr nicotinamide pro,hyp,his biotin vaI,tyr,met pantothenate phe,a]a,glu p-aminobenzoic trp,arg,lys acid asp,asn,gln f o l i c acid lipoic acid
B~,B2,B~,B~a
Glucose (2%) was used as the carbon source in all combinations . Simple combinations contained only one of the numbered chemicals. Complexity was increased by the separate addition of one inorganic source and/or one sulphur source to each of the trace elements . To the combinations 3+5+9+10+12 and 4+5+9+10+12 each of the vitamins and~or amino acids were added i n all possible con~binati ons
FIGURE
,
0.1
i . The growth of Zymomonas mobilis ATCC 10988 in the potentially best chemically defined minimal media (a,b,d,e,f) compared with that of the complete medium (c) .
,o
~tlH3~ (~ -
o
,t
d
TIME ,t(h)
. . . . . . . TIME, t ('h)
8" "o,
0.1
m--m : first subculturin g , e--e : second subculturing . All media contained 2% glucose and chemicals of Table 1 as follows : a= 4+5+9+10 ; b= 3+5+9%10+12 d = 4+5+9+12 ; e = b or f with the addition of pantothenate and f= 4+5+9+10+12 .
675
Table 2 shows the growth requirements of s t r a i n s 10988 and CUI in r e l a t i o n to the pantothenate auxotrophy . Both s t r a i n s grow normally in presence of valine . No growth i s observed on medium which lacks pantothenate or on medium supplemented w i t h pyruvate . TABLE 2 . Growth of Zvmomonas mobi!is strains on the chemically defined MM, CM, and ap'proprialely supplemented media
I~
CM
I*
2*
I*
MM-pan 2"
I*
MM-pan+val MM-pan+pyr
2*
I*
2"
2*
I*
10988 CUI
0.23 0.28 0.24 0 . 3 3
0.26 0.33 0 . 0 2 0 . 0 4 0.05 0.1 0.24 0.34 0.025 0.065 0.09 0 . 2 2
0 . 0 2 5 0.03 0 . 0 3 0.05
11163
0.24 0 . 3 8
0 . 2 6 0.4
0.12
D.12
CP4
0.23 0 . 3 0
0.25 0.34
0 . 1 6 0 . 1 9 0.18 0 . 3 0
0 . 2 1 D.22 0.4
0.24
0 . 1 7 0.32
*I = growth estimated after i day , 2= growth estimated after 8 days. Growth w a s estimated by O.D. measurements and are given as the mean of five replicas. The standard devia%ion was less Than 9 0.005 ~ pan= pantothenate (0.Spg.ml -I) val= valine (2.5-5 ~g.ml -I) , pyr= pyruvate (0.25-0.5 g.mi -I) .
The k i n e t i c s of ethanol production of the s t r a i n s 10988 , CUT , CP4 and 11163 grown on the described MM in the presence of various i n i t i a l glucose concentrations were estimated . AICC 10988 , 11163 and CUI produce ethanol at s i m i l a r rates on CM (Drainas et a l , 1984) . CUT shows the same reduction of ethanol production in the presence of 101 and 20% i n i t i a l glucose ( f i g . 2) as described f o r CM (Drainas et a l , 1984) . Similar r e s u l t s were obtained f o r the CP4 s t r a i n which appears to be the most productive s t r a i n (Table 3) . This r e s u l t i s consistent with the reported p r o d u c t i v i t i e s of CP4 grown on CM (Skotnicki et a l , 1981) I
3O0f ZOOI
I
II1
4
~
I
J
~
P
9
I
I
I
I
a
FIGURE a~a a
1o01.
i
8
Kinetics of ethanol production in Zymomonas mobilis strains on MM under various initial glucose concentrations .
I
70 ~
so
~
30
2
9"~ 26 8
g
9 ,O 9
A
A
I, D ~
~
s
I"
b
9 ,s 9 : ATCC 10988
A
I&
1
8
A
I
.
