Cloning and expression of a Bacillus α-glucosidase gene in Escherichia coli, Zymomonas mobilis and Pseudomonas putida

Journal of Biotechnology, 29 (1993) 189-203

189

© 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00

BIOTEC 00868

Cloning and expression of a Bacillus a-glucosidase gene in Escherichia coli, Zymomonas mobil& and Pseudomonas putida A n e t a T. Strzelecki

a, A m a n d a E. G o o d m a n b, Peter L. Rogers c and John M. Watson d

a Centrefor Early Human Development, Monash Medical Clayton, Victoria, Australia b School of Microbiology and Immunology, and c Department of Biotechnology, University of New South Wales, P.O. Box I, Kensington, N.S.W. 2033, Australia d CSIRO Division of Plant Industry, P.O. Box 1600, Canberra, ACT 2601, Australia

(Received 17 July 1992; accepted 31 August 1992)

Summary A gene bank constructed from the Gram-positive bacterium, Bacillus brevis 27-7 ( N R R L B-4389) was screened by a fluorimetric plate assay method to identify a-glucosidase production. A B. brevis-derived recombinant plasmid, which produced the highest levels of a-glucosidase activity and appeared stable in Escherichia coli, was designated pNSW350. E. coli RRl(pNSW350) was used to optimise conditions for the a-glucosidase assay in E. coli. The enzyme assay results showed that the properties of a-glucosidase in R R l ( p N S W 3 5 0 ) and in B. brevis were similar, with the p H range being broader in RR1(pNSW350) than in B. brevis. The cloned a-glucosidase gene from B. brevis was contained on a 4.5 kb B a m H I / H i n d I I I fragment of B. brevis D N A and was found to be induced in E. coli by maltose. The o~-glucosidase gene was subcloned into the vector pRK404 and the resultant broad-host-range recombinant plasmid was mobilised into Zym o m o n a s mobilis and Pseudomonas putida, a-Glucosidase activity was detected in Z y m o m o n a s and Pseudomonas transconjugants. Pseudomonas transconjugants were able to grow on maltose as the sole carbon source, while Z. mobilis transconjugants failed to grow. Enzyme assays showed that Pseudomomas transconjugants exhibited a 1.3-fold higher level of a-glucosidase activity and Correspondence to: A.T. Strzelecki, Centre for Early Human Development, Monash Medical Centre,

Clayton Road, Clayton, Vic. 3168, Australia.

190

Zymomonas transconjugants a 0.6-fold lower level of activity than found in B. brevis when grown in the presence of maltose, a-Glucosidase activity was induced by maltose in both Zymomonas and Pseudomonas transconjugants. a-Glucosidase; Maltose; Zymomonas mobilis; Bacillus brevis; Pseudomonas putida

Introduction

Increased industrial use of renewable raw materials is essential to conserve resources in individual areas and to produce biodegradable products to relieve pressure on the environment. Carbohydrates derived from starch should be regarded as a significant source of raw materials to substitute for petrochemical products. One current area of considerable interest is the production of ethanol from renewable resources for use as a fuel substitute. Starch is a major carbohydrate of plants and is one of the most widely distributed substances in nature. Starch, as hydrolysed corn, has been used for ethanol production in the US and pilot-scale cassava trials have been carried out in Australia. Traditionally, starch is liquefied by acid or enzyme hydrolysis for 60-80 h before saccharification (to glucose) followed by a batch process. The linear component of starch yields almost 100% maltose with enzymatic treatment (Brautlecht, 1953). Alpha-glucosidase can be used alone or in conjunction with glucoamlyase for the hydrolysis of maltose and maltodextrins to produce high-dextrose syrups or crystalline o-glucose (Kennedy et al., 1988). In combination with glucoamylase, a-glucosidase might be used to resolve the problem of the re-formation of glucosidic linkages that occurs when high concentrations of dextrose are produced. The final stage of starch metabolism in many microorganisms is catalysed by a-glucosidase. This enzyme hydrolyses the (1,4)-a- a n d / o r (1,6)-a-linkages in the oligosaccharides remaining after the degradation of starch by amylases. The use of such enzymes in starch hydrolysis, however, is costly (Kennedy et al., 1988). Traditionally, the microorganism used in glucose bioprocesses has been yeast. However, other microorganisms, in particular the bacterium Zymomonas mobilis, produce ethanol from glucose as efficiently as yeast. Z. mobilis is an anaerobic, aerotolerant, Gram-negative bacterium which is capable of degrading glucose, sucrose and fructose to ethanol plus carbon dioxide with few other by-products (Swings and DeLey, 1977). Z. mobilis can produce almost theoretical amounts of ethanol from glucose and has thus attracted much attention as a possible alternative to yeasts for producing industrial bioalcohol (Rogers et al., 1979). To be a commercially viable alternative, however, Z. mobilis must be productive whilst growing on cheap and readily available natural substrates. We are aiming to extend the range of degradable substrates of Z. mobilis to include maltose. As an initial step towards this goal we now describe the cloning and expression of an a-glucosidase gene isolated from the Gram-positive bac-

