CURRENT MICROBIOLOGY VOI. 25 (1992), pp. 279-282
Current Microbiology 9 Springer-Verlag New York Inc. 1992
Molecular Cloning and Expression of Bacillus subtilis bgIS Gene in Saccharomyces cerevisiae Yongqing Chen, Xinqi Huang] Daxin Song, Fan Yang, and Weijun Zheng 1 Department of Microbiology and Microbial Technology, Fudan University, Shanghai, P.R.C.
Abstract. A 2.7-kb EcoRI DNA fragment carrying a Bacillus subtilis endo-/3-1,3-1,4-glucanase gene (bglS) from the E. coli plasmid pFG1 was cloned into an Escherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transform Saccharomyces cerevisiae, and the bglS gene was expressed. Variation between levels of bglS gene expression in S. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found in B. subtilis, but the expression level of bglS gene in S. cerevisiae (YCSH) was much lower than that in E. coli (YCSH).
/3-1,3-1.4-Glucan is the main component of the barley endosperm cell wall, accounting for about 10-14% of the total sugar in barley. A high proportion of/3-1,3-1,4-glucan in barley will increase the viscosities of wort, slow filtration rates, and even cause undesirable hazes and precipitates during beer fermentations. Although Saccharomyces cerevisiae can produce several kinds of/3-glucanases, none of them is able to degrade the/3-1,3-1,4-mixed linkage glucan . In 1984, E. Hinchliff  reported having cloned and expressed the/~-l,3-1,4-glucanase gene in S. cerevisiae. We have isolated a 7.1-kb EcoRI chromosomal DNA fragment from Bacillus subtilis 1.88, which carries the/3-1,3-1,4-glucanase gene (bglS), and have determined the location of the bglS gene in a 2.7-kb EcoRI DNA fragment after subcloning . In this paper, we describe the cloning and expression of the 2.7-kb DNA fragment in S. cerevisiae.
group. YFD24(Ap r, ura § including the Yeast 2u plasmid sequence) was an E. coli/yeast shuttle plasmid vector supplied by the Institute of Genetics.
Materials and Methods
Construction of shuttle vector and transformation ofS. cerevisiae. Restriction digestion, DNA ligation, and transformation were performed according to the methods described by Maniatis et al.  and Hisao .
Bacterial strains and plasmids. Cloning was carried out in E. coli MC1061(F- araD139, A(ara leu)7996AlacY74 galU galK hsr-hsm+strA) and S. cerevisiae Y33(his a d e - u r a - l e u - ) . Both strains were supplied by the Institute of Genetics (Fudan University, Shanghai, PRC). E. coli plasmid pFG1 (pBR325 + 7.1-kb EcoRI DNA fragment) was the bgls cloning vector constructed by our research 1 Present address: Genetic Engineering Institution of Yunnan Academy of Agriculture Science, Kunming, P.R.C.
Media. E. coli medium, LB medium, and others were the same as in Song et al. . For yeast medium, including minimal medium and selective medium, refer to Hinchcliff and Box . The detection medium for/3-1,3-1,4-glucanase activity in yeast was (per liter): peptone 20 g, yeast extract 10 g, barley fl-glucan 1 g, dH20 1000 ml, pH 6.0, agar 15 g. Reagent. Reagents for DNA manipulation were bought from Shanghai Sino-American Biotechnology Corp., and barley/3-glucan was bought from Sigma Corp. Preparation of plasmid DNA. Plasmid DNA was prepared by the method of Birnboim and Doly . Preparation of 2.7-kb EcoRl D N A fragment. After complete digestion of pFGI by EcoRI, the 2.7-kb EcoRI DNA fragment containing the bgIS gene was collected according to the method described by Maniatis et al. .
Detection and assay of ll-l,3-1,4-glncanase activity. S. cerevisiae colonies were screened for fl-glucanase activity by detection plates. After S. cerevisiae colonies had grown on detection plates for 3-4 days at 30~ the plates were stained with 0.1% (w/v) Congo red, and clear halos around colonies were indicative of/3glucanase activity. Quantitative assay offl-glucanase activity was performed as reported by Chen et al.  (use of dinitrosalicyclic
Address reprint requests to: Dr. Yongqing Chen, Department of Microbiology, Fudan University, Shanghai 200433, People's Republic of China.
CURRENT MICROBIOLOGYVol. 25 (1992)
carrying the bglS gene. Of approximately 1000 transformants screened for /3-glucanase activity, 10 showed clear degradation halos. All l0 harbored hybrid plasmids, designated YCSHI-10 respectively. The clear degradation halos produced by strains harboring YCSH were smaller than those of bglS gene donor Bacillus subtilis, but larger than those of strains harboring pFG1. We divided the 10 recombinants into two groups according to the measure of the degradation halos, and that indicated the differences in their enzyme productivity. We chose one strain from each group (YCSH1 and YCSH5) for further analysis.