16
;
o , A , n : CUT
~
..A
glucose glucose glucose
b~ A O
.
: 2% :10% :20%
9
i;o
24
OD O~ ,
:,o:.
9
32
B
m
40
48
|
,'
56
*
64
Timehours Table 3 shows the y i e l d s of ethanol production in c o r r e l a t i o n with the amounts of glucose consumed and t o t a l protein produced at the end of the growth . Ethanol y i e l d s of 10988 , 11163 and CUT on MM in the presence of 2%,
676
TABLE
3 . Ethanol production in cells glucose concentrations
%Glc
mg protein
grown on M M under v a r i o u s
mg EtOH/mg 20.11 1.0 15.9• 40.7~1.0 28.1 ~1.0
mg Glc/mg
initial
mg EtOH/mg Glc
ATCC 10988 CUI CP4 11163
2 2 2 2
1.79 2.44 1.98 2.29
50.1 • 1.0 38.7• 92.5_+1.0 89.8• 1.0
0.40 0.41 0.44 0.31
ATCC 10988 CUI CP4 11163
10 10 10 10
1.84 0.90 1.57 1.54
130.7 16.6 248.4 126.3
• i 0.5 • !0.5
302.1 200.0 496.8 280.0
• • 2.0 i2.0 •
0.43 0.08 0.5[) 0.45
ATCC 10988 CUI CP4 11163
20 20 20 20
1.52 0.20 1.78 0.25
103.9• 9.0 10.2 236.9 • 105.5 •
346.5 225.0 526.0 340.3
• • • 12.0
0.30 0.04 0.45 0.31
G r o w t h time : 22h on 2 % glucose , 66h on 10% and 20% glucose . mg of e t h a n o l (ETOH) and glucose (Glc) are the mean of six replicas . The standard deviation was less than 0,02 .
TABLE
q .
Enzyme activities of Zymomonas nitrogen sources . Growth conditions GS NH4CI NAN0, L-glutamine L-g] utamate L-asparagine L-aspartate (~M .
1.228 0.294 2.244 0.960 2.000 I. 389 0.495
Inoculum Cultures
mobilis ATCC 10988 on various
Enzyme activities OTC Asn-ase 0.118 0.041 0. 108 0.061 0.104 0.079 0.046
0.190 0.178 0.185 0.210 0.326 0.215 0.215
: 5ml liquid culture grown on C M washed twice w i t h M M w i t h o u t n i t r o g e n . : 5 0 m l M M w i t h o u t n i t r o g e n , s u p p l e m e n t e d with 1 0 m M o f the appropriate nitrogen source N starved cultures : Cells grown on ammonia were harvested at late e x p o n e n t i a l phase , washed twice in -N m e d i u m and r e s u s p e n d e d in -N medium for three hours . S = glutamine synthetase , ~moles glutamate hydroxymate x min -I x mg protein -l. OTC = ornithine t r a n s c a r b a m y l a s e , D moles citrulline x min -I x mg p r o t e i n " I Asn-ase = aminohydrolase activity of a s p a r a g i n a s e , ~moles asparagine x min -i x mg protein -i . The standard d e v i a t i o n obtained from six replica~ was less than 0.010 .
