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Genetic manipulation system in propionibacteria
JOURNALOFBIOSCIENCE ANDBIOENGINEERING Vol. 93, No. 1, l-8. 2002
REVIEW Genetic Manipulation System in Propionibacteria PORNPIMON
KIATPAPAN’
AND
YOSHIKATSU
MUROOKA2*
Biochemistry Unit, School of Science, Rangsit University, Patumthani 12000, Thailand’ and Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita, Osaka 5650871, Japan’ Received 5 September 2001iAccepted 20 September 2001
Members of the genus Propionibacterium are widely used in the production of vitamin B,,, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propionibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibacterium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in L freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin B,, biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in E!freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probiotics and the vitamin B,, biosynthetic pathway. [Key words: propionibacteria, vitamin B,,]
I.
PROPERTIES
5-aminolevulinic
acid, ALA synthase, cholesterol
OF PROPIONIBACTERIA
Classification of propionibacteria Propionibacteria are gram-positive, nonmotile, non-spore-forming, facultatively anaerobic, and rodlike bacteria. The G+C content of their DNA is in the range of 53-67 mol% (1). The genus Propionibacterium is classified into two groups, the classical or dairy propionibacteria and the acne group or cutaneous propionibacteria. The main habitat of cutaneous propionibacteria is human skin, whereas that of dairy or classical propionibacteria are cheese and milk. On the basis of DNA homology and similarities in cell wall composition (2), dairy or classical propionibacteria are classified into six species (3): Propionibacterium freudenreichii, P jensenii, I? theonii, l? acidipropionici, P coccoides, and p cyclohexanicum. Similarly, cutaneous bacteria are classified into six species: p acnes, I? avidium, I? granulosum, I! lymphophilums, P propionicum, and Propioniferax innocua. Cutaneous propionibacteria are also known as anaerobic corynefotms or anaerobic diphtheroides. In contrast with cutaneous propionibacteria, classical propionibacteria do not produce indole and cannot liquefy gelatin. Other than these, the two groups cannot be clearly distinguished based on other physiological characteristics. * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-7416
fax: +81-(0)6-6879-7418
oxidase, expression vector,
Thus, classical and cutaneous propionibacteria differ principally based on their typical natural habitats. Metabolites from propionibacteria Classical or dairy propionibacteria are an industrially important group of microorganisms that have been widely used for the production of vitamin B,,, tetrapyrrole compounds and propionic acid, and in bread making, in the cheese industry, as starter culture for ensilage, and in some pharmaceutical preparations (2, 4-6). Propionic acid, a primary metabolite produced by propionibacteria, has various industrial uses including the production of cellulose plastics, herbicides and perfumes (6). Propionic acid is also an important mold inhibitor and can be used as food and feed preservative. There is considerable evidence that dairy propionibacteria have probiotic effects determined based on their production of propionic acid, bacteriocins, propionicins and vitamin B,,, stimulation of growth of other beneficial bacteria and ability to survive gastric digestion (7-10). Recently, it has been shown that propionibacteria, particularly I? acidipropionici and I? jreudenreichii, can produce and liberate NO by the reduction of nitrate or nitrite (11). In cheese ripening, secondary proteolysis is an important process in which bacteria play a fundamental role in the release of small amount of peptides and amino acids and the formation of flavor compounds. Characterization of enzymes was often carried out using strains of l? freudenreichii subsp. shermanii, due to their major importance as a
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KIATPAPAN
J. BIOSCI. BIOENG.,
AND MUROOKA
starter: for Swiss-type cheese making, which revealed the existence of amino-, carboxyl-, oligo- and endopeptidases in the ripening process (12). A few peptidase genes such as pip have been sequenced from propionibacteria. The pip gene encodes a proline iminopeptidase in p freudenreichii ATCC9617 (13). An esterase of Z? freudenreichii subsp. shermanii was cloned (Annu and Soile, 2001, abst. p. 18, 3rd Inter. Symp. Propionibacteria, Zurich). This enzyme degrades short-chain fatty acid substrates such as p-nitorophenyl butyrate. Furthermore, the contribution of propionibacteria to the formation of free amino acids in cheese has been confirmed at the genetic level (Rossi, 2001, abst. p. 18-19, 3rd Inter. Symp. Propionibacteria, Zurich). II.
DEVELOPMENT OF HOST-VECTOR IN PROPIONIBACTERIA
SYSTEM
Classical propionibacteria are considered to be nonpathogenie organisms. They are attractive as host bacteria for constructing a heterologous gene expression system. Although mutagenesis has been frequently applied to propionibacteria, genetic engineering is still at its early stage (2, 14, 15). Thus, the development of a genetic manipulation system in propionibacteria will enable the production of heterologous proteins and bacteriocins for use in the food industry. Gene cloning may facilitate genetic studies and the production of porphyrins, tetrapyrrole compounds and vitamin B,, in propionibacteria. The lack of genetic manipulation studies in propionibacteria may be due to the following: the high GC content of the organisms, lack of information on plasmid sequence determination, lack of available plasmid vector for gene transfer, the presence of a strong restriction modification system in propionibacteria and lack of a suitable selective marker. Plasmids in propionibacteria Screening and characterization of endogenous plasmids in propionibacteria were first reported in 1990 (Table 1, 16). About 20% of propionibacteria were found to carry one or two plasmids. The plasmid profiles were characterized and distinguished based on size and a restriction map. Plasmids ranged in size from 4.4 Mdal to 119 Mdal or higher and were named pRGO1 TABLE Plasmid
1.
Plasmids
Size (Mda)
pRG0 1
4.4
in Propionibacterium Species
Strain
II acidipropionici
El7 E214 ATCC4875 ATCC14072 PS18 PS49 PP798 El.l.l PJ54 93 93 5932, F32 5932, F32 13 13
&?fieudenreichii
pRG02
6.3 6.3 25 30 5.6 4.4 35
pRG03 pRG07 pRG04 pRG06 pRG0 1 pRGO5 a Reference
( 16).
strains”
T jensenii P: jensenii p freudenreichii p fieudenreichii p freudenreichii FYj?eudenreichii p jensenii F! jensenii
through pRGO7. Z? freudenreichii strains contained the greatest diversity of plasmid profiles while l? acidipropionici strains contained only the 4.4-Mdal plasmid (16). The 4.4-Mdal plasmid, pRG0 1, was commonly found in strains of p ncidipropionici, P fieudenreichii, and t! jensenii. Plasmids pLME106 (6.9 kb) and pLME108 (3.6 kb) were found in I? jensenii and I! fieudenreichii, respectively (17). Small endogenous plasmids, ~545 and ~546, that were found in strains of 19 fieudenreichii LMG 16545 and F! freudenreichii subsp. freudenreichii LMG 16546, respectively, had the same size of 3.6 kb and were closely related to each other, but not to plasmid pRGO1 (18). The nucleotide sequences of plasmids pRGO1 (accession no. AB007909) (19), pLME106 (accession no. AJ250233) (17), pLME 108 (accession no. AJ006662) and ~545 (accession no. AF29 175 1) (18) were determined. Notably, plasmid pLME106 of l? jensenii had 100% nucleotide sequence identity to plasmid pRGO1 of p acidipropionici (20), whereas sequence analysis of orfl and orf2, encoding replication proteins, RepA and RepB of plasmid pRG0 1 showed 46% and 53% identities to those of plasmid ~545, respectively. RepA protein had homology to the theta replicase found in several gram-positive bacteria of high GC content, indicating that plasmids pRGO1, pLME106 and ~545 may replicate via the theta-type replication. In addition, the RepB protein may be responsible for initiating plasmid replication (17-22). Shuttle vectors construction and transformation The early study on transformation in propionibacteria using protoplast transformation and electroporation was reported (23). The broad-host-range plasmid vector pGK12 transformed propionibacteria at a transformation frequency of 6.5 x 10’. The low efficiency of transformation in propionibacteria using the broad-host-range plasmid pGK12, which is widely used in gram-positive bacteria, suggests the plasmid instability of the rolling-circle-type replication in propionibacteria that carry the plasmid of the theta-type replication. Shuttle vectors for use in propionibacteria have recently been developed using a propionibacterial replicon and appropriate selection markers (18, 19). To construct an efficient shuttle vector, several antibiotic resistance genes as selective markers were tested. The hygromycin B resistance gene (hygB’) from Streptomyces hygroscopicus was found to be a suitable marker in both E. coli and propionibacteria. The promoter of the hygB gene was reported to be expressed in several gram-positive bacteria of high GC content, such as mycobacteria (24) and Streptomyces sp., and in E. coli (25,26). A useful shuttle vector, pPK705, was constructed using the pRGO1 replicon, pUCl8, and the Streptomyces hygB’ gene as a drug marker (19) (Fig. 2). Transformation by electroporation was developed using propionibacterial competent cells prepared in 10% glycerol so that the cells could be stored at -80°C for several months. To overcome the high restriction modification system in propionibacteria, the shuttle vector pPK705 was prepared from p freudenreichii IF012426. The high transformation efficiencies, ranging from about lo6 to lo7 cfu/ug of DNA, were obtained in p pentosaceum HUT8606, I? freudenreichii subsp. shermanii
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GENE MANIPULATION IN PROPIONIBACTERIA
IF012426 and HUT 8612, and p freudenreichii ATCC 49 15. I? freudenreichii subsp. freudenreichii IF0 12424 and l? freudenreichii subsp. shermanii P93, which carry endogenous plasmids pRG03 and pRG07 (16), were also transformed, but showed a 1O*-to 103-fold lower efficiency. Jore et al. (18) also constructed a shuttle vector using the ~545 replicon, pBR322, and the erythromycin resistance gene from Saccharopolyspora erythraea to generate pBRESP36A. They tested several sources of the erythromycin resistance gene and found that this gene from Streptomyces aureus was nonfunctional in p freudenreichii (van Luijk et al., 2001, abst. p. 13,3rd Inter. Symp. Propionibacteria, Zurich). The shuttle vector pBRESP36A transformed p freudenreichii strains ATCC 6207, VTBl and LMG 16545 at an efficiency of about 10’ cfu/ug of DNA. However, the shuttle vector pBRESP36A did not transform strains of I? acidipropionici DSM 13572 and DSM 20727, i? jensenii DSM 20535, and p theonii DSM 20276 (18) in contrast with the shuttle vector pPK705 (19). Another shuttle vector based on the plasmid pLME108, isolated from Pr freudenreichii subsp. shermanii DF2, was constructed (Stierli et al., 2001, abst. p. 12, 3rd Inter. Symp. Propionibacteia, Zurich). The success in developing a host vector system in propionibacteria may be due to the use of the replicon from an endogenous plasmid and the Streptomyces hygB’ or the Saccharopolyspora erm6 gene as the selection marker (18, 19). The existence of a strong restriction-modification system in propionibacteria was overcome using the vector prepared from Propionibacterium cells.