191 terium Bacillus brevis 27-7 (Wang et al., 1976) into the Gram-negative bacteria E. coli, Z. mobilis and P. putida.

Materials and Methods

Bacterial strains and plasmids. Escherichia coli strains used were RR1 (Bolivar et al., 1977) and HB101 (Boyer and Roulland-Dussoix, 1969). The Zymomonas mobilis strain used was ZM6100 (Goodman et al., 1984). The Pseudomonas putida strain was PP1-8 (Wong and Dunn, 1974). The Bacillus brevis strain was 27-7 (NRRL B-4389; Wang et al., 1976). The plasmids used as cloning vectors were pUCll8 (ApR; from Vieira and Messing, 1987) and pRK404 (TcR; Ditta et al., 1985). The mobilising plasmid pRK2013 (KmR)is described by Figurski and Helinski (1979). The plasmids pNSW350, pNSW355 and pNSW358 are described in the text. Media. E.coli strains were grown in Luria broth (Miller, 1972) with shaking at 37°C or on modified Kim and Wimpenny (KW) medium containing (per 1): (NH4)2504, 4 g; MgSOn.7HzO, 0.1 g; CaCI2.2H20, 0.1 g; yeast extract, 0.5 g; 1.64% FeEDTA, 2 ml; carboxymethyl cellulose (CMC), 5 g; and agar, 15 g. (Kim and Wimpenny, 1981). Antibiotics were added in the following concentrations (Ixg ml-1): ampicillin (Ap), 200 and tetracycline (Tc), 20. Z. mobilis strains were grown statically in rich medium (RM; Goodman et al., 1982) at 30°C or in minimal medium (BM; Goodman et al., 1982). Antibiotics were added in the following concentrations (~g ml-a): Tc, 20; trimethiprim (Tp), 10. P. putida (Rp R) strains were grown in LB at 30°C with shaking at 200 rpm or on KW plates. Antibiotics were added in the following concentrations (~g ml-a): Tc, 20 and rifampicin (Rp), 100. B. brevis cultures were grown in LB at 30°C in Erlenmeyer flasks and were Shaken at 200 rpm. Solid medium was LB containing Difco Bacto agar (15 g 1-1; Detroit, MI). DNA methods Plasmid DNA was isolated from 5 ml overnight cultures according to the sodium dodecyl sulfate (SDS)-alkaline method described by Nester (1981). DNA separation was achieved using a 20 cm horizontal gel apparatus. The agarose (SeaKem LE, FMC Corp.) concentration was 0.5% or 0.8% and gels were run at 3 V cm -1. Restriction enzymes and T4 DNA ligase were obtained from Boehringer-Mannheim and used according to the manufacturer's instructions. The transformation method used in this study has been described previously (Browne et al., 1984) and the conjugation method used was that described by Goodman et al., 1984. DNA was labelled by nick translation (Rigby et al., 1977) and Southern blot hybridisation was carried out using the method of Southern (1975) as modified by Smith and Summers (1980) to achieve bidirectional transfer.

192

Determination of protein For strains ZM6100, HB101, RR1, B. brevis and P. putida standard curves of absorption at 600 nm versus protein concentration, using the Folin-Ciocalteu reagent and the method reported by Lowry et al. (1951) were determined.

