Kazuhisa Hatakeyama

Genomic organization of the biotin biosynthetic genes of coryneform bacteria: Cloning and sequencing of the bioA-bioD genes from Brevibacterium flavum

Three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum and Corynebacterium glutamicum have been shown to be able to convert 7-keto-8-aminopelargonic acid to biotin through a biotin synthetic pathway identical to that from Escherichia coli (Hatakeyama et al., DNA Sequence, in press, 1993). We report in this paper the cloning and sequencing of the biotin biosynthetic genes encoding the 7,8-diaminopelargonic acid aminotransferase (bioA) and the dethiobiotin synthetase (bioD) of B. flavum MJ233, by complementation of E. coli bioA and bioD mutants. Both bioA and bioD genes from B. flavum were located on a 4.0-kb SalI DNA fragment. Nucleotide sequence analysis of this fragment revealed that these genes consist of a 1272 bp and a 675 bp open reading frame, respectively. The deduced amino acid sequence of the 7,8-diaminopelargonic acid aminotransferase (BioA) is 51.3% and 31.9% identical to that of the E. coli and Bacillus spaericus bioA gene products, respectively. The deduced amino acid sequence of the dethiobiotin synthetase (BioD) is 25.9% and 32.7% identical to that of the E. coli and B. sphaericus bioD gene products, respectively. In addition, the genomic organization of the bioA, bioB and bioD genes in B. flavum has been shown to be different from that in E. coli and B. sphaericus.

Electrotransformation of intact cells of Brevibacterium flavum MJ-233

SummaryElectroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per μg DNA was 5×104 when the cell suspension was pulsed at a cell density of 1×1010/ml and at a DNA amount of 1.0μg.

Analysis of the biotin biosynthesis pathway in coryneform bacteria: Cloning and sequencing of the bioB gene from Brevibacterium flavum

The biotin biosynthetic pathway of three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium glutamicum were analysed by cross-feeding experiments using several Escherichia coli biotin-requiring mutants. The three strains of coryneform bacteria tested were able to convert 7-keto-8-aminopelargonic acid to biotin, through a biotin synthetic pathway identical to that from E. coli. The biotin biosynthetic gene, bioB, of B. flavum was cloned by phenotypic complementation of E. coli bioB mutants. The bioB gene was located on a 1.7 kb HindIII-SacI DNA fragment. Nucleotide sequence analysis of this fragment revealed that the bioB gene of B. flavum consists of a 1005 bp open reading frame. Its deduced amino acid sequence is 35.7% and 31.5% identical to that of the E. coli and Bacillus sphaericus bioB gene products, respectively. B. flavum mutants obtained by in vivo disruption of the bioB gene lost their ability to grow on minimal medium containing dethiobiotin, indicating that the bioB gene product is necessary for the conversion of dethiobiotin to biotin.

Analysis of biotin biosynthesis pathway in coryneform bacteria: Brevibacterium flavum.

Publisher Summary This chapter examines the coryneform bacterium, Brevibacterium flavum MJ233, for use as a bioconverter to produce amino acids and organic acids. The chapter also develops a new bioprocess using biotin removal from a minimal medium as a means to repress cellular division. The biosynthetic pathway of biotin is investigated mainly by using strains of Escherichia coli ( E. Coli ). The biotin biosynthetic genes are organized in an operon in E. coli , but in two different clusters in Bacillus sphaericus. Among the enzymes of the pathway, all that are involved in dethiobiotin synthesis from pimelic acid are presented—pimelyl–CoA synthetase, 7-keto-8-aminopelargonic acid synthetase, 7,8-diaminopelargonic acid aminotransferase, and dethiobiotin synthase. However, the final step in biotin synthesis, the conversion of dethiobiotin to biotin involves a flavodoxin, ferredoxin in addition to the bioB gene product. The lack of biotin biosynthesis is a taxonomical characteristic of the glutamic acid-producing group of coryneform bacteria. However, there have been no practical data regarding the deleted steps of biotin biosynthesis. The chapter reveals the deleted steps of the biotin biosynthetic pathway in several coryneform bacteria by using cross-feeding experiments with E. coil bio mutants. In addition, biotin biosynthetic genes are cloned and sequenced from a strain of coryneform bacterium, Brevibacterium flavum, MJ233.