III.
PRODUCTION OF CHOLESTEROL IN PROPIONIBACTERIA
3
OXIDASE
A gene encoding cholesterol oxidase (choA) from Streptomyces (27) and its modified form expressed in E. coli (28) have been widely used as a diagnostic tool for cholesterol degradation in several species of bacteria including LactobaciZZus casei (29) and Streptococcus thermophilus (30). Streptomyces choA could be expressed in E. coli under the control of propionibacterial promoters (3 1) at the same level as that under the Zac or tat promoter. Studies of selective markers used in constructing the shuttle vector pPK705 for propionibacteria indicated that the promoter sequence might play a significant role in gene expression in gram-positive bacteria of high GC content. Native promoter fragments, Pl, P4, P8, and P116, from PI freudenreichii subsp. shermanii IF012424 were isolated using the pCVE1 promoter probe vector (28) carrying the choA gene (Fig. 1). The expression vector for Streptomyces choA in propionibacteria was developed (31) (Fig. 2). The Streptomyces choA gene was expressed in PI freudenreichii subsp. shermanii under the control of promoters Pl, P4, and P8 but not promoter P116, suggesting that sequences of some promoter regions are recognized by a sigma factor of RNA polymerase of both E. coli and Propionibacterium. The production of cholesterol oxidase by recombinant ? fieudenreichii subsp. shermanii was studied (3 1). The cholesterol oxidase activity of recombinant I? freudenreichii subsp. shermanii, which harbored the plasmid pPK705COl and carried choA under the control of promoter Pl, was approximately 6.0 U/ml of culture broth after 3 to 4 days of
Choromosomal DNA from P. freudenreichii suhsp. shermanii IF012424
I NdelE.coli DH5 a
Ch ChoA
FIG. 1. Strategy for screening of promoter regions from p fieudenreichii subsp. shermanii IF012424. Chromosomal DNA fragments of about 0.5 kb were inserted at the multiple cloning site of pCVE1. E. coli DH5a was transformed by the recombinant pCVE 1 to generate ampicillin-resisL__L_-?I _L_1__1___1 _..:>___ ___:*:.._ _&._:-_
4
KIATPAPAN AND MUROOKA
.J. BIOSCI. BIOENG.,
Hind111
Hind111
For propionibacteria
NdeI
-For
Expression vector
EcoR1For propionibacteria FIG. 2. Construction of an expression vector used in propionibacteria. An expression vector was constructed from a promoter probe vector, pCVE1, with a promoter fragment from P@eudenreichii subsp. shermanii IF012424. The cholesterol oxidase gene (choA) from Streptomyces sp. was used as a reuorter gene. The three stoo codons renresent three different frames of stop codons for terminating translation, but not transcription allowing recognition orits own SD sequeke and the itart codon of choA.
culture. The maximum cholesterol oxidase activity of recombinant l? jkeudenreichii subsp. shermanii cultured without HygB was similar to or higher than that cultured with HygB, indicating that the plasmid was stably maintained in recombinant propionibacteria for 8 d without selective pressure. The stable production of enzyme for one week confirmed that p fieudenreichii is a promising host for the expression of novel heterologous gene products. IV. PRODUCTION OF ALA IN I! FREUDENREICHII SUBSP. SHEIMANII Genes involved in ALA and vitamin B,, biosynthesis 5-Aminolevulinic acid (ALA) is known as a common precursor of heme, tetrapyrroles, porphyrins, and vitamin B,, in all living organisms. The biosynthesis of tetrapyrroles by all microorganisms starts with the formation of ALA and proceeds through the formation of porphobilinogen (PBG) and uroporphobilinogen (UPB). ALA has a potential to be widely used as a biodegradable herbicide and insecticide, and a therapeutic drug for cancer as well as a growth hormone for plants (32). Study on ALA production in propionibacteria by recombinant DNA technology should be useful in future studies on the production of vitamin B,, by Propionibacterium. ALA is synthesized via either of two pathways, the C4 pathway (Shemin pathway) and the C5 pathway (Fig. 3). In animals, yeast, fungi and certain bacteria, including Rhodobacter species and Rhizobium species, ALA is formed by the condensation of glycine and succinyl CoA
(C4 pathway) catalyzed by ALA synthase (EC 2.3.1.37) (33, 34). The C5 pathway has been reported in plants (35) and in some bacteria including E. coli (36-38), Salmonella typhimurium (39), archaebacteria and anaerobic bacteria (40,41). In the C5 pathway, ALA is formed from glutamate by a series of reactions that include the activation of glutamate by ligation to tRNA, reduction of the activation of glutamate to yield glutamyl 1-semialdehyde (GSA) catalyzed by an NAD(P)H-dependent reductase and transamination of GSA to form ALA catalyzed by a GSA 2,1-aminotransferase to form ALA. The genomic library from P: freudenreichii was constructed to isolate genes involved in tetrapyrrole biosynthesis (42). The hem L gene that encodes GSA 2,1-aminotransferase was identified via complementation of an ALA-deficient mutant (hemL) of E. coli (43). This result suggests that ALA in propionibacteria is synthesized via the C5 pathway which is supported by the result using a 14C-labeled substrate (44). However, recent report of analysis using 13C-labeled glucose suggests that ALA in l? freudenreichii is synthesized via both the C4 and C5 pathways (45). However, until now, there is no report of hemA encoding ALA synthase in propionibacteria. ALA is metabolized to porphobilinogen (PBG), a precursor of tetrapyrrole biosynthesis, by PBG synthase (or ALA dehydratase). The hemB gene encoding PBG synthase in p1freudenreichii was cloned by complementation to the hemB mutant of E. coli (42). The regions upstream and downstream of the hemB gene were sequenced, and it was found that the hemY gene en-
GENE MANIPULATION IN PROPIONIBACTERIA
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5
Hemes Chloropllyus FIG. 3. Biosynthesis pathway of tetrapyrrole, vitamin B,,, heme and chlorophyll via ALA. coding protoporphyrinogen oxidase and the hemH gene encoding ferrochelatase located immediately upstream of the hemB gene (46). Between the hemYHB and hemL genes, two open reading frames were found and named hemX and hemR, respectively. These two genes appear to be involved in hem transport and regulation of the hem gene expression. Using the genomic library from l? fieudenreichii, cobA, a gene encoding uroporphyrinogen III methyltransferase was identified (47). Uroporphyrinogen III methyltransferase of R fieudenreichii catalyzes not only the addition of two methyl groups to uroporphyrinogen III to yield the early vitamin B,, intermediate, precorrin-2, but also the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B,, pathway due to its overmethylation property. The enzyme catalyzes the addition of three methyl groups to proporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of natural products, e.g., factors Sl (48) and S3 (49), previously isolated from this organism. A gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport
protein considered to be part of the cobalt transport system involved in vitamin B,, synthesis (47). These two genes do not appear to constitute part of an extensive cobalamin operon. Expression of heterologous hemA gene and ALA accumulation in propionibacteria The expression of the heterologous gene may require an efficient gene cloning system, stability of plasmid-carrying foreign DNA and production of the foreign protein. To overproduce ALA in propionibacteria via the C4 pathway, which is a one-step synthesis process from glycine, the expression vectors pKHEM0 1and pKHEM04 were constructed using hemA encoding ALA synthase from Rhodobacter sphaeroides (50), propionibacterial promoters, P 1 and P4, and the shuttle vector pPK705 (Fig. 2, 5 1). Promoters Pl and P4 controlled the production of ALA and PBG in recombinant E. coli at the same level (Table 2). The production level of ALA and PBG in recombinant propionibacteria under the control of promoter P4 (pKHEMO4) was twice that under the control of promoter Pl (pKHEMOl), indicating that promoter P4 was more effective in controlling the expression of Rhodobacter
TABLE 2. Production of ALA and PBG by E. coli DH5a and l? fieudenreichii subsp. shermanii IF012426 carrying various plasmids Addition of Bacterium
Plasmid
E. coli”
pPK705 (he&) pKHEM0 1 (P l-hen&) pKHEM04 (P4-hemA+) pPK705 (hemA) pKHEMO1 (Pl-he&+) pKHEM04 (P4-hen&) pKHEM04 (P4-hemA+) pKHEM04 (P4-hen&+) pKHEM04 (P4-herd’)
p1fieudenreichil*
Production of
glycine (30 mM)
levulinic acid (1 mM)
-
-
_ _ _ + + + + ampicillin. Pl- and P4-her&+ in parentheses
PBG (mM) 0.03 0.55 0.58 0.04 0.07 0.21 1.04 0.23 0.23
0.1 9.1 9.7 1.0 2.2 5.0 7.9 6.6 8.3 indicate that the hemA gene was
a Cells were grown for 24 h in LB medium containing 100 &ml under the control of the Pl and P4 promoters, respectively. b Cells were grown for 72 h in GYT medium with 0.5% (NH,),SO, and 10 mg/l CoCl,.6H,O in the presence or absence of glycine or levulinic acid.
6
KIATPAPAN
J. BIOSCI. BIOENG.,
AND MUROOKA -13
-ml0 .. .. -.
2 s f B
-.5
3 .
Time (d) FIG. 4. Time course of production of ALA and PBG in recombinant P fieudenreichii subsp. shermaniiIF012426 carrying pKHEM04 (51).
hemA than promoter Pl in propionibacteria. The effects of glycine, a substrate of ALA synthesis, and levulinic acid, a competitive inhibitor of PBG synthase, on ALA synthesis were studied. It was found that levulinic acid inhibited propionibacterial cell growth and resulted in a small increase in ALA, although PBG synthesis was also inhibited. However, in the presence of 1 mM levulinic acid and 30 mM glycine, the production of ALA per cell increased to 8.3 mM. In the fermentation experiment, ALA production increased linearly for 36 h and gradually increased for 6 d (Fig. 4). PBG gradually decreased after 4 d of cultivation.This is the first report that the production of ALA by I! freudenreichii subsp. shermanii is increased by heterologous gene cloning in propionibacteria, although ALA production via the C4 pathway in a recombinant E. coli (52) and mutant R. sphaeroides (53) strains has been reported. The production of ALA and heme via the C, pathway in E. coli has also been reported (54). The production of ALA by R jkeudenreichii subsp. shermanii was comparable with that by the recombinant E. coli. CONCLUSION Propionibacteria isolated from cheese and milk, which are known as dairy or classical propionibacteria, are nonpathogenic organisms and thus are attractive for use as a host bacterium in genetic engineering. A genetic manipulation system should provide a powerful tool for cloning an interesting gene and its expression in propionibacteria. Although there have been several attempts to develop a gene manipulation system in propionibacteria, no report of the system has been published. In 2000, a host-vector system in propionibacteria was developed in several laboratories and thus the genetic manipulation in propionibacteria became possible. In this review, production of cholesterol oxidase and ALA using an expression vector in propionibacteria was demonstrated. Study on ALA production in propionibacteria by recombinant DNA technology should be useful in future studies on the production of vitamin B,, by Propi-
onibacterium. The genes involved in vitamin B,, production and their expression to overproduce vitamin B,, or tetrapyrrole compounds are under investigation in author’s laboratory. Molecular breeding of propionibacterial strains with some useful genes will also have a potential use in probiotic studies and in the dairy food industry. REFERENCES 1. Sneath, P.H.A., Mair, N.S., Sharpe, M.E., and Holt, J. G.: Bergey’s manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore, Md. (1986). 2. Johnson, J. L. and Cummins, C. S.: Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachniupropionica. J. Bacterial., 109, 1047-1066 (1972). 3. Vorobjeva, L. I.: Propionibacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands (1999). 4. Florent, J. and Ninet, L.: Vitamin B12, p. 497-5 19. In Peppler, H. J. and Perlman, D. (ed.), Microbial technology, 2nd ed., vol. 1. Academic Press, New York (1979). 5. Langsrud, T. and Reinbold, G. W.: Flavor development and microbiology of Swiss cheese - a review. II. Starters, manufacturing processes and procedures. J. Milk Food Technol., 36,53 l-542 (1973). 6. Playne, M. J.: Propionic and butyric acids, p. 731-759. In Moo-Young, M. (ed.), Comprehensive biotechnology, vol. 3. Pergamon Press, Oxford, United Kingdom (1985). 7. Ko, II. L., Pulverer, G., and Jeljaszewicz, J.: Propionicins, bacteriocin produced by Propionibacterium avidum. Zentralbl Bakteriol., 241, 325-328 (1978). 8. Grinstead, D.A. and Barefoot, S. F.: Jenseniin 4 a heat stable bacteriocin produced by Propionibacterium jensenii P126. Appl. Environ. Microbial., 58,215-220 (1992). 9. Lyon, W. J. and Glatz, B.A.: Isolation and purification of propionicin PLG- 1, a bacteriocin produced by a strain of Propionibacterium theonii. Appl. Environ. Microbial., 59, 83-88 (1993). 10. Fay, T., Langsrud, T., Nes, I. F., and Holo, H.: Biochemical and genetic characterization of propionicin Tl, a new bacteriocin from Propionibacterium theonii. Appl. Environ. Microbiol., 66,4230-4236 (2000). 11. Avice, J. C., Ourry, A., Laine, P., Roland, N., Louahlia, S., Roussel, E., Brookes, S., and Boucaud, J.: A rapid and reliable method for NO quantification and 15N0/14NO determination using isotope ratio mass spectrometry: an application for the detection of NO synthesis in propionibacteria. Rapid communication in mass spectrometry, 13(Issue 12), 1197-1200 (1999). 12. Gagnaire, V., Molle, D., Sorhuang, T., and Lerje, J.: Peptidases of dairy propionic acid bacteria. Le Lait, 79, 43-57 (1999). 13. Leehouts, K., Bolhuis, A., Boot, J., Deutz, I., Toon, M., Verema, G., Kok, J., and Ledeboer, A.: Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis. Appl. Environ. Microbial.,
64,473&4742 (1998). 14. Hotherr, L. A. and Glatz, B.A.: Mutagenesis of strains of Propionibacterium produce cold sensitive mutants. J. Dairy Sci., 66,2482-2487 (1983). 15. Paik, H. D. and Glatz, B. A.: Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidopropionici. Appl. Microbial. Biotechnol., 42, 22-27 (1994). 16. Rehberger, T. G. and Glatz, B.A.: Characterization of Propionibacterium plasmids. Appl. Environ. Microbial., 56, 864-871 (1990).