Alpha-glucosidase assay Alpha-glucosidase activity was estimated by measuring the p-nitrophenol (pNP) released from p-nitrophenyl-a-D-glucoside (pNPG; Sigma Chemical Co., USA). Each strain tested was grown overnight in the appropriate medium. The cells were centrifuged, washed and recentrifuged. The cells were then lysed and 1 ml of buffer was added to the lysed cells which were vortexed. One ml of lysed cells (or diluted lysed cells) was added to 1 ml of phosphate buffer (0.5 M K H z P O 4 " NaOH, pH 6.5) and then 1 ml of 0.1% pNPG was added to start the reaction. The assay was carried out at 50°C in a water bath without shaking. The reaction was stopped by the addition of 2 ml of 1 M NazCO 3. Standards consisted of various known concentrations of pNP in buffer, incubated at 50°C and stopped by the addition of NazCO 3. All samples were centrifuged to remove any cell debris before being read at 400 nm on a Pye Unicam SP6-550 spectrophometer. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 nmol of pNP per min. The limits of sensitivity were 0.1 mU ml-1 culture fluid for extracellular fractions and 0.01 m U m l -1 of culture for cell-associated fractions. The specific activity was defined as enzyme activity per mg protein.

4-Methylumbelliferyl-a-D glucoside (a-MUG) plate overlay method A plate assay method for the screening of Cellulomonas isolates for/3-glucosidase activity (J.M. Watson, unpublished) was adapted for use with a-glucosidase by replacing /3-MUG with a-MUG. Colonies were grown on KW or BM plates containing appropriate antibiotics, and in some cases IPTG (120 ixg ml-l), and maltose. The plates were incubated at either 30°C or 37°C, as appropriate, and colonies appeared the next day. Plates were left incubating for 3 d to allow some autolysis before being tested for enzyme activity. The plates were then overlayed with 0.7% agarose dissolved in phosphate buffer (0.05 M KH2PO4-NaOH pH 7.0) containing 0.4 mg ml-1 a-MUG. The overlay was allowed to set and the plates were then incubated at 37°C for 30 min to 6 h and examined under UV (302 nm) light. Colonies which possessed a-glucosidase activity exhibited fluorescence due to the production of 4-methylumbelliferone (MU).

Results

Cloning of an a-glucosidase gene from Bacillus brevis The B. brevis gene bank was constructed from B. brevis strain 27-7 from which the a-glucosidase enzyme had previously been isolated, purified and characterised (McWethy and Hartman, 1979). The vector pUC118 (Vieira and Messing, 1987)

193 was linearised with AccI, treated with calf alkaline phosphatase (CAP) and ligated, in an equimolar ratio, to ClaI-digested bacterial genomic DNA (AccI and ClaI produce complementary cohesive ends). Ligation of AccI and ClaI ends result in the loss of both enzyme recognition sites. The ligation mixture was transformed into E. coli RR1 and transformants were selected on LB.Ap20o plates. All transformants arising from the ligation mixture were pooled and stored at - 80°C in LB containing 80% glycerol. The gene bank of B. breuis DNA cloned in E. coli RRI contained an estimated 2-5 x 105 transformants. The AccI site in p U C l l 8 is in the 5' end of the lacZ a fragment and thus genes cloned into this site, in the correct orientation, may be expressed from the lac promoter. Screening the gene bank for a-glucosidase activity was achieved by a plate overlay method using the synthetic substrate 4-methylumbelliferyl-a-D-glucoside (a-MUG). This method only detects those colonies which possess a-glucosidase activity and, although E. coli can grow on maltose, it does not show any fluorescence using this method. The gene bank was diluted in saline phosphate buffer and plated onto KWAp medium (containing IPTG) for single colonies. Fifteen positive colonies were detected out of about 2.2 x 104 screened. Figure 1 shows positive E. coli colonies fluorescing under UV light after being tested by the a-MUG overlay method.