T4 DNA Ligase E
I (13~lkl=) ECoRI
I ,14 DNA
Fig. 1. Construction of an E. coli/yeast shuttle hybrid plasmid YCSH. - - B . subtilis chromosomal DNA; ~ yeast 2p.; ~ URA + 3; - - vector DNA sequences. Restriction sites for the enzyme: E, EcoRI, B, BamHI; H, HindII; P, PstI.
acid reagent for the determinationof reducing sugar). The substrate concentration was 2 mg/ml. One unit of enzyme activity was definedas the amountof enzymecapableof liberating 1nmol reducingsugarper minute(withglucoseas reference)at 40~ pH 7.0.
Results and Discussion Construction of plasmid YCSH (bglS). The EcoRI DNA fragment was "shot-gun" cloned into plasmid pBR325. The 7.1-kb insert was shown to direct the synthesis of /3-glucanase in Escherichia coli MC1061. We designated the hybrid plasmid pFG1. The 2.7-kb EcoRI DNA fragment isolated from pFG 1 plasmid was ligated with YFD24 plasmid DNA after YED24 was digested with the same restriction endonuclease (Fig. 1) and was introduced into E. coli MC1061. Transformants were obtained on the LB plates containing ampicillin. Then the transformants were cultured on/3-glucanase activity detection plates, and those which could produce clear halos were strains harboring the hybrid shuttle vector
Expression ofbglS gene on YCSH inE. coil Qualitative assay on/3-glucanase activity detection plates showed that YCSH1 and YCSH5 differed in their /3-glucanase production. The E. coli strains harboring hybrids were inoculated into LB broth containing ampicillin (100/~g/ml) respectively. After shaking at 37~ for 24 h (bglS gene donor strain B. subtilis 1.88 as reference), the cultures were filtered and prepared for quantitative assay for /3-1,3-1,4glucanase activity, and the results were summarized in Table 1. The table showed that the expression level of bglS gene in E. coli (YCSH) was only 1.2-5.3% of that of the donor strain, but it was two to four times higher than that of E. coli (pFG1), and the enzyme productivity in E. coli MC1061(YCSH1) was 2.07fold of that in E. coli MC1061(YCSH5). Restriction digestion map analysis indicated the insert orientations of the 2.7-kb DNA fragment in hybrid plasmid YCSH1 and YCSH5 were opposite. From the results above, we can see that gene sources, characteristics of plasmid vector, length of cloning fragment, and insert orientation all can affect the expression level of the gene. Comparison of fl-l,3-1,4-glucanase from E. coli MC1061(YCSH) with that from B. subtilis. In order to confirm that the/3-1,3-l,4-glucanase encoded by shuttle plasmid YCSH was the product of bglS gene from B. subtilis 1.88, we assayed for the enzyme specificity and optimal pH of the/3-glucanase from both E. coli MCI061(YCSH) and B. subtilis 1.88 under the same conditions (Table 2). Twenty-fourhour reactions of different substrates showed the two/3-glucanases had identical enzyme specificities: both can only degrade the/3-1,3-1,4-glucoside bond linking barley/3-glucan and lichenan, and can not react on other kinds of linkage. The pH analysis also showed that the two had similar pH curves, and both
Y. Chen et al.: B. subtilis Gene in S. cerevisiae Table 1. Expression of a B. subtilis b g l S gene in E. coli E. coli
MC1601 (YCSH 1)
Enzyme activity (U/ml)
Table 2. The specificity of/3-1,3-1,4-glucanase in B. subtilis and E. coli transformants
Table 3. Expression of/3-1,3-1,4-glucanase gene activity in S. cerevisiae
Reducing sugars (mg/ml/24 h)
Enzyme activity (U/ml):
6.207 3.778 0 0 0
1.89 1.48 0 0 0
/3-Glucan (barley) Lichenan CM-Cellulose Cellulose Soluble starch
/3-1,3-1,4 /3-1,3-1,4 /3-1,4 /3-1,4 c~-1,4-1,6
/3-1,2 0 c~-1,6-B-1,4 0
had their optimal pH at about 7.0. From the facts above, we can conclude that the enzyme characteristics of the two/3-glucanases were identical, which indicates that the bglS gene carried by E. coli MC1061 was from B. subtilis and that both strains produced the same/3-glucanase.