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17. Miescher, S., Sterli, M. P., Teuber, M., and Meile, L.: Propionicin SMl, a bacteriocin from Propionibacterium jensenii DFl: isolation and characterization of the protein and its gene. Syst. Appl. Microbial., 23, 174-184 (2000). 18. Jore, J. P. M., Lujik van, N., Luiten, R G. M., Werf van der M. J., and Pouwels, P. II.: Efficient transformation system for Propionibacterium freudenreichii based on a novel vector. Appl. Environ. Microbial., 67,499-503 (2001). 19. Kiatpapan, P., Hashimoto, Y., Nakamura, H., Piao, Y-Z., Yamashita, M., Ono, H., and Murooka, Y.: Characterization of pRG0 1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria. Appl. Environ. Microbial., 66, 46884695 (2000). 20. Kiatpapan, P., Hashimoto, Y., Nakamura, H., Piao, Y-Z., Yamashita, M., 0110, H., and Murooka, Y.: Characterization of pRG0 1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria (Author correction). Appl. Environ. Microbiol., 67, 1024 (2001). 21. Ankri, S., Bouvier, I., Reyes, O., Predalu, F., and Leblon, G.: A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum. Plasmid, 36, 3Wl (1996). 22. Stolt, P. and Stoker, N. G.: Protein-DNA interactions in the ori region of the Mycobacterium fort&urn plasmid pAL5000. J. Bacterial., 178,6693-6700 (1996). 23. Luchansky, J.B., Muriana, P.M., and Klaenhammer, T. R: Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. Mol. Microbial., 2, 637446 (1988). 24. Garbe, R.T., Barathi, J., Barnini, S., Zbang, Y., AbouZeid, C., Tang, D., Mukherjee, R., and Young, B. D.: Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology, 140, 133-l 38 (1994). 25. Pernodet, J. L.: Antibiotic resistant gene cassettes derived from the interposon for use in E. coli and Streptomyces. Gene, 190,315-317(1997). 26. Pulido, D., Zalacain, M., and Jimenez, A.: The hyg gene promoter from Streptomyces hygroscopicus: a novel form of Streptomyces promoter. Biochim. Biophys. Res. Commun., 150,270-274 (1988). 27. Murooka, Y., Ishizaki, T., Nimi, O., and Maekawa, N.: Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702. Appl. Environ. Microbial., 52, 1382-1385 (1986). 28. Nomura, N., Choi, K.P., Yamashita, M., Yamamoto, H., and Murooka, Y.: Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria. J. Ferment. Bioeng., 79,410-416 (1995). 29. Somkuti, G.A., Daniel, K. Y., Solaiman, D. K., and Steinberg, D. H.: Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei. Appl. Microbial. Biotechnol., 37,330-334 (1992). 30. Somkuti, G. A., Solaiman, D. K., and Steinberg, D. H.: Native promoter-plasmid vector system for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus. Plasmid, 33, 7-14 (1995). 31. Kiatpapan, P., Yamashita, M., Kawaraichi, N., Yasuda, T., and Murooka, Y.: Heterologous expression of a gene for cholesterol oxidase in probiotic strains of Lactobacillus plantarum and Propionibacterium freudenreichii under the control of native promoters. J. Biosci. Bioeng., 92,459465 (2001). 32. Nishikawa, S. and Murooka, Y.: 5-Aminolevulinic acid (ALA): the production by fermentation and agricultural and biochemical applications, p. 149-170. In Harding, S. E. (ed.), Biotechnology and genetic engineering reviews, vol. 18,
GENE MANIPULATION IN PROPIONIBACTERIA
7
Chapter 7. Intercept, Andover, UK (2001). 33. Brown, C. E., Shemin, D., and Katz, J. J.: Nuclear magnetic resonance studies of the biosynthesis of vitamin B,,. J. Biol. Chem., 248,8015-8021 (1973). 34. Granick, S. and Beale, S. I.: Hemes, chlorophylls and related compounds: biosynthesis and metabolic regulation. Adv. Enzymol., 46, 33-203 (1978). 35. Beale, S. I. and Castelfranco, P. A.: The biosynthesis of 6aminolevulinic acid in higher plants. II. Formation of 14C 6aminolevulinic acid from labeled precursors in greening plant tissues. Plant Physiol., 53,297-303 (1974). 36. Ikemi, M., Murakami, K, Hashimoto, M., and Murooka, Y.: Cloning and characterization of genes involved in the biosynthesis of &aminolevulinic acid in Escherichia coli. Gene, 121, 127-132 (1992). 37. Ilaq, L. L., Jahn, D., Eggertsson, G., and Soll, D.: The Escherichia coli hemL gene encodes glutamate l-semialdehyde aminotransferase. J. Bacterial., 173,3408-3413 (1991). 38. Li, J. M., Russel, C. S., and Cosloy, D.: Cloning and structure of the hemA gene of Escherichia coli K12. Gene, 82, 209-217 (1989). 39. Elliott, T. and Roth, J. R.: Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis. Mol. Gen. Genet.. 216. 303-314 (19891. 40. Avissar, Y. J., O;me&d, J. G., ahd Beale, S. I.: Distribution of &aminolevulinic acid biosynthesis pathways among phototrophic bacterial groups. Arch. Microbial., 151, 5 13-519 (1989). 41. Fugino, E, Fugino, T., Karita, S., Sakka, K., and Ohmiya, K.: Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui. J. Bacterial., 177, 5 169-5 175 (1995). 42. Hashimoto, Y., Yamashita, M., Ono, H., and Murooka, Y.: Characterization of the hemB gene encoding b-aminolevulinic acid dehydratase from Propionibacterium fieudenreichii. J. Ferment. Bioeng., 82, 93-100 (1996). 43. Murakami, K., Hashimoto, Y., and Murooka, Y.: Cloning and characterization of the gene encoding glutamate l-semialdehyde 2,1-aminomutase, which is involved in &aminolevulinic acid synthesis in Propionibacterium freudenreichii. Appl. Environ. Microbial., 59,347-350 (1993). 44. Scott, A. I., Townsend, C. A., Okuda, K., and Kajiwara, M.: Biosynthesis of corrins. I. Experiment with [‘4C]porphobilinogen and [“‘Cluroporphyrinogens. J. Am. Chem. Sot., 96, 8054-8069 (1974). 45. Iida, K. and Kajiwara, M.: Evaluation of biosynthetic pathways to &aminolevulinic acid in based on biosynthesis of vitamin B12. Biochemistry, 39, 3666-3670 (2000). 46. Hashimoto, Y., Yamashita, M., and Murooka, Y.: The Propionibacterium fieudenreichii hemYHBXU gene cluster, which encodes enzymes and a regulator involved in the biosynthetic pathway from glutamate to protoheme. Appl. Microbiol. Biotecbnol., 47, 385-392 (1997). 47. Sattler, I., Roessuner, C. A., Stolowich, N. J., Hardin, S. H., Harris-Haller, L. W., Yokubaitis, N. T., Murooka, Y., Hashimoto, Y., and Scott, A. I.: Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium ffeudenreichii (shermanii). J. Bacterial., 177, 1564-1569 (1995). 48. Muller, G., Schmiedl, J., Sawidis, I., Wirth, G., Scott, A. I., Santander, P. J., Williams, H. J., Stolowich, N. J., and Kriemler, H. P.: Factor Sl, a natural corphin from Propionibacterium shermanii. J. Am. Chem. Sot., 109, 6902-6904 (1987). 49. Muller, G., Schmiedl, J., Schneider, R., Worner, G., Scott, A. I., Williams, H. J., Santander, P. J., Stolowich, N. J., Fagerness, P. E., Mackenzie, N. E., and Kriemler, H. P.: Structure of Factor S3, a metabolite of Propionibacterium shermanii derived from uroporphyrinogen I. J. Am. Chem.
8
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Sot., 108,7875-7877 (1986). 50. Neidle, E. L. and Kaplan, S.: Expression of the Rhodobacter sphaeroides hem4 and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes. J. Bacterial., 175, 22922303 (1993). 5 1. Kiatpapan, P. and Murooka, Y.: Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium fieudenreichii. Appl. Microbial. Biotechnol., 56, 144-149 (2001). 52. Werf, M. J. and Zeikus, J. G.: 5Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaero-
J. Broscr. BIOENG.,
ides hemA gene. Appl. Environ. Microbial., 62, 356&3566 (1996). 53. Nishikawa, S., Watanabe, K., Tanaka, T., Miyachi, N., Hotta, Y., and Murooka, Y.: Rhodobacter sphaeroides mutants which accumulate 5aminolevulinic acid under aerobic and dark conditions. J. Biosci. Bioeng., 87,798-804 (1999). 54. Verderber, E., Lucast, L. J., van Dehy, J. A., Cozart, P., Etter, J. B., and Best, E. A.: Role of the hemA gene product and &aminolevulinic acid in regulation of Escherichiu coli heme synthesis. J. Bacterial., 179,4583-4590 (1997).