Characterisation of clones expressing a-glucosidase activity The 15 isolates showing a-glucosidase activity were tested further to determine the recombinant plasmid stability in each isolate and to quantitate the amount of enzyme produced. The isolates were purified by three successive single-colony isolations on KWAp IPTG plates and were then tested for a-glucosidase activity after three days incubation using the a-MUG overlay method. Of the initial 15 positive isolates tested, four showed fluorescence after 30 min, another four showed faint fluorescence after 4.5 h incubation while the remaining isolates no longer showed any fluorescent colonies. Only the four isolates showing fluorescence after 30 min were investigated further. To quantitate the amount of a-glucosidase enzyme produced by each of the four isolates, a standard method for a-glucosidase estimation was used (see Materials and Methods). The results are shown in Table 1. E. coli RR1 did not possess any detectable a-glucosidase activity and therefore provided an appropriate background in which to assay positive clones. The B. brevis strain 27-7, from which the gene bank was made, possessed a-glucosidase activity as expected and showed a 1.9-fold increase in a-glucosidase activity with the addition of maltose suggesting induction. The four selected isolates showed varying degrees of a-glucosidase activity and, with the exception of isolate 4, were all higher than that of B. brevis. In general, these E. coli isolates exhibited increased enzyme activity when grown in the presence of IPTG and a decrease in activity if glucose as well as IPTG was added. Three of the four isolates showed increased activity in the presence of maltose, although for isolates 1 and 2 the increase in activity was not as high as was observed when IPTG was added. These results show that isolate 1 had the highest a-glucosidase specific activity under all conditions tested. This

194

Fig. 1. Positive E. coli RR1 clones obtained from B. brevis gene bank showingfluorescenceunder UV light after being tested using the a-MUG overlaymethod. isolate was chosen for further investigation. The plasmid containing the a-glucosidase gene in this isolate was designated pNSW350. Characterisation of a-glucosidase activity in E. coli RR1 (pNSW350) compared with that in B. brevis The a-glucosidase assay was optimised for E. coli RRl(pNSW350), and compared to that for B. brevis. This involved determining the optima for temperature, pH and efficiency of cell lysis. R R I ( p N S W 350) cells were grown in LB + I P T G and B. brevis cells were grown in LB + Mal. The first parameter to be examined was temperature. The cells were lysed using toluene for RRl(pNSW350) and glass beads for B. brevis and resuspended in phosphate buffer pH 7.0. The results are shown in Fig. 2. For both RR1 (pNSW350) and B. brevis the optimum temperature for a-glucosidase activity was 50°C, giving specific activities of 150 and 2.8 nmol mg-1 min-a protein, respectively, while enzyme activity declined markedly in both

195 TABLE 1 Specific activity of a-glucosidase in various strains Strain

R R I isolate 1 2 3 4 RRI (pNSW350) b RRI (pNSW352) b HB101 (pNSW358) b ZM6100 (pNSW358) b PP1-8 (pNSW358) b B. brevis b RRI HB101 (pRK404) ZM6100 (pRK404) PP1-8 (pRK404)

Average a-glucosidase specific activity a No addition

+ Mal

+ IPTG

+ IPTG GLU

SD c

51 7.4 2.8 1.2 48 45 51 2.3 6.5 1.3 < 0.1 < 0.1 < 0.1 < 0.1

73 16 16 1.0 110 106 94 4 9 2.5 < 0.1 < 0.1 < 0.1 < 0.1

138 40 17 37 226 49 59 2.9 7.3 1.7 < 0.1 < 0.1 < 0.1 < 0.1

33 11 7.7 0.1 NT NT NT 0.5 1.2 NT -

2.6 2 0.5 1.5 5 2 1.5 0.3 0.4 0.3 -

-

-

a Average obtained from three or four individual assays. Specific activity (nmol m g - 1 min-1 protein). b assays under optimised conditions. NT, not tested. c SD c, + / _ greatest standard deviation obtained for each strain grown in various media. Growth media: E. coil and B. brevis strains were grown in LB; Z. mobilis and P. putida strains were grown in RM.

S

140

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,

20

i

30

.

i

i

40

50

Temperature •

E.coli

.

0

60

70

(oC) o----

B.brevis

Fig. 2. Alpha-glucosidase activity estimated at different temperatures for E. coli RRl(pNSW350) and B. brevis. Average Specific Activity = average obtained from four individual assays (nmol nag-1 m i n - 1 protein).