677
10% and 20% i n i t i a l glucose are s i m i l a r to y i e l d s found f o r CM (Drainas et a l , 1984) . CP4 appears to have the best y i e l d . These data s t r o n g l y i n d i c a t e t h a t the e s t a b l i s h e d MM does not a f f e c t the ethanol p r o d u c t i v i t i e s and y i e l d s nf most of Z.mobilis s t r a i n s tested . The MM described in t h i s work is a!so s u i t a b l e to study the r e g u l a t i o n of enzyme a c t i v i t i e s in Z.mobilis . B i o s y n t h e t i c and c a t a b o l i c enzymes of amino a c i d metabolism were assayed under various growth c o n d i t i o n s i n c l u d i n g the a d d i t i o n of amino acids and inorganic n i t r o g e n s a l t s as sole N sources (Table 4). S p e c i f i c a c t i v i t i e s of glutamine synthetase , o r n i t h i n e transcarbamylase and asparaginase were estimated . A c t i v i t i e s of glutamate dehydrogenase , glutamate synthase , a s p a r t y l t r a n s f e r a s e a c t i v i t y of asparaginase and arginase were not detectable by the used methodology . The lack of arginase a c t i v i t y , however , is c o n s i s t e n t with the i n a b i l i t y of Z.mobilis to u t i l i z e a r g i n i n e as sole N source . As i t appears on Table 4 , there are i n d i c a t i o n s of i n d u c t i o n and repression c o n d i t i o n s of enzyme a c t i v i t y . Glutamine synthetase and o r n i t h i n e transcarbamylase a c t i v i t i e s are higher when c e l l s grow on the amino acids included on Table 4 , as well as on ammonia , but they are low when c e l l s grow on n i t r a t e and CM . Asparginase appears to be higher when c e l l s grow on asparagine as sole N source . These r e s u l t s support the s u i t a b i l i t y of the present MM in s t u d i e s of the r e g u l a t i o n of amino acid metabolism in Z.mobilis . Acknowledgement : This work was supported by the National Programme f o r Research and Technology (PAET 83) , M i n i s t r y of Research and Technology and the European Commission grants GBI-3-O9~-GR , GBI-3-O99-GR (Biomolecular Engineering Program). REFERENCES Belaich ,J.P. and Senez ,J.C. (1965) . J . B a c t e r i o l . 89 , 1195-1200 Davies ,R.H. (1965) . Biochim.Biophys.Acta 107 , 54-58 Davies ,R.H. and Mora ,J. (1968) . J . B a c t e r i o l . 96 , 383-388 Drainas ,C., Kinghorn ,J.R. and Pateman ,J.A. (1977). J . g e n . M i c r o b i o l . 98, 493-501 Drainas ,C., S l a t e r ,A.A., Coggins , L . , Montague , P . , Costa ,R.G. Ledingham , W.M. and Kinghorn ,J.R. (1983) . B i o t e c h n o l . L e t t . 5 , 405-408 Drainas ,C., Typas ,M.A. and Kinghorn ,J.R. (1984) . B i o t e c h n o l . L e t t . 6 , 37-42 F o r r e s t ,W.W. (1969) . Microbial growth . In : The 19th Symposium of the SQciety f o r General Microbiology , P.Meadow and S . J . P i r t ,eds. pp. 65-86 Goodman ,A.E., Rogers ,P.L. and Skotnicki ,M.L. (1982) . A p p l . E n v i r o n . M i c r o b i o l . 44 , 496-498 Karsch , T . , Stahl ,U. and Esser ,K. (1983) . E u r . J . A p p l . M i c r o b i o l . B i o t e c h n o l . 18 , 387-391 Kinghorn ,J.R. and Pateman ,J.A. (1973) . J . g e n . M i c r o b i o l . 78 , 39-46 Pateman ,J.A. (1969) . Biochem.J. 115 , 769-775 Rogers , P . L . , Lee ,K.J. and Tribe ,D.E. (1979). B i o t e c h n o l . L e t t . I , 165-170 Skotnicki ,M.L., Lee , K . J . , Tribe ,D.E. and Rogers ,P.L. (1981) . Appl.Environ.Microbiol. 41 , 889-893 Swings ,J. and De Ley ,J. (1977) . B a c t e r i o l . R e v . 41 , 1-46 Tonomura ,K., Kurose ,N., Konishi ,S. and Kawasaki ,H. (1982) . Agr.Biol.Chem.Tokyo 46 (11) 2851-2853 Van Pee ,W., Vahlaar ,M. and Swings ,J. (1974) .Acad.R.Sci.Outre-Mer (Brussels) Bull.Seances (2) , 2D6-211
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