Letters in Applied Microbiology 1990, 11, 214-219
Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactu~~cillus plantavum S . JONES & P.J. W A R N E RBiotechnology Centre, Cranfeld Institute of Technology, Bedford M K 4 3 OAL, U K Received 29 June 1990 and accepted 3 July 1990
S . & WARNER P.J. , 1990. Cloning and expression of alpha-amylase from Bacillus arnyloliquefaciens in a stable plasmid vector in Lactobacillus planfururn. Letters in Applied Microhiology 11,21&219.
Lactohacihs plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus arnyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations
Bacteria of the genus Lactobacillus are used widely in the preparation of fermented meats, vegetables and dairy products. Lactobacillus plantarum is also used in temperature regions to aid the preservation of fodder crops as silage (Woolford 1984). These processes may be optimized, and new uses for the organisms may be found, as a result of genetic manipulation. Relatively little is known about the genetics of the lactobacilli. High frequency transformation has been achieved only recently; a number of species have been transformed reproducibly using electroporation, at efficiencies of up to lo5 transformants/pg DNA (Chassy 1987; Luchansky et al. 1988; Badii et al. 1989). However, the capacity of these organisms to express heterologous genes, apart from those encoding antibiotic resistance and plasmid replication, has not been extensively studied. In particular studies involving expression of metabolic enzymes has been limited (Bates et al. 1989; Jossen et al. 1989). The ability of lactobacilli to express heterologous proteins, in particular enzymes which enable growth on cheaper substrates would have considerable commercial potential, especially if they are secreted. For example the
ability to secrete amylases may enable the substrate range of the organisms to be extended to polysaccharides. In addition, alpha-amylase activity is easily assayed and has been used successfully as a model protein in research on the mechanism of protein secretion as well as on the relationship between protein structure and stability (Yuuki et al. 1985). Since Lactobacillus plantarum is unable to degrade starch, the acquisition of alpha-amylase activity would be beneficial, both for academic studies and commercial applications. Here, we report the molecular cloning and functional expression of an alpha-amylase gene from Bacillus amyloliquefaciens in Lactobacillus plantarum NCDOll93, using the plasmid vector pIL253. Materials and Methods GROWTH A N D MAINTENANCE OF CULTURES
Bacterial strains and plasmids used are listed in Table 1. Lactobacillus strains were cultured on MRSB (DeMan et al. 1960) medium at 37°C. Bacillus
Amylase in Lactobacillus Table 1. Strains and plasmids used in this study
Bacteria Lactobacillus plantarum 1 193 Bacillus suhtilis lA510
Cryptic plasmids 11.5 kb/19.5 kb Plasmid free Restriction Modification
4.8 kb, Erm' 11.6 kb, Cam', Amy+ 7.0 kb, Cam', Amy'
Simon & Chopin 1988 G. Steinborn This study
Plasmids plL253 pSB6 pCIT602
Abbreviations: NCFB, National Collection of Food Bacteria; BGSC, Bucillus Genetic Stock Center, Ohio State University, Ohio, USA; Erm', erythromycin resistance; Cam', chlordmphenicol resistance; Amy+, alpha-amylase positive.
strains were cultured on LB (Maniatis et al. 1982) at 37"C, with shaking. Lactococcus loctis IL1403 (pIL253) was cultured on M17 medium (Terzaghi et al. 1975), in which lactose was replaced with glucose (0.5% w/v), at 30°C. Media were solidified with agar (Oxoid no. 3, 1.5% wiv) where required.
selected on DM3 agar (Chang & Cohen 1979), containing erythromycin (5 pg/ml), incubated at 37°C for 48 h. Lactobacillus plantarum was transformed via electroporation as described previously (Badii et at. 1989). Transformants were selected on MRS agar (MRSA) containing erythromycin (10 pg/ml) and lincomycin (100 Pgiml).