196

300

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100

0

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Fig. 3. Alpha-glucosidase activity estimated at various pH for E. coil RRl(pNSW350) and B. breuis. Average SpecificActivity = average obtained from four individual assays (nmol mg- 1 min - 1 protein). strains at 60°C. Interestingly, the activity profile over the range of temperatures tested was found to be the same in the two strains, although expression was about 50-fold higher in RRl(pNSW350) than in B. brevis. The next p a r a m e t e r to be examined was pH. Determining the p H optimum involved changing the p H of the buffer in which the ceils were resuspended and also the p H of the buffer added to the enzyme assay. Cells were lysed using toluene and incubated at 50°C. The results are shown in Fig. 3. For both strains the optimum p H was 6.5, giving specific activities of 250 and 2.4 nmol m g - 1 m i n - 1 protein for R R l ( p N S W 3 5 0 ) and B. brecis, respectively. At p H values greater than 7.0, no activity was detected in B. brevis whereas activity in RR1(pNSW350) was only reduced about 2.5 fold from the maximum at p H 6.5. The three methods of lysing cells examined were toluene treatment, vortexing with glass beads or sonication. A 10 ml volume of culture was centrifuged and the cells were washed in phosphate buffer, and pelleted again. Toluene (25 txl) was added to the cell pellet of one tube and vortexed for 5 min, then 1 ml of phosphate buffer was added and vortexed for a further 5 min. In a second tube, glass beads (Sigma 75-150 txm) were added to the cell pellet and vortexed for 5 min followed by the addition of 1 ml of phosphate buffer and a further 5 min of vortexing. To the third tube, 1 ml of phosphate buffer was added and the cell pellet was resuspended before being sonicated using a Branson sonifer (USA) for 10 min, at 42% output in 15 s bursts interrupted by 15 s pauses to allow cooling in an

197 ice-water bath. The suspensions were then assayed at 37°C and 50°C for RRl(pNSW350) and B. brevis, respectively, in phosphate buffer, pH 6.5. For RRl(pNSW350), all three methods of cell lysis resulted in essentially the same level of a-glucosidase activity. The average specific activities obtained were 141, 140 and 136 nmol mg-1 min-1 protein for toluene treatment, glass beads and sonication, respectively. For B. brevis, the most efficient method of cell lysis was achieved by vortexing with glass beads which gave an average specific activity of 6.5 nmol mg-a min-1 protein. Lysis by sonication yielded the lowest level of activity, 2.1 nmol mg -1 min -1 protein, while toluene treatment yielded an activity of 4.0 nmol mg- 1 min- 1 protein. The above results indicated that the properties of a-glucosidase in RR1 (pNSW350) and B. brevis were similar. Both RRl(pNSW350) and B. brevis produced an a-glucosidase which had a temperature optimum of 50°C and a pH optimum of pH 6.5. The most efficient method of lysing was the use of glass beads for B. brevis ceils whereas for toluene treatment it was RRl(pNSW350).

Genetic analysis and subcloning Restriction digests were carried out on plasmid pNSW350 isolated from RRl(pNSW350) and the size of the DNA insert in pUCll8 was calculated to be approximately 4.5 kb (data not shown). The 4.5 kb BamHI/HindIII fragment was isolated from an agarose gel, labelled with a32p-dATP and hybridised to a Southern blot of genomic digests of B. brevis and E. coli DNA. The cloned 4.5 kb fragment showed homology with B. brevis genomic DNA but not with E. coli genomic DNA. Activity of the endogenous a-glucosidase promoter Since a-glucosidase levels in the four RRI isolates were increased by the addition of IPTG (Table 1), it appeared that the gene was expressed from the lac promoter in pUC118. As levels were also increased by the addition of maltose (Table 1) it was probable that the a-glucosidase gene was also able to be expressed from its own endogenous promoter. To test this, the 4.5 kb BamHI/HindIII fragment from pNSW350 was subcloned into pUCll9, which is identical to pUCll8 except that the orientation of the polylinker (containing the multiple restriction enzyme cloning sites) is reversed (Vieira and Messing, 1987). RR1 transformants were selected on LBAP20o plates, and after transfer to KWAp200 plates were tested for fluorescence. Varying degrees of fluorescence were noted. Plasmid DNA from a brightly fluorescing colony, was analysed and found to contain the 4.5 kb BamHI/HindIII a-glucosidase-encoding fragment plus the 3.2 kb pUCll9 fragment. This plasmid was designated pNSW352. The a-glucosidase activity of RRI(pNSW352) was examined. From Table 1, it can be seen that expression of t~-glucosidase in RRI(pNSW352) was not increased by IPTG but was increased by maltose, and to the same level as that of RRI(pNSW350). It appeared, therefore, that the promoter of the B. brevis a-glucosidase gene was present on the 4.5 kb BamHI/HindIII fragment and was functional in E. coli.

