Mutagenetic study of a novel inosine monophosphate dehydrogenase from Bacillus amyloliquefaciens and its possible application in guanosine production.

In this study, the amino acid sequence of inosine monophosphate dehydrogenase (IMPDH) from a guanosine-overproducing strain Bacillus amyloliquefaciens TA208 was found to be highly conserved comparing to its analogue in B. amyloliquefaciens FZB42, only with two substitutions of serine 166 to proline and glutamic acid 481 to lysine. To speculate on the effects of these variation sites, two reverse site-directed mutants P166S and K481E, as well as one deletion mutant IMPDHΔCBS, were characterised. According to the kinetic analysis of these enzymes, site-481 is a key mutation site to affect the nicotinamide adenine dinucleotide (NAD+) affinity, which accounted for the higher catalytic efficiency of IMPDH. On the contrary, mutants P166S and IMPDHΔCBS did not show better catalytic activity compared to normal IMPDH. Moreover, the overexpression of IMPDH-encoding gene guaB in B. amyloliquefaciens TA208 could improve the total production of guanosine up to 13.5 g L-1, which was 20.02% higher than that of the original strain.

[Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens].

OBJECTIVE: To study the effects of overexpression of key enzyme genes (prs, purF and guaB) on guanosine production in Bacillus amyloliquefaciens TA208. METHODS: The prs, purF, guaB and prs-purF genes were inserted into constructed expression plasmid PBE43. All these constructed plasmids were electroporated into B. amyloliquefaciens TA208. The transcriptional level of various genes in the resulting strains was tested by real-time quantitative PCR. The activity of inosine 5'-monophosphate dehydrogenase in the resulting strains was detected. Finally, cell growth, glucose consumption and guanosine production of 4 engineering strains along with control strain were examined. RESULTS: The transcriptional analysis showed that overexpression of prs, purF and guaB gene accompanied by their own transcription level up-regulated. Overexpression of prs or purF genes alone slightly down-regulated the transcriptional level of purine operon, but overexpression of guaB gene independently did not disturb the transcription of prs gene and purine operon. Enzyme activity analysis showed that overexpression of prs or purF gene did not change the activity of inosine 5'-monophosphate dehydrogenase and its activity increased by 126% through overexpression of guaB gene. Finally, by fermentation flask test, we found that overexpression of prs and purF gene alone could not promote guanosine accumulation. However, overexpression of guaB gene resulted in an increase in the production of guanosine, which was 20.7% higher than the control strain. The guanosine concentration and the conversion ratio from glucose to guanosine in the host strain containing co-expression plasmid were 14.4% and 6.8% higher than the control strain. CONCLUSION: Overexpression of guaB gene could enhance the guanosine yield in the culture broth. However, for prs and purF gene, only co-expression of them could lead to a significant improvement of guanosine production in B. amyloliquefaciens. It should provide a valuable insight into the construction of industrially important strains for guanosine production by metabolic engineering.

Low-molecular-mass purine nucleoside phosphorylase: characterization and application in enzymatic synthesis of nucleoside antiviral drugs.

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillus subtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni(2+)-NTA chromatography. Compared with other rPNPs, PNP(816) was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in k (cat)/K (m) using inosine as the substrate at 37°C. The PNP(816) or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.