I S O L A T I O N Of. P L A S M I D D N A
Plasmid DNA was isolated from preparations of Lactobacillus plantarum as previously described (Anderson & McKay 1983). Large scale plasmid preparations from Lactococcus lactis were as previously described (Gasson 1983). Large scale and rapid preparations of plasmids from Bacillus were performed by the Birnboim and Doly method (Birnboim & Doly 1979), with modifications (Maniatis et al. 1982), followed by purification with ethidium bromidecaesium chloride density gradient centrifugation. M O L E C ' U LA K C I . O N I N G
Restriction endonucleases (Bethesda Research Laboratories) and T4 DNA ligase were used according to the manufacturer's recommendations. All other molecular cloning techniques were performcd as described (Maniatis et al. 1982). After excision from agarose gels, DNA fragments were treated with Gene CleanTM(Bio 101 Inc.). Bacillus subtilis was transformed with plasmid DNA via protoplast formation and regeneration (Chang & Cohen 1979). Transformants were
DETECTION O F A L P H A - A M Y L A S E
Bacillus subtilis colonies were tested for alphaamylase production by replicating on to a nitrocellulose filter which was then placed, colony side down, on a starch plate (phosphate buffer (40 mmol/l, pH 6.0)), containing soluble starch (1% w/v), agar (1.5% w/v) and erythromycin (5 pg/ml). After overnight incubation (37"C), the nitrocellulose filter was removed and the plate was stained blue with I,-KI solution (20 mmol/l). Colonies producing alpha-amylase were surrounded by a clear halo. For detecting alpha-amylase production by Lactobacillus plantarum colonies, the procedure was similar, but MRSA plates containing soluble starch (1% w/v), erythromycin (10 pg/ml) and lincomycin (100 pg/ml) were used. Alpha-amylase activity in culture supernatants was determined as follows. Organisms were harvested by centrifugation after 24 h or 48 h growth and the supernatant fluid was collected. Aliquots (200-1000 p l ) were used in the PhadebasTMtest system (Pharmacia Ltd.), which was used as directed by the manufacturers. Southern hybridization was performed as previously described (Southern 1975). Probes
S . Jones and P . J . Warner
were labelled with digoxygenin (Boehringer Mannheim) and hybridization was detected as a colour reaction, based on the digoxygeninantidigoxygenin ELISA.
was isolated from its B. subtilis host as a 2.2 kb Bcl 1-Barn H1 fragment. This was ligated to the Lactococcus vector pIL253 (Simon & Chopin 1988), linearized with Barn H1 to yield plasmid pCIT602 (Fig. 1). The ligated vector was used to transform protoplasts of a plasmid free strain of B. subtilis, which showed no alpha-amylase activity. Transformants were selected with erythromycin and screened for amylase production. Plasmid DNA was isolated from alpha-
Results and Discussion
The alpha-amylase gene from B. arnyloliquefaciens was obtained as a cloned fragment in plasmid pSB6 (Kunze et al. 1988). The plasmid
I O 3-
B c l I - BarnHI
pCIT602 7.0 kb
Fig. 1. Construction of the recombinant plasmid, pCIT602, carrying the alpha-amylase gene. Plasmid pSB6 was digested with Bcl I-Barn HI and the alpha-amylase encoding 2.2 kb fragment was ligated to pIL253 linearized with Barn H 1. Abbreviations: MLS, macrolides, lincosamides, streptogramin B resistance; REP, replication region; Cam', chloramphenicol resistance; PRS,polyrestriction site.
Amylase in Lactobacillus amylase producing recombinants and subjected to agarose gel electrophoresis, which revealed the presence of a recombinant plasmid of the predictcd size (approximately 7 kb). This plasmid, designated pCIT602, was used to transform Lactobacillus plantarum NCDOll93, a strain previously shown to undergo transformation at relatively high frequencies (Badii et ul. 1989). Plasmid pCIT602 was introduced into Lactobacillus plantarum NCDOll93, using electroporation, at a relatively high frequency (5.08 x lo5 transformants/pg DNA). Transformants were selected using erythromycin and lincomycin; use of the latter reduces the frequency of false positives (Jossen et al. 1989). They were then screened for alpha-amylase activity and examination of the plasmids present confirmed the presence of pCIT602 in these transformants, in addition to the 11.5 kb and 19.5 kb plasmids resident in NCDO1193 (Luiz-Barba and Warner, unpublished observations). The presence of the alpha-amylase gene in Lactobacillus pluntarum transformants was confirmed by Southern hybridization (Fig. 2). Total DNA from the alpha-amylase positive recombinants of both €3. subtitis and Lactobacillus plantarum was probed with the 2.2 kb fragment encoding alpha-amylase, from pSB6. The probe hybridized pCIT602 DNA from both B. subtilis and Lactobacillus plantarum transformants. There was no evidence of chromosomal integration in either species, since the probe only hybridized to bands corresponding to a 7 kb plasmid. The probe failed to hybridize to controls comprising Lactobacillus plantarum NCDOll93 and bacteriophage lambda DNA digested with Hin d 111. Preliminary experiments have been carried out to compare the efficiency of expression and secretion of alpha-amylase in 8. subtilis and Laclobucillus plantarum. Culture supernatant fluids from overnight cultures of the two organisms, harbouring pCIT602, were assayed for alpha-amylase and the results are shown in Table 2. In order to ensure that the acidity of MRSB after growth of Lactobacillus plantarum was not resulting in loss of activity, the experiment was repeated using MRSB buffered with malate (100 mmol/l), which maintained the pH at 6.5. However, although this resulted in a slight increase of alpha-amylase activity, the
Table 2. Alpha-amylase activity
supernatants of Lactobacillus plantarum and Bacillus suhtilis harbouring pCIT602 Units of alpha-amylase activity/litre of supernatant Period of incubation of culture (h)
Bacillus suhtilis 1A510
ND, not determined.