REVIEW Genetic Manipulation System in Propionibacteria PORNPIMON
KIATPAPAN’
AND
YOSHIKATSU
MUROOKA2*
Biochemistry Unit, School of Science, Rangsit University, Patumthani 12000, Thailand’ and Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita, Osaka 5650871, Japan’ Received 5 September 2001iAccepted 20 September 2001
Members of the genus Propionibacterium are widely used in the production of vitamin B,,, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propionibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibacterium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in L freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin B,, biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in E!freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probiotics and the vitamin B,, biosynthetic pathway. [Key words: propionibacteria, vitamin B,,]
I.
PROPERTIES
5-aminolevulinic
acid, ALA synthase, cholesterol
OF PROPIONIBACTERIA
Classification of propionibacteria Propionibacteria are gram-positive, nonmotile, non-spore-forming, facultatively anaerobic, and rodlike bacteria. The G+C content of their DNA is in the range of 53-67 mol% (1). The genus Propionibacterium is classified into two groups, the classical or dairy propionibacteria and the acne group or cutaneous propionibacteria. The main habitat of cutaneous propionibacteria is human skin, whereas that of dairy or classical propionibacteria are cheese and milk. On the basis of DNA homology and similarities in cell wall composition (2), dairy or classical propionibacteria are classified into six species (3): Propionibacterium freudenreichii, P jensenii, I? theonii, l? acidipropionici, P coccoides, and p cyclohexanicum. Similarly, cutaneous bacteria are classified into six species: p acnes, I? avidium, I? granulosum, I! lymphophilums, P propionicum, and Propioniferax innocua. Cutaneous propionibacteria are also known as anaerobic corynefotms or anaerobic diphtheroides. In contrast with cutaneous propionibacteria, classical propionibacteria do not produce indole and cannot liquefy gelatin. Other than these, the two groups cannot be clearly distinguished based on other physiological characteristics. * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-7416
fax: +81-(0)6-6879-7418
oxidase, expression vector,
Thus, classical and cutaneous propionibacteria differ principally based on their typical natural habitats. Metabolites from propionibacteria Classical or dairy propionibacteria are an industrially important group of microorganisms that have been widely used for the production of vitamin B,,, tetrapyrrole compounds and propionic acid, and in bread making, in the cheese industry, as starter culture for ensilage, and in some pharmaceutical preparations (2, 4-6). Propionic acid, a primary metabolite produced by propionibacteria, has various industrial uses including the production of cellulose plastics, herbicides and perfumes (6). Propionic acid is also an important mold inhibitor and can be used as food and feed preservative. There is considerable evidence that dairy propionibacteria have probiotic effects determined based on their production of propionic acid, bacteriocins, propionicins and vitamin B,,, stimulation of growth of other beneficial bacteria and ability to survive gastric digestion (7-10). Recently, it has been shown that propionibacteria, particularly I? acidipropionici and I? jreudenreichii, can produce and liberate NO by the reduction of nitrate or nitrite (11). In cheese ripening, secondary proteolysis is an important process in which bacteria play a fundamental role in the release of small amount of peptides and amino acids and the formation of flavor compounds. Characterization of enzymes was often carried out using strains of l? freudenreichii subsp. shermanii, due to their major importance as a
2
KIATPAPAN
J. BIOSCI. BIOENG.,
AND MUROOKA
starter: for Swiss-type cheese making, which revealed the existence of amino-, carboxyl-, oligo- and endopeptidases in the ripening process (12). A few peptidase genes such as pip have been sequenced from propionibacteria. The pip gene encodes a proline iminopeptidase in p freudenreichii ATCC9617 (13). An esterase of Z? freudenreichii subsp. shermanii was cloned (Annu and Soile, 2001, abst. p. 18, 3rd Inter. Symp. Propionibacteria, Zurich). This enzyme degrades short-chain fatty acid substrates such as p-nitorophenyl butyrate. Furthermore, the contribution of propionibacteria to the formation of free amino acids in cheese has been confirmed at the genetic level (Rossi, 2001, abst. p. 18-19, 3rd Inter. Symp. Propionibacteria, Zurich). II.
DEVELOPMENT OF HOST-VECTOR IN PROPIONIBACTERIA
SYSTEM
Classical propionibacteria are considered to be nonpathogenie organisms. They are attractive as host bacteria for constructing a heterologous gene expression system. Although mutagenesis has been frequently applied to propionibacteria, genetic engineering is still at its early stage (2, 14, 15). Thus, the development of a genetic manipulation system in propionibacteria will enable the production of heterologous proteins and bacteriocins for use in the food industry. Gene cloning may facilitate genetic studies and the production of porphyrins, tetrapyrrole compounds and vitamin B,, in propionibacteria. The lack of genetic manipulation studies in propionibacteria may be due to the following: the high GC content of the organisms, lack of information on plasmid sequence determination, lack of available plasmid vector for gene transfer, the presence of a strong restriction modification system in propionibacteria and lack of a suitable selective marker. Plasmids in propionibacteria Screening and characterization of endogenous plasmids in propionibacteria were first reported in 1990 (Table 1, 16). About 20% of propionibacteria were found to carry one or two plasmids. The plasmid profiles were characterized and distinguished based on size and a restriction map. Plasmids ranged in size from 4.4 Mdal to 119 Mdal or higher and were named pRGO1 TABLE Plasmid
1.
Plasmids
Size (Mda)
pRG0 1
4.4
in Propionibacterium Species
Strain
II acidipropionici
El7 E214 ATCC4875 ATCC14072 PS18 PS49 PP798 El.l.l PJ54 93 93 5932, F32 5932, F32 13 13
&?fieudenreichii
pRG02
6.3 6.3 25 30 5.6 4.4 35
pRG03 pRG07 pRG04 pRG06 pRG0 1 pRGO5 a Reference
( 16).
strains”
T jensenii P: jensenii p freudenreichii p fieudenreichii p freudenreichii FYj?eudenreichii p jensenii F! jensenii
through pRGO7. Z? freudenreichii strains contained the greatest diversity of plasmid profiles while l? acidipropionici strains contained only the 4.4-Mdal plasmid (16). The 4.4-Mdal plasmid, pRG0 1, was commonly found in strains of p ncidipropionici, P fieudenreichii, and t! jensenii. Plasmids pLME106 (6.9 kb) and pLME108 (3.6 kb) were found in I? jensenii and I! fieudenreichii, respectively (17). Small endogenous plasmids, ~545 and ~546, that were found in strains of 19 fieudenreichii LMG 16545 and F! freudenreichii subsp. freudenreichii LMG 16546, respectively, had the same size of 3.6 kb and were closely related to each other, but not to plasmid pRGO1 (18). The nucleotide sequences of plasmids pRGO1 (accession no. AB007909) (19), pLME106 (accession no. AJ250233) (17), pLME 108 (accession no. AJ006662) and ~545 (accession no. AF29 175 1) (18) were determined. Notably, plasmid pLME106 of l? jensenii had 100% nucleotide sequence identity to plasmid pRGO1 of p acidipropionici (20), whereas sequence analysis of orfl and orf2, encoding replication proteins, RepA and RepB of plasmid pRG0 1 showed 46% and 53% identities to those of plasmid ~545, respectively. RepA protein had homology to the theta replicase found in several gram-positive bacteria of high GC content, indicating that plasmids pRGO1, pLME106 and ~545 may replicate via the theta-type replication. In addition, the RepB protein may be responsible for initiating plasmid replication (17-22). Shuttle vectors construction and transformation The early study on transformation in propionibacteria using protoplast transformation and electroporation was reported (23). The broad-host-range plasmid vector pGK12 transformed propionibacteria at a transformation frequency of 6.5 x 10’. The low efficiency of transformation in propionibacteria using the broad-host-range plasmid pGK12, which is widely used in gram-positive bacteria, suggests the plasmid instability of the rolling-circle-type replication in propionibacteria that carry the plasmid of the theta-type replication. Shuttle vectors for use in propionibacteria have recently been developed using a propionibacterial replicon and appropriate selection markers (18, 19). To construct an efficient shuttle vector, several antibiotic resistance genes as selective markers were tested. The hygromycin B resistance gene (hygB’) from Streptomyces hygroscopicus was found to be a suitable marker in both E. coli and propionibacteria. The promoter of the hygB gene was reported to be expressed in several gram-positive bacteria of high GC content, such as mycobacteria (24) and Streptomyces sp., and in E. coli (25,26). A useful shuttle vector, pPK705, was constructed using the pRGO1 replicon, pUCl8, and the Streptomyces hygB’ gene as a drug marker (19) (Fig. 2). Transformation by electroporation was developed using propionibacterial competent cells prepared in 10% glycerol so that the cells could be stored at -80°C for several months. To overcome the high restriction modification system in propionibacteria, the shuttle vector pPK705 was prepared from p freudenreichii IF012426. The high transformation efficiencies, ranging from about lo6 to lo7 cfu/ug of DNA, were obtained in p pentosaceum HUT8606, I? freudenreichii subsp. shermanii
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GENE MANIPULATION IN PROPIONIBACTERIA
IF012426 and HUT 8612, and p freudenreichii ATCC 49 15. I? freudenreichii subsp. freudenreichii IF0 12424 and l? freudenreichii subsp. shermanii P93, which carry endogenous plasmids pRG03 and pRG07 (16), were also transformed, but showed a 1O*-to 103-fold lower efficiency. Jore et al. (18) also constructed a shuttle vector using the ~545 replicon, pBR322, and the erythromycin resistance gene from Saccharopolyspora erythraea to generate pBRESP36A. They tested several sources of the erythromycin resistance gene and found that this gene from Streptomyces aureus was nonfunctional in p freudenreichii (van Luijk et al., 2001, abst. p. 13,3rd Inter. Symp. Propionibacteria, Zurich). The shuttle vector pBRESP36A transformed p freudenreichii strains ATCC 6207, VTBl and LMG 16545 at an efficiency of about 10’ cfu/ug of DNA. However, the shuttle vector pBRESP36A did not transform strains of I? acidipropionici DSM 13572 and DSM 20727, i? jensenii DSM 20535, and p theonii DSM 20276 (18) in contrast with the shuttle vector pPK705 (19). Another shuttle vector based on the plasmid pLME108, isolated from Pr freudenreichii subsp. shermanii DF2, was constructed (Stierli et al., 2001, abst. p. 12, 3rd Inter. Symp. Propionibacteia, Zurich). The success in developing a host vector system in propionibacteria may be due to the use of the replicon from an endogenous plasmid and the Streptomyces hygB’ or the Saccharopolyspora erm6 gene as the selection marker (18, 19). The existence of a strong restriction-modification system in propionibacteria was overcome using the vector prepared from Propionibacterium cells.