198 The pUC vectors, or any pBR322-derived vectors, cannot be maintained in Z. mobilis and thus could not be used to transfer the a-glucosidase encoding gene to Z. mobilis. Therefore, the a-glucosidase gene from pNSW350 was subcloned into the broad-host-range vector pRK404 (Ditta et al., 1985) which is capable of replication in a range of Gram-negative bacterial species including Z. mobilis (Liu et al., 1988). Construction of broad-host-range recombinant plasmids containing the B. brevis a-glucosidase gene The 4.5 kb fragment containing the a-glucosidase gene was ligated to pRK404 restricted with BamHI and HindIII and transformed into E. coli HB101. Transformants were selected on LBTc20 plates. Fifty transformants were patched onto KWTc20 plates and were tested for a-glucosidase activity using the a-MUG overlay method. All 50 transformants were fluorescent under UV light. DNA from six transformants was isolated and digested with BamHI and HindIII. Gel analysis of these digests showed that the transformants contained recombinant plasmids which corresponded to the vector pRK404 and the 4.5 kb fragment containing the B. brevis a-glucosidase gene. A Southern blot of BamHI and HindIII digested plasmid DNA from four transformants was probed with the original 4.5 kb BamHI-HindIII fragment from pNSW350. Homology was observed between the probe fragment and the corresponding 4.5 kb fragment in each of the transformants (Fig. 4). All transformants, showed a-glucosidase activity when tested using the a-MUG overlay method. Two isolates (containing plasmids designated pNSW353 and 358) were examined. The amount of enzyme expressed was quantitated using the a-glucosidase assay as optimised for RR1 (pNSW350). HB101(pRK404) was included as a control and, as expected, no a-glucosidase activity was detected. Both clones showed a-glucosidase activity, when grown in LBTcMal, of 56 and 94 nmol rain-1 mg-~ protein for HB101(pNSW353) and HB101(pNSW358), respectively. Since pNSW358 showed a higher a-glucosidase activity, it was transferred to Z. mobilis and P. putida for further investigation. Transfer of the a-glucosidase gene to Z. mobilis and P. putida The recombinant plasmid pNSW358 was mobilised into Z. mobilis and P. putida by filter mating using HB101(pRK2013) as the mobilising strain. Transconjugants were isolated on RMTpTc plates for Z. mobilis and LBRpTc plates for P. putida. Twenty Z. mobilis transconjugants and five P. putida transconjugants were purified by single colony isolation and all were found to maintain resistance to Tc. These transconjugants were patched onto BMTpTc plates for Z. mobilis and KWRpTc plates for P. putida. After 3-4 days incubation at 30°C, the isolates were tested for a-glucosidase activity using the a-MUG overlay method. Seventy percent of the Z. mobilis transconjugants showed varying degrees of fluorescence under UV light with 30% showing no fluorescence. All P. putida transconjugants showed fluorescence under UV light. The Z. mobilis transconjugants were streaked onto minimal medium containing maltose as the sole carbon source. None of the

199 1

2

3

4

5

6

7 1

A

2

3

4

5

6

7

B

Fig. 4. Gel electrophoresis and hybridisation of plasmid preparations from E. coli transformants containing the a-glucosidase gene subcloned into pRK404. (A) Agarose gel. From left: lanes 1-4, BamHl and H/ndlII double digest of HB101 transformants; lane 6, BamHI and HindllI double digest of pRK404; lane 7, HindlII digest of A DNA. Gel conditions: 1% horizontal agarose gel, 40 V, 40 mA, 16 h. (B) Hybridisation of a Southern blot of the gel shown in A with the 4.5 kb fragment containing the a-glucosidase gene isolated from pNSW350. t r a n s c o n j u g a n t s was able to grow o n these plates. T h e P. putida t r a n s c o n j u g a n t s were also streaked o n t o m i n i m a l m e d i u m c o n t a i n i n g maltose as the sole c a r b o n source. All five P. putida t r a n s c o n j u g a n t s were able to grow o n m a l t o s e as a sole c a r b o n source. As controls, P. putida PP1-8(pRK404) a n d P. putida PP1-8 R p R were streaked o n t o the same m e d i u m , b u t w i t h o u t the a d d i t i o n of Tc in the case of PP1-8 R p R. N e i t h e r of these two strains was able to grow o n m i n i m a l m e d i u m with m a l t o s e as the sole c a r b o n source.

