Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene

Abstract

Maltose metabolism in Lactococcus lactis involves the conversion of /?-glucose l-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible /?-phosphoglucomutase (8-PGM). The gene encoding /?-PGM (pgmB) was cloned from a genomic library of L. lactis using antibodies. The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found. The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified /?-PGM (25 kDa). A short sequence at the N-terminus was found to be similar to known metalbinding domains. The expression of /?-PGM in L. lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium. This indicates that &PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates. The specific activity of /?-PGM during fermentation was dependent on the maltose concentration in the medium. The maximum specific activity of /?-PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0 8 to 11-0 g I-'. Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased. The specific activity of a-PGM responded similarly to &PGM, but the magnitude of the response was lower. Preferential sugar utilization and a-and /?-PGM suppression was observed when L. lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar. In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.

A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active site

“…Primers (5Ј-GAA TTC CAT ATG TTT AAA GCA GTA TTG-3Ј and 5Ј-CCG CTC GAG TTA TTT TTG CTT TTG AAG-3Ј) were used to introduce a NdeI and a XhoI restriction site at the beginning and end of the gene, respectively. After digestion, the PCR product was cloned in the pET-22b(ϩ) expression vector (Novagen, San Diego, CA) giving the expected published sequence (35). Transformed Escherichia coli BL21(DE3) with this plasmid were used to overexpress ␤-PGM.…”

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

“…Since the phosphorolysis of glucose, lactose and other carbon sources usually yields α-glucose-1P, α-PGM could be a keyenzyme in sugar-nucleotide biosynthesis. However, to our knowledge, only the gene encoding β-PGM has been cloned from L. lactis (Qian et al 1997).…”

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

“…Product formation was homolactic. The similarity in the behavior of the two strains indicates that the genetic manipulation had no major effect on the cells when grown on glucose or lactose, which was expected as there is no detectable ␤-PGM activity in glucose-or lactose-cultured wild-type cells (28).…”

“…The glucose formed enters the glycolysis by glucokinase while ␤-G1P is converted to glucose 6-phosphate by ␤-phosphoglucomutase (␤-PGM) before entering the glycolysis (29). ␤-PGM is repressed by glucose and lactose and induced by maltose and trehalose (28), suggesting that trehalose is also catabolized by a phosphorylase, analogous to observations in Euglena gracilis (2) and in Micrococcus varians (16). However, the initial catabolism of maltose and trehalose is still not well established in L. lactis, and the possibility of alternative pathways has not been investigated.…”