III.
PRODUCTION OF CHOLESTEROL IN PROPIONIBACTERIA
3
OXIDASE
A gene encoding cholesterol oxidase (choA) from Streptomyces (27) and its modified form expressed in E. coli (28) have been widely used as a diagnostic tool for cholesterol degradation in several species of bacteria including LactobaciZZus casei (29) and Streptococcus thermophilus (30). Streptomyces choA could be expressed in E. coli under the control of propionibacterial promoters (3 1) at the same level as that under the Zac or tat promoter. Studies of selective markers used in constructing the shuttle vector pPK705 for propionibacteria indicated that the promoter sequence might play a significant role in gene expression in gram-positive bacteria of high GC content. Native promoter fragments, Pl, P4, P8, and P116, from PI freudenreichii subsp. shermanii IF012424 were isolated using the pCVE1 promoter probe vector (28) carrying the choA gene (Fig. 1). The expression vector for Streptomyces choA in propionibacteria was developed (31) (Fig. 2). The Streptomyces choA gene was expressed in PI freudenreichii subsp. shermanii under the control of promoters Pl, P4, and P8 but not promoter P116, suggesting that sequences of some promoter regions are recognized by a sigma factor of RNA polymerase of both E. coli and Propionibacterium. The production of cholesterol oxidase by recombinant ? fieudenreichii subsp. shermanii was studied (3 1). The cholesterol oxidase activity of recombinant I? freudenreichii subsp. shermanii, which harbored the plasmid pPK705COl and carried choA under the control of promoter Pl, was approximately 6.0 U/ml of culture broth after 3 to 4 days of
Choromosomal DNA from P. freudenreichii suhsp. shermanii IF012424
I NdelE.coli DH5 a
Ch ChoA
FIG. 1. Strategy for screening of promoter regions from p fieudenreichii subsp. shermanii IF012424. Chromosomal DNA fragments of about 0.5 kb were inserted at the multiple cloning site of pCVE1. E. coli DH5a was transformed by the recombinant pCVE 1 to generate ampicillin-resisL__L_-?I _L_1__1___1 _..:>___ ___:*:.._ _&._:-_
4
KIATPAPAN AND MUROOKA
.J. BIOSCI. BIOENG.,
Hind111
Hind111
For propionibacteria
NdeI
-For
Expression vector
EcoR1For propionibacteria FIG. 2. Construction of an expression vector used in propionibacteria. An expression vector was constructed from a promoter probe vector, pCVE1, with a promoter fragment from P@eudenreichii subsp. shermanii IF012424. The cholesterol oxidase gene (choA) from Streptomyces sp. was used as a reuorter gene. The three stoo codons renresent three different frames of stop codons for terminating translation, but not transcription allowing recognition orits own SD sequeke and the itart codon of choA.
culture. The maximum cholesterol oxidase activity of recombinant l? jkeudenreichii subsp. shermanii cultured without HygB was similar to or higher than that cultured with HygB, indicating that the plasmid was stably maintained in recombinant propionibacteria for 8 d without selective pressure. The stable production of enzyme for one week confirmed that p fieudenreichii is a promising host for the expression of novel heterologous gene products. IV. PRODUCTION OF ALA IN I! FREUDENREICHII SUBSP. SHEIMANII Genes involved in ALA and vitamin B,, biosynthesis 5-Aminolevulinic acid (ALA) is known as a common precursor of heme, tetrapyrroles, porphyrins, and vitamin B,, in all living organisms. The biosynthesis of tetrapyrroles by all microorganisms starts with the formation of ALA and proceeds through the formation of porphobilinogen (PBG) and uroporphobilinogen (UPB). ALA has a potential to be widely used as a biodegradable herbicide and insecticide, and a therapeutic drug for cancer as well as a growth hormone for plants (32). Study on ALA production in propionibacteria by recombinant DNA technology should be useful in future studies on the production of vitamin B,, by Propionibacterium. ALA is synthesized via either of two pathways, the C4 pathway (Shemin pathway) and the C5 pathway (Fig. 3). In animals, yeast, fungi and certain bacteria, including Rhodobacter species and Rhizobium species, ALA is formed by the condensation of glycine and succinyl CoA
(C4 pathway) catalyzed by ALA synthase (EC 2.3.1.37) (33, 34). The C5 pathway has been reported in plants (35) and in some bacteria including E. coli (36-38), Salmonella typhimurium (39), archaebacteria and anaerobic bacteria (40,41). In the C5 pathway, ALA is formed from glutamate by a series of reactions that include the activation of glutamate by ligation to tRNA, reduction of the activation of glutamate to yield glutamyl 1-semialdehyde (GSA) catalyzed by an NAD(P)H-dependent reductase and transamination of GSA to form ALA catalyzed by a GSA 2,1-aminotransferase to form ALA. The genomic library from P: freudenreichii was constructed to isolate genes involved in tetrapyrrole biosynthesis (42). The hem L gene that encodes GSA 2,1-aminotransferase was identified via complementation of an ALA-deficient mutant (hemL) of E. coli (43). This result suggests that ALA in propionibacteria is synthesized via the C5 pathway which is supported by the result using a 14C-labeled substrate (44). However, recent report of analysis using 13C-labeled glucose suggests that ALA in l? freudenreichii is synthesized via both the C4 and C5 pathways (45). However, until now, there is no report of hemA encoding ALA synthase in propionibacteria. ALA is metabolized to porphobilinogen (PBG), a precursor of tetrapyrrole biosynthesis, by PBG synthase (or ALA dehydratase). The hemB gene encoding PBG synthase in p1freudenreichii was cloned by complementation to the hemB mutant of E. coli (42). The regions upstream and downstream of the hemB gene were sequenced, and it was found that the hemY gene en-
GENE MANIPULATION IN PROPIONIBACTERIA
VOL. 93,2002
5
Hemes Chloropllyus FIG. 3. Biosynthesis pathway of tetrapyrrole, vitamin B,,, heme and chlorophyll via ALA. coding protoporphyrinogen oxidase and the hemH gene encoding ferrochelatase located immediately upstream of the hemB gene (46). Between the hemYHB and hemL genes, two open reading frames were found and named hemX and hemR, respectively. These two genes appear to be involved in hem transport and regulation of the hem gene expression. Using the genomic library from l? fieudenreichii, cobA, a gene encoding uroporphyrinogen III methyltransferase was identified (47). Uroporphyrinogen III methyltransferase of R fieudenreichii catalyzes not only the addition of two methyl groups to uroporphyrinogen III to yield the early vitamin B,, intermediate, precorrin-2, but also the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B,, pathway due to its overmethylation property. The enzyme catalyzes the addition of three methyl groups to proporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of natural products, e.g., factors Sl (48) and S3 (49), previously isolated from this organism. A gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport
protein considered to be part of the cobalt transport system involved in vitamin B,, synthesis (47). These two genes do not appear to constitute part of an extensive cobalamin operon. Expression of heterologous hemA gene and ALA accumulation in propionibacteria The expression of the heterologous gene may require an efficient gene cloning system, stability of plasmid-carrying foreign DNA and production of the foreign protein. To overproduce ALA in propionibacteria via the C4 pathway, which is a one-step synthesis process from glycine, the expression vectors pKHEM0 1and pKHEM04 were constructed using hemA encoding ALA synthase from Rhodobacter sphaeroides (50), propionibacterial promoters, P 1 and P4, and the shuttle vector pPK705 (Fig. 2, 5 1). Promoters Pl and P4 controlled the production of ALA and PBG in recombinant E. coli at the same level (Table 2). The production level of ALA and PBG in recombinant propionibacteria under the control of promoter P4 (pKHEMO4) was twice that under the control of promoter Pl (pKHEMOl), indicating that promoter P4 was more effective in controlling the expression of Rhodobacter
TABLE 2. Production of ALA and PBG by E. coli DH5a and l? fieudenreichii subsp. shermanii IF012426 carrying various plasmids Addition of Bacterium
Plasmid
E. coli”
pPK705 (he&) pKHEM0 1 (P l-hen&) pKHEM04 (P4-hemA+) pPK705 (hemA) pKHEMO1 (Pl-he&+) pKHEM04 (P4-hen&) pKHEM04 (P4-hemA+) pKHEM04 (P4-hen&+) pKHEM04 (P4-herd’)
p1fieudenreichil*
Production of
glycine (30 mM)
levulinic acid (1 mM)
-
-
_ _ _ + + + + ampicillin. Pl- and P4-her&+ in parentheses
PBG (mM) 0.03 0.55 0.58 0.04 0.07 0.21 1.04 0.23 0.23
0.1 9.1 9.7 1.0 2.2 5.0 7.9 6.6 8.3 indicate that the hemA gene was
a Cells were grown for 24 h in LB medium containing 100 &ml under the control of the Pl and P4 promoters, respectively. b Cells were grown for 72 h in GYT medium with 0.5% (NH,),SO, and 10 mg/l CoCl,.6H,O in the presence or absence of glycine or levulinic acid.
