Alginate is synthesized by the gram-negative bacteria Azotobacter vinelandii. Alginate appears as an important component in both active vegetative cells and in cysts, which is one of the bacteriums life cycle forms.
Transketolase is an essential enzyme in the non-oxidative branch of the pentose phosphate pathway. Transketolase is responsible for the conversion of D-erythrose 4-phosphate to Dfructose 6-phosphate which is the starting precursor in alginate biosynthesis. Mutant and wild-type transketolase have been widely studied in Escherichia coli, this work focused on studying three transketolase genes from A. vinelandii.
In this study, three transketolase genes (tktA1, tktA2 and tktA3) from A. vinelandii were mutated by in frame deletions of about 1200 bp and each were cloned into a suicide vector containing the spectinomycin resistance gene and the sacB gene of Bacillus subtilis as selective markers.
The tktA1 and tktA2 genes were also individually cloned into a transposon vector under control of the Pm-promoter. A previous master student had constructed a transposon containing tktA3.
The three chromosomal copies of genes were to be inactivated by homologous gene replacement in A. vinelandii ATCC12518. These deletion mutants should then be complemented by the transposon encoded genes. However, even after several experiments no deletion mutants were obtained.
The second part of this work focused on studying the three wild-type transketolase genes (tktA1, tktA2 and tktA3) of A. vinelandii by cloning, identification, purification and activity analyses in E. coli. The open reading frames of the three transketolase genes tktA1, tktA2 and tktA3 were cloned and overexpressed from the Pm-promoter in an RK2 based expression vector E. coli S17.1 λ pir were used as host since transformants of RV308 containing these plasmids could not be obtained.
Thus, the three proteins were produced in E. coli S17.1 λ pir. No protein activity was found from any of three expression strains. The SDS-PAGE of sonicated cells showed bands of 74.6 kDa and 73.8 kDa corresponding tktA2 and tktA3. The three transketolase proteins were purified from sonicated cells using FPLC and the obtained fraction were analysed by SDSPAGE. Protein bands with the expected molecular weight for Tkt2 and Tkt3 were obtained. When these extracts were purified by FPLC, this protein band was only found in the void fraction. The TktA1 protein band was not observed neither using the sonicated cells nor in the fractions obtained from the FPLC.