activity observed in Lactobacillus plantarum supernatants remains only a fraction of those observed in B. subtilis. This paper describes the use of pIL253 to introduce alpha-amylase activity into Lactobacillus pfantarum. It demonstrates that this vector, designed for gene cloning in lactococci, can be used equally efficiently in the lactobacilli. The choice of vector is of great importance in gene cloning in lactic acid bacteria. Many small Gram-positive plasmids replicate via singlestranded DNA and these are subject to instability when foreign DNA is inserted. In this study, plasmid pGK12 (Kok et al. 1984) was originally used as a cloning vector, because it could be efficiently transformed into Luctobacillus plantarum (Badii et al. 1989). Recombinants carrying the alpha-amylase gene were observed, but proved to be extremely unstable, despite repeated attempts at isolation. Plasmid pIL253 was subsequently employed because it is known not to replicate via single-stranded DNA, since it is derived from the plasmid pAMB1. The recombinant derived here, pCIT602 can be subcultured repeatedly on starch containing medium with no loss of the plasmid; its stability in the absence of selection is currently under investigation. Although alpha-amylase was detected in culture supernatants of Lactobacillus plantarum NCD01193 (pCIT602), the level of expression is certainly much lower than that observed in B. subtilis and appearance of enzyme activity may result from lysis of, or leakage from the organisms, rather than true secretion. The effect of malate, which stabilizes the pH appears to result in slightly enhanced activity in Lactobacillus plantarum (pCIT602), which probably reflects the pH optimum of the enzyme, in the region of pH 6-7. Levels of expression in Lacto-
S. Jones and P . J . Warner that a starch degrading cassette, comprising alpha- and beta-amylase activities and a debranching enzyme such as pullulanase would enable lactobacilli to completely degrade a cheap and readily available substrate such as starch to maltose and glucose, conferring an obvious economic advantage. Expression of novel heterologous genes in Lactobacillus will undoubtedly spread beyond increased metabolic diversity, to enable, for instance, generation of strains able to utilize waste products, produce high value products and variants with specific and optimized gut adherence as probiotics. S.J. is supported by an AFRC Research Grant. The authors thank G. Steinborn, J. Kok and A. Chopin for strains and plasmids.
Fig. 2. Southern hybridisation of Lactobacillus plantarum transformants containing pCIT602. The probe was the 2.2 kb Bcl 1-Bum HI fragment encoding alpha-amylase, from pSB6. Tracks are as follows: (A) bacteriophage lambda DNA digested with Hin d 111; (B) rapid plasmid preparation of DNA from wild type Lactobacillus plantarum NCDOll93; (C&D) rapid plasmid preparation of DNA from Lactobacillus plantarum NCDO1193 (pCIT602); (E) CsCl purified plasmid DNA from Bacillus subtilis 1A510 (pCIT602). In the latter track, no covalently closed circular DNA is evident, due to damage by nicking, but the larger bands are often seen with this preparation and represent concatameric forms.
bacillus plantarum are currently under investigation, and we are currently determining the promoter and signal sequences of the alphaamylase gene in order to confirm its similarity to those previously elucidated. Attempts will then be made to improve expression in Lactobacillus planlarum by optimizing the promoter sequence for this organism. The alpha-amylase cassette has application for the study and optimization of expression of heterologous genes in Lactobacillus. Such studies will pave the way for improvement of existing lactic acid fermentations, by, for example, obviating the need for addition of expensive substrates. In future it is envisaged
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