Quantitation of a-glucosidase activity in Pseudomonas and Zymomonas strains possessing the B. brevis a-glucosidase gene T h e a-glucosidase activity of PP1-8(pNSW358) a n d Z M 6 1 0 0 ( p N S W 3 5 8 ) was assayed after six successive single-colony purifications. T h e strains to be tested

200 were grown from a single colony from the sixth purification and were all a-MUG +. These colonies were grown overnight in broth at 30°C, statically for Z. mobilis strains and on an orbital shaker (200 rpm) for Pseudomonas strains. ZM6100(pRK404) and PP1-8(pRK404) were included as controls. The results, shown in Table 1, indicated that when maltose was added to the growth medium, the level of a-glucosidase activity was slightly increased. An approx. 1.5-fold increase in a-glucosidase activity was observed in both ZM6100 and PP1-8 transconjugants. The addition of IPTG caused little difference in the activity from either transconjugant strain. Glucose addition to the medium showed an inhibitory effect with the levels of a-glucosidase activity being depressed in both ZM6100 and PP1-8 transconjugant strains.

Discussion

An a-glucosidase gene from the Gram-positive bacterium B. brevis was cloned and expressed in the Gram-negative bacteria E. coli, P. putida and Z. mobilis. While other genes from Gram-positive bacteria have been cloned and expressed in E. coli (see for example, Fouet et al., 1982; Joyet et al., 1984; Hoshiko et al., 1987), this is the first report of the expression of an a-glucosidase gene in the above organisms, and, to our knowledge, the first report of the cloning of an a-glucosidase gene from B. brevis. While a-glucosidase expression was maintained in strains containing the plasmids pNSW350, pNSW352 and pNSW358, it was noted that at each manipulation step individual transformants or transconjugants showed different levels of enzyme activity, or sometimes no activity, as determined by the a-MUG plate assay. We are investigating further the stability of these plasmids, and the expression of a-glucosidase, in Z. mobilis and P. putida. There may be inherent problems due to instability of recombinant plasmids containing genes such as a-glucosidase, since we also identified a-glucosidase activity in other E. coli gene banks containing cloned DNA fragments derived from the DNA of B. subtilis, P. fluorescens, Xanthomonas and Cellulomonas spp. In the process of purifying these potential isolates, however, the a-glucosidase activity was quickly lost and no recombinant plasmids bearing a-glucosidase genes could be isolated. While the temperature profile of the cloned a-glucosidase was the same in E. coli as in the native host, the pH profile was slightly different. The optima of each, however, were the same in both hosts and the same as that reported by McWethy and Hartmen (1979). The property of temperature tolerance probably resides in the structure of the protein itself, but that of pH dependence may be affected by the host organism to a small degree. Joyet et al. (1984) found that the temperature and pH profiles of the a-amylase gene product from B. lichenifomis cloned in E. coli were the same in both hosts. It will be interesting to examine these properties for the a-glucosidase enzyme produced in Z. mobilis and P. putida. The levels of specific enzyme activity determined by the recombinant plasmids in the different hosts were interesting. For pNSW350 in E. coli, expression was directed by both the lac promoter of the vector as well as the endogenous