6
KIATPAPAN
J. BIOSCI. BIOENG.,
AND MUROOKA -13
-ml0 .. .. -.
2 s f B
-.5
3 .
Time (d) FIG. 4. Time course of production of ALA and PBG in recombinant P fieudenreichii subsp. shermaniiIF012426 carrying pKHEM04 (51).
hemA than promoter Pl in propionibacteria. The effects of glycine, a substrate of ALA synthesis, and levulinic acid, a competitive inhibitor of PBG synthase, on ALA synthesis were studied. It was found that levulinic acid inhibited propionibacterial cell growth and resulted in a small increase in ALA, although PBG synthesis was also inhibited. However, in the presence of 1 mM levulinic acid and 30 mM glycine, the production of ALA per cell increased to 8.3 mM. In the fermentation experiment, ALA production increased linearly for 36 h and gradually increased for 6 d (Fig. 4). PBG gradually decreased after 4 d of cultivation.This is the first report that the production of ALA by I! freudenreichii subsp. shermanii is increased by heterologous gene cloning in propionibacteria, although ALA production via the C4 pathway in a recombinant E. coli (52) and mutant R. sphaeroides (53) strains has been reported. The production of ALA and heme via the C, pathway in E. coli has also been reported (54). The production of ALA by R jkeudenreichii subsp. shermanii was comparable with that by the recombinant E. coli. CONCLUSION Propionibacteria isolated from cheese and milk, which are known as dairy or classical propionibacteria, are nonpathogenic organisms and thus are attractive for use as a host bacterium in genetic engineering. A genetic manipulation system should provide a powerful tool for cloning an interesting gene and its expression in propionibacteria. Although there have been several attempts to develop a gene manipulation system in propionibacteria, no report of the system has been published. In 2000, a host-vector system in propionibacteria was developed in several laboratories and thus the genetic manipulation in propionibacteria became possible. In this review, production of cholesterol oxidase and ALA using an expression vector in propionibacteria was demonstrated. Study on ALA production in propionibacteria by recombinant DNA technology should be useful in future studies on the production of vitamin B,, by Propi-
onibacterium. The genes involved in vitamin B,, production and their expression to overproduce vitamin B,, or tetrapyrrole compounds are under investigation in author’s laboratory. Molecular breeding of propionibacterial strains with some useful genes will also have a potential use in probiotic studies and in the dairy food industry. REFERENCES 1. Sneath, P.H.A., Mair, N.S., Sharpe, M.E., and Holt, J. G.: Bergey’s manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore, Md. (1986). 2. Johnson, J. L. and Cummins, C. S.: Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachniupropionica. J. Bacterial., 109, 1047-1066 (1972). 3. Vorobjeva, L. I.: Propionibacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands (1999). 4. Florent, J. and Ninet, L.: Vitamin B12, p. 497-5 19. In Peppler, H. J. and Perlman, D. (ed.), Microbial technology, 2nd ed., vol. 1. Academic Press, New York (1979). 5. Langsrud, T. and Reinbold, G. W.: Flavor development and microbiology of Swiss cheese - a review. II. Starters, manufacturing processes and procedures. J. Milk Food Technol., 36,53 l-542 (1973). 6. Playne, M. J.: Propionic and butyric acids, p. 731-759. In Moo-Young, M. (ed.), Comprehensive biotechnology, vol. 3. Pergamon Press, Oxford, United Kingdom (1985). 7. Ko, II. L., Pulverer, G., and Jeljaszewicz, J.: Propionicins, bacteriocin produced by Propionibacterium avidum. Zentralbl Bakteriol., 241, 325-328 (1978). 8. Grinstead, D.A. and Barefoot, S. F.: Jenseniin 4 a heat stable bacteriocin produced by Propionibacterium jensenii P126. Appl. Environ. Microbial., 58,215-220 (1992). 9. Lyon, W. J. and Glatz, B.A.: Isolation and purification of propionicin PLG- 1, a bacteriocin produced by a strain of Propionibacterium theonii. Appl. Environ. Microbial., 59, 83-88 (1993). 10. Fay, T., Langsrud, T., Nes, I. F., and Holo, H.: Biochemical and genetic characterization of propionicin Tl, a new bacteriocin from Propionibacterium theonii. Appl. Environ. Microbiol., 66,4230-4236 (2000). 11. Avice, J. C., Ourry, A., Laine, P., Roland, N., Louahlia, S., Roussel, E., Brookes, S., and Boucaud, J.: A rapid and reliable method for NO quantification and 15N0/14NO determination using isotope ratio mass spectrometry: an application for the detection of NO synthesis in propionibacteria. Rapid communication in mass spectrometry, 13(Issue 12), 1197-1200 (1999). 12. Gagnaire, V., Molle, D., Sorhuang, T., and Lerje, J.: Peptidases of dairy propionic acid bacteria. Le Lait, 79, 43-57 (1999). 13. Leehouts, K., Bolhuis, A., Boot, J., Deutz, I., Toon, M., Verema, G., Kok, J., and Ledeboer, A.: Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis. Appl. Environ. Microbial.,
64,473&4742 (1998). 14. Hotherr, L. A. and Glatz, B.A.: Mutagenesis of strains of Propionibacterium produce cold sensitive mutants. J. Dairy Sci., 66,2482-2487 (1983). 15. Paik, H. D. and Glatz, B. A.: Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidopropionici. Appl. Microbial. Biotechnol., 42, 22-27 (1994). 16. Rehberger, T. G. and Glatz, B.A.: Characterization of Propionibacterium plasmids. Appl. Environ. Microbial., 56, 864-871 (1990).