Cloning and expression of hybrid plasmid YCSH in Saccharomyces cerevisiae. An E. coli/yeast shuttle vector YCSH was constructed in order to introduce the B. subtilis /3-1,3-1,4-glucanase gene into S. cerevisiae. The shuttle plasmid carried the ura + gene, while the recipient S. cerevisiae was the ura- auxotroph, so transformed S. cerevisiae could be selected on the ura- medium. After YCSH plasmid DNA was isolated from E. coli MC1061(YCSH), S. cerevisiae was transformed by the LiAc method, and transformants were selected on ura- medium. The transformation efficiency was about 102-103 transformants per/zg plasmid DNA. The S. cerevisiae transformants were analyzed for proof that they carried the YCSH plasmid. The transformants were inoculated on /3-1,3-1,4glucanase activity detection plates. After incubation at 30~ for 3-4 days, the plates were stained with 0.1% Congo red, and transformant colonies produced clear halos with diameters of several
millimeters. With an improved rapid plasmid DNA isolation method, YCSH plasmid DNA was isolated from the transformants, and after EcoRI digestion, two bands were obtained during electrophoresis: a linear YFD24 DNA band and a 2.7kb DNA band. Plasmid DNA isolated from S. cerevisiae transformants was also used to transform E. coli MCI06I. The re-transformants were resistant to both ampicillin and tetracycline, and the re-transformants grown on/3-1,3-1,4-glucanase activity detection plates produced clear degradation halos of about the same size as those produced by previous E. coli (YCSH). This shows that shuttle vector YCSH (bglS) was really harboring in S. cerevisiae Y33(YCSH). Quantitative assay indicated that the bglS gene on plasmids YCSHI and YCSH5 had different expression levels in S. cerevisiae. The strains Y33(YFD24), Y33(YCSH1), and Y33(YCSH5) were inoculated respectively in the ura- culture to grow to log phase by shaking at 30~ Then the cells were collected by centrifugation. The cells were ground at 0~ until 70% of the cells were broken. After a second centrifugation, the cell extracts were taken for quantitative assay of /3-glucanase activity (Table 3). The results showed that /~-glucanase activity could be detected in the S. cerevisiae Y33(YCSH) cell extract, with its level equivalent to that reported by Hinchliffe . The bglS gene product activity in Y33(YCSH1) was still 2.3-fold higher than that in Y33(YCSH5). But as shown in Tables 2 and 3, the expression level of the bglS gene in S. cerevisiae was far lower than the expression level in E. coli by the same vector.
282 Effect of insert orientation on expression of bglS gene. Previous reports did not agree on this question. Cantwell et al. suggested that the expression level of the bgIS gene was not influenced by the 4kb EcoRI insert's orientation in plasmid pJG82, but Hinchliffe's work showed  that the difference in enzyme production was up to 1.6-fold when a 3.5kb EcoRI DNA fragment had two orientations in plasmid pEH13, and our results were similar to the latter. According to our work, whether the bgIS gene was expressed or not was not determined by the orientations of the cloning insert, but the expression level was affected by insert orientations. This may lead to the conclusion that the bglS gene probably had its own transcription promoter while the bglS gene expression level was affected by the vector expression system. For a cloned gene with its own promoter, the effect may be dependent on the length of the insert, insert position, vector, and properties of the host cell. Gene expression is a very complicated, multistep, regulated process, and expression of a foreign gene adds more to this complexity. Expression of a foreign gene is related to species difference of genes from different sources, coordination of a foreign gene with the host cell's replication, transcription, translation, post-translational modification, and other regulation systems. The source of the transcription promoter, its structure and strength, plasmid copy number in the host cell, and the capability of the host cell's replication, transcription, and translation enzyme systems to recognize and operate on the foreign gene expression signal all directly influence the gene's expression level. This is the case with the cloned B. subtilis bglS gene in our experiment: the expression level of bglS gene is lower in E. coli and S. cerevisiae than in B. subtilis. Gantwell et al.  improved a B. subtilis bgl gene's expression in S. cerevisiae by 1000 times by changing its promoter. This may be an important way to improve expression of foreign genes. In order to make the new S. cerevisiae strain carrying the bglS gene suitable for industrial applica-
CURRENT MICROBIOLOGY Vol. 25 (1992)
tion, we must solve the problem of secretion of the bgIS gene product. In Hinchliffe's report in 1984 (6), no/3-1,3-1,4-glucanase activity was detected in the culture of S. cerevisiae carrying the bgl gene, and there was only low enzyme activity in cell extract. In our experiment, we not only detected/3-glucanase activity in the cell extract, but also found clear halos around S. cerevisiae colonies carrying the bglS gene on the /3-glucanase activity detection plates. The results indicated that, although the bglS gene expression level was low in S. cerevisiae, a portion of/3glucanase was still secreted outside the cell membrane. Our further research is still under way.
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