Chapter 14
Production of 5-Aminolevulinic Acid and VitaminB12Using Metabolic Engineering of Propionibacterium freudenreichii Pornpimon Kiatpapan1, Nitjakarn Kanamnuay2, Boonsri Jongserijit2, Yong Zhe Piao3, Mitsuo Yamashita3, and Yoshikatsu Murooka3,* 1BiochemistryUnit, School of Science, Rangsit University, Patumthani 12000, Thailand 2Departmentof Biology, Faculty of Science, Silpakorn University, Nakorn, Pathom 73000, Thailand 3Departmentof Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565-0871, Japan
Metabolic engineering of Propionibacterium sp. by expression of genes involved in vitamin B12 biosynthesis pathway is reviewed. The Rhodobacter hemA encoding 5-aminolevulinic acid (ALA) synthase and Propionibacterium hemBandcobA encoding A L A dehydratase and uroporphyrinogen III methyltransferase, respectively, were expressed in P. freudenreichii subsp. shermanii IFO12426 using vector pPK705 and Propionibacterium promoters. Productions of A L A and vitamin B12 were studied in strain IFO12426 carrying plasmid-contained hemA, hemB,orcobA. Production of A L A and vitamin B12 were 4- and 2-fold enhanced, respectively, in recombinant Propionibacterium.
© 2004 American Chemical Society
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222 Vitamin B is one of the most complicated non-polymeric molecules biosynthesized in cells and is present in organisms belonging to the three kingdoms, eubacteria, archaebacteria, and eukaryotes. Vitamin B12, including its coenzyme form, either deoxya denos yl coba la m in or methylcobalamin, is an important coenzyme that is used as a cofactor ina number of enzyme-catalyzed rearrangement and methylation reactions and is able to donate a large chemical potential to its protein counterpart by utilizing the central cobalt atom to perform chemical reactions. Exclusively microorganisms synthesize vitamin B i . Animals requiring this vitamin meet this need by food intake or by absorption of the vitamin produced by animal intestinal microorganisms. Humans, unable to absorb vitamin Β12 produced in the large intestine, are dependent on food intake for this vitamin. In humans, a deficiency of vitamin Bj causes pernicious anemia and peripheral neurological disorder. The commercial production of vitamin Β12 is currently carried out by microbial fermentation using strains of Propionibacterium or Pseudomonas. Several review articles for vitamin Bu synthesis have been published (1-6). However, no scientific work for vitamin Β12 production with genetically engineered strains has been reported except some patents (7, 8). Here, we will review our recent works of production of 5aminolevulinic acid (ALA) and vitamin B by genetically engineered Propionibacterium freudenreichii. In all organisms, the synthesis of ternes, cobalamin, chlorophylls and vitamin B | starts with the formation of 5-aminoIevulirac acid (ALA) and proceeds through the formation of porphobilinogen (PBG) and uroporphobilinogen (UPB). A L A is a useful metabolite since it is utilized as a biodegradable herbicide, insecticide, for photodynamic cancer therapy, or plant growth hormone (9). A L A is synthesized by either of two pathways, the C (Shemin pathway) and the C pathways (Fig. 1). In aerobic and aerotolerant microorganisms studied thus far, A L A is formed by the condensation of glycine and succinyl CoA (Shemin pathway) catalyzed by A L A synthase. The CS pathway has been reported in archeabacteria, anaerobic bacteria, and the facultative anaerobe Propionibderium shermanii (10). A L A is synthesized from glutamate by a series of reactions, which include the activation of glutamate by its ligation to tRNA, reduction of the activated glutamate by an NADPH dependent reductase to yield glutamatel-semialdehyde (GSA) and transamination of GSA by a GSA 2,1-aminotransferase to form A L A . Genes involved in A L A and vitamin B12 biosynthesis have been studied in Propinibacteriumfreudenreichii(11). The hemh gene that encodes GSA 2,1aminotransferase was identified by complementation of an ALA-deficieni mutant (hemh) of Ε coli (10). The hemB gene encoding PBG synthase (ALA dehydratase) in P. freudenreichii was cloned by complementation to the hemB mutant of E. coli (12). The cobA gene encoding uroporphyrinogen III methyltransferase was identified and overexpressed in Ε coli (13). To date, no hemA gene encoding A L A synthase in propionibacteria has been reported. Overexpression of genes involved in the biosynthetic pathway of vitamin Β12 should facilitate the production of A L A and vitamin B . Success in genetic ï2
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223 manipulation and gene expression of Propionibacterium opens the possibility of genetic study and molecular breeding of propionibacteria (14).
Development of an expression vector in propionibacteria
To construct an expression vector for heterologous genes in propionibacteria, an appropriate vector and promoter are required. Recently, shuttle vectors for shuttling between Propionibacterium and Escherichia coli were constructed using propioni bacterial repli con and an appropriate selection marker. Vector pPK705 was constructed by us (15) from endogenous plasmid pRGOl from P. acidipropionici, pUC18 (16) and the Streptomyces hygromycin Β resistant gene (17). Another vector pBRESP36A was constiucted using the p545 repli con, pBR322 and the erythromycin resistant gene from Saccharopolyspora erythraea (18). The vector pFK 705 could transform several species of Propionibacterium at high efficiency using the vector prepared from Propionibacterium cells to overcome a high restriction-modification system in propionibacteria (15). The vector pBRESP36A transformed only P. freudenreichii at high efficiency (18). The vector pPK705 was developed to express heterologous genes in propionibacteria. Several native promoters from P. freudenreichii subsp. shermanii IFO12424 were isolated using a pCVEl promoter probe vector containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene (19\ since promoters from gram-negative bacteria, such as lac or tac, could not drive any gene in propionibacteria These propionibacterial promoters expressed the Streptomyces choA gene in E. coli (20). Two of four strong promoters, which expressed the Streptomyces choA gene in P. freudenreichii subsp. shermanii, were selected to construct an expression vector for use with propionibacteria. Preferably, the gene to be expressed should be a gram-positive bacterium of high GC content Thus, the expression vectors, P I N and P4N, were constructed using propionibacterial promoters, PI and P4, three frame stop codons from pCVEl and the ribosomebinding site (SD sequence) and the start codon of choA which contains the synthesized Ncol restriction enzyme site. A n interesting gene to be expressed could be inserted at the Ncol site fused to the start codon of choA. Transcription of a heterologous gene in propionibacteria occurs through the SD sequence and start codon of Streptomyces choA (21).
Production of 5-aminolevulinic acid (ALA) The expression of the Streptomyces choA gene in P. freudenrichii subsp. shermanii under the control of propionibacterial promoter led to construction of
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