VOL. 93,2002
17. Miescher, S., Sterli, M. P., Teuber, M., and Meile, L.: Propionicin SMl, a bacteriocin from Propionibacterium jensenii DFl: isolation and characterization of the protein and its gene. Syst. Appl. Microbial., 23, 174-184 (2000). 18. Jore, J. P. M., Lujik van, N., Luiten, R G. M., Werf van der M. J., and Pouwels, P. II.: Efficient transformation system for Propionibacterium freudenreichii based on a novel vector. Appl. Environ. Microbial., 67,499-503 (2001). 19. Kiatpapan, P., Hashimoto, Y., Nakamura, H., Piao, Y-Z., Yamashita, M., Ono, H., and Murooka, Y.: Characterization of pRG0 1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria. Appl. Environ. Microbial., 66, 46884695 (2000). 20. Kiatpapan, P., Hashimoto, Y., Nakamura, H., Piao, Y-Z., Yamashita, M., 0110, H., and Murooka, Y.: Characterization of pRG0 1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria (Author correction). Appl. Environ. Microbiol., 67, 1024 (2001). 21. Ankri, S., Bouvier, I., Reyes, O., Predalu, F., and Leblon, G.: A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum. Plasmid, 36, 3Wl (1996). 22. Stolt, P. and Stoker, N. G.: Protein-DNA interactions in the ori region of the Mycobacterium fort&urn plasmid pAL5000. J. Bacterial., 178,6693-6700 (1996). 23. Luchansky, J.B., Muriana, P.M., and Klaenhammer, T. R: Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. Mol. Microbial., 2, 637446 (1988). 24. Garbe, R.T., Barathi, J., Barnini, S., Zbang, Y., AbouZeid, C., Tang, D., Mukherjee, R., and Young, B. D.: Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology, 140, 133-l 38 (1994). 25. Pernodet, J. L.: Antibiotic resistant gene cassettes derived from the interposon for use in E. coli and Streptomyces. Gene, 190,315-317(1997). 26. Pulido, D., Zalacain, M., and Jimenez, A.: The hyg gene promoter from Streptomyces hygroscopicus: a novel form of Streptomyces promoter. Biochim. Biophys. Res. Commun., 150,270-274 (1988). 27. Murooka, Y., Ishizaki, T., Nimi, O., and Maekawa, N.: Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702. Appl. Environ. Microbial., 52, 1382-1385 (1986). 28. Nomura, N., Choi, K.P., Yamashita, M., Yamamoto, H., and Murooka, Y.: Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria. J. Ferment. Bioeng., 79,410-416 (1995). 29. Somkuti, G.A., Daniel, K. Y., Solaiman, D. K., and Steinberg, D. H.: Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei. Appl. Microbial. Biotechnol., 37,330-334 (1992). 30. Somkuti, G. A., Solaiman, D. K., and Steinberg, D. H.: Native promoter-plasmid vector system for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus. Plasmid, 33, 7-14 (1995). 31. Kiatpapan, P., Yamashita, M., Kawaraichi, N., Yasuda, T., and Murooka, Y.: Heterologous expression of a gene for cholesterol oxidase in probiotic strains of Lactobacillus plantarum and Propionibacterium freudenreichii under the control of native promoters. J. Biosci. Bioeng., 92,459465 (2001). 32. Nishikawa, S. and Murooka, Y.: 5-Aminolevulinic acid (ALA): the production by fermentation and agricultural and biochemical applications, p. 149-170. In Harding, S. E. (ed.), Biotechnology and genetic engineering reviews, vol. 18,
GENE MANIPULATION IN PROPIONIBACTERIA
7
Chapter 7. Intercept, Andover, UK (2001). 33. Brown, C. E., Shemin, D., and Katz, J. J.: Nuclear magnetic resonance studies of the biosynthesis of vitamin B,,. J. Biol. Chem., 248,8015-8021 (1973). 34. Granick, S. and Beale, S. I.: Hemes, chlorophylls and related compounds: biosynthesis and metabolic regulation. Adv. Enzymol., 46, 33-203 (1978). 35. Beale, S. I. and Castelfranco, P. A.: The biosynthesis of 6aminolevulinic acid in higher plants. II. Formation of 14C 6aminolevulinic acid from labeled precursors in greening plant tissues. Plant Physiol., 53,297-303 (1974). 36. Ikemi, M., Murakami, K, Hashimoto, M., and Murooka, Y.: Cloning and characterization of genes involved in the biosynthesis of &aminolevulinic acid in Escherichia coli. Gene, 121, 127-132 (1992). 37. Ilaq, L. L., Jahn, D., Eggertsson, G., and Soll, D.: The Escherichia coli hemL gene encodes glutamate l-semialdehyde aminotransferase. J. Bacterial., 173,3408-3413 (1991). 38. Li, J. M., Russel, C. S., and Cosloy, D.: Cloning and structure of the hemA gene of Escherichia coli K12. Gene, 82, 209-217 (1989). 39. Elliott, T. and Roth, J. R.: Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis. Mol. Gen. Genet.. 216. 303-314 (19891. 40. Avissar, Y. J., O;me&d, J. G., ahd Beale, S. I.: Distribution of &aminolevulinic acid biosynthesis pathways among phototrophic bacterial groups. Arch. Microbial., 151, 5 13-519 (1989). 41. Fugino, E, Fugino, T., Karita, S., Sakka, K., and Ohmiya, K.: Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui. J. Bacterial., 177, 5 169-5 175 (1995). 42. Hashimoto, Y., Yamashita, M., Ono, H., and Murooka, Y.: Characterization of the hemB gene encoding b-aminolevulinic acid dehydratase from Propionibacterium fieudenreichii. J. Ferment. Bioeng., 82, 93-100 (1996). 43. Murakami, K., Hashimoto, Y., and Murooka, Y.: Cloning and characterization of the gene encoding glutamate l-semialdehyde 2,1-aminomutase, which is involved in &aminolevulinic acid synthesis in Propionibacterium freudenreichii. Appl. Environ. Microbial., 59,347-350 (1993). 44. Scott, A. I., Townsend, C. A., Okuda, K., and Kajiwara, M.: Biosynthesis of corrins. I. Experiment with [‘4C]porphobilinogen and [“‘Cluroporphyrinogens. J. Am. Chem. Sot., 96, 8054-8069 (1974). 45. Iida, K. and Kajiwara, M.: Evaluation of biosynthetic pathways to &aminolevulinic acid in based on biosynthesis of vitamin B12. Biochemistry, 39, 3666-3670 (2000). 46. Hashimoto, Y., Yamashita, M., and Murooka, Y.: The Propionibacterium fieudenreichii hemYHBXU gene cluster, which encodes enzymes and a regulator involved in the biosynthetic pathway from glutamate to protoheme. Appl. Microbiol. Biotecbnol., 47, 385-392 (1997). 47. Sattler, I., Roessuner, C. A., Stolowich, N. J., Hardin, S. H., Harris-Haller, L. W., Yokubaitis, N. T., Murooka, Y., Hashimoto, Y., and Scott, A. I.: Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium ffeudenreichii (shermanii). J. Bacterial., 177, 1564-1569 (1995). 48. Muller, G., Schmiedl, J., Sawidis, I., Wirth, G., Scott, A. I., Santander, P. J., Williams, H. J., Stolowich, N. J., and Kriemler, H. P.: Factor Sl, a natural corphin from Propionibacterium shermanii. J. Am. Chem. Sot., 109, 6902-6904 (1987). 49. Muller, G., Schmiedl, J., Schneider, R., Worner, G., Scott, A. I., Williams, H. J., Santander, P. J., Stolowich, N. J., Fagerness, P. E., Mackenzie, N. E., and Kriemler, H. P.: Structure of Factor S3, a metabolite of Propionibacterium shermanii derived from uroporphyrinogen I. J. Am. Chem.
8
KIATPAPAN AND IvIUROOK.4
Sot., 108,7875-7877 (1986). 50. Neidle, E. L. and Kaplan, S.: Expression of the Rhodobacter sphaeroides hem4 and hemT genes, encoding two 5-aminolevulinic acid synthase isozymes. J. Bacterial., 175, 22922303 (1993). 5 1. Kiatpapan, P. and Murooka, Y.: Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium fieudenreichii. Appl. Microbial. Biotechnol., 56, 144-149 (2001). 52. Werf, M. J. and Zeikus, J. G.: 5Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaero-
J. Broscr. BIOENG.,
ides hemA gene. Appl. Environ. Microbial., 62, 356&3566 (1996). 53. Nishikawa, S., Watanabe, K., Tanaka, T., Miyachi, N., Hotta, Y., and Murooka, Y.: Rhodobacter sphaeroides mutants which accumulate 5aminolevulinic acid under aerobic and dark conditions. J. Biosci. Bioeng., 87,798-804 (1999). 54. Verderber, E., Lucast, L. J., van Dehy, J. A., Cozart, P., Etter, J. B., and Best, E. A.: Role of the hemA gene product and &aminolevulinic acid in regulation of Escherichiu coli heme synthesis. J. Bacterial., 179,4583-4590 (1997).