201 promoter of the a-glucosidase gene, since levels were increased by the addition of either I P T G or maltose. When the vector and a-glucosidase gene were arranged in the opposite orientation, as in pNSW352, expression was no longer controlled by the lac promoter, but was driven by the endogenous promoter. In E. coli, the cloned a-glucosidase gene was expressed from its own promoter at a level about 50-fold higher than that found in its native host, B. brevis. This may have been due to the higher copy number of recombinant plasmids in the E. coli isolates, a n d / o r to a lack of negative regulation, possibly present in B. brevis and lacking in E. coli. In E. coli, the plasmids pNSW352 and pNSW358 produced similar levels of a-glucosidase and activity was increased by the addition of maltose but not by IPTG. In both of these plasmids (pNSW352 is a p U C l l 9 derivative and pNSW358 is a pRK404 derivative) the lac promoter is adjacent to the HindIII end of the 4.5 kb B a m H I / H i n d I I I fragment encoding the a-glucosidase gene, whereas in pNSW350 (a pUC118 derivative) the lac promoter is adjacent to the B a m H I end. Thus, transcription of the a-glucosidase gene must proceed in the direction from the B a m H I end towards the HindIII end of the fragment. When pNSW358 was transferred to Z. mobilis and P. putida, the level of a-glucosidase activity was not affected by the addition of IPTG, as expected. An increase in enzyme activity of about 2-fold, however, was caused by the addition of maltose to cultures of these pNSW358 transconjugants, similar to that seen in E. coli (pNSW358) and in the original host B. brevis. This indicates that maltose was being taken up by the cells and inducing expression of the cloned a-glucosidase gene. P. putida (pNSW358) was able to grow on maltose whereas the parent P. putida strain was not; Z. mobilis (pNSW358) was unable to grow on maltose as the sole carbon source. Although Z. mobilis (pNSW358) produced less a-glucosidase than either P. putida or E. coli hosts containing pNSW358, it is unlikely that its inability to grow on maltose was due to low enzyme levels, since these were of the same order of magnitude to those produced in the original host B. brevis. It is more likely that Z. mobilis lacks a transport mechanism for maltose and that gene(s) for maltose transport were not encoded on the cloned fragment. This was found to be the case for a sucrase gene cloned from B. subtilis into E. coli (Fouet et al., 1982). The recombinant E. coli, while expressing sucrase activity, was unable to transport sucrose into the cell, and was thus unable to grow on sucrose as sole carbon source (Fouet et al., 1982). It has been reported that the uptake of glucose by Z. mobilis requires no energy, and that a facilitated-diffusion system is involved (Romano, 1986). It is possible that the substrate transport system(s) in Z. mobilis is either primitive or deficient since no growth on any substrate other than glucose, fructose and sucrose has been achieved despite transfer to this organism of genes coding for enzymes involved in the catabolism of other sugars - cellobiose (Misawa and Nakamura, 1989; S u e t al., 1989), xylose (Liu et al., 1988), galactose (Goodman, 1986) and lactose (Carey et al., 1983; Goodman et al., 1984). To date this has frustrated attempts to increase the substrate range of this organism to produce ethanol from cheap substrates. Although ethanol has been produced by recombinant Z. mobilis strains from

202 cellobiose (Su et al., 1989), galactose (Goodman, 1986), and lactose (Goodman et al., 1984) the process using intact cells is too slow to be commercially useful. Since maltose induced the a-glucosidase gene in whole cells of Z. mobilis (pNSW358) some maltose was obviously entering the ceils. Our preliminary data suggest that ethanol is being produced by ZM6100(pNSW358) ceils and this is currently being investigated further. Improving maltose transport into Z. mobilis might be achieved either by mutation and selection or by cloning in the appropriate transport genes, as suggested by Fouet et al. (1982) for a sucrase gene cloned in E. coli. It is not known why the levels of a-glucosidase produced by Z. mobilis (pNSW358) and P. putida (pNSW358) were lower than those produced by HB101 (pNSW358). This could be due to different efficiencies of expression of the gene in these strains. Genes for other catabolic enzymes cloned and expressed in Z. mobilis have generally shown a 10- to 20-fold lower expression in this host than in the corresponding recombinant E. coli host (Goodman et al., 1984; Liu et al., 1988; Su et al., 1989). Work is in progress to place such catabolic genes under the control of strong Z. mobilis promoters which have recently been isolated (Dunn, personal communication).

Acknowledgements We wish to thank P. Hartman for the kind gift of the B. brevis 27-7 strain containing the a-glucosidase gene. Part of this work was supported by a grant from the National Energy Research, Development and Demonstration Council ( N E R D D C ) of Australia.

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