Anaerobic Growth of Escherichia coli on Glycerol by Importing Genes of the dha Regulon from Klebsiella pneumoniae

Abstract

The dha regulon of Klebsiella pneumoniae specifying fermentative dissimilation of glycerol was mobilized by the broad-host-range plasmid RP4:mini Mu and introduced conjugatively into Escherichia coli. The recipient E. coli was enabled to grow anaerobically on glycerol without added hydrogen acceptors, although its cell yield was less than that of K. pneumoniae. The reduced cell yield was probably due to the lack of the coenzyme-B12-dependent glycerol dehydratase of the dha system. This enzyme initiates the first step in an auxiliary pathway for disposal of the extra reducing equivalents from glycerol. The lack of this enzyme would also account for the absence of 1,3-propanediol (a hallmark fermentation product of glycerol) in the spent culture medium. In a control experiment, a large quantity of this compound was detected in a similar culture medium following the growth of K. pneumoniae. The other three known enzymes of the dha system, glycerol dehydrogenase, dihydroxyacetone kinase and 1,3-propanediol oxidoreductase, however, were synthesized at levels comparable to those found in K. pneumoniae. Regulation of the dha system in E. coli appeared to follow the same pattern as in K. pneumoniae: the three acquired enzymes were induced by glycerol, catabolite repressed by glucose, and glycerol dehydrogenase was post-translationally inactivated during the shift from anaerobic to aerobic growth. The means by which the E. coli recipient can achieve redox balance without formation of 1,3-propanediol during anaerobic growth on glycerol remains to be discovered.

Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

“…In Listeria, efficient utilization of glycerol and expression of glycerol utilization genes depend on the alternative sigma factor B (1). The Bacillus subtilis glycerol utilization genes are induced by glycerol 3-phosphate, which involves the glycerol 3-phosphate-activated antiterminator GlpP, and they (34,36,39,49). In E. coli, the glycerol 3-phosphate-dependent regulator GlpR represses transcription of the three glycerol utilization operons, which are also controlled by cyclic AMPcyclic AMP receptor, ArcA-ArcB, and FNR (36).…”

Fructose-6-phosphate Aldolase Is a Novel Class I Aldolase from Escherichia coli and Is Related to a Novel Group of Bacterial Transaldolases

“…As well, an NADH-dependent glycerol dehydrogenase could withdraw dihydroxyacetone from the reaction. A glycerol dehydrogenase is known from E. coli, and in this context, it is of interest that the encoding gldA gene (34,35) lies immediately downstream (overlapping for 28 bp in the 3Ј region) of the talC gene of E. coli that encodes this second fructose-6-P aldolase. This chromosomal location indicates that both talC and gldA are part of an operon and may serve in a metabolic pathway that handles dihydroxyacetone.…”

Identification and Characterization of Coenzyme B 12 -Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

“…The plasmids used in this study are shown in Table 1. The E. coli strains DH5␣ (4), ECL707 (43), and BL21 (Invitrogen, Karlsruhe, Germany) were used as hosts for the cloning experiments, the activitybased screening procedures, and production of the dehydratases, respectively. Salmonella enterica serovar Typhimurium TA100 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).…”

“…Thus, pAK1 and the recombinant plasmids of the libraries could be stably cotransformed to E. coli cells, and cotransformants were readily selected by resistance to both chloramphenicol and ampicillin. The E. coli strain ECL707 (43), which lacks the activities of the genes responsible for aerobic glycerol breakdown by E. coli, was used as the host for pAK1. As expected, E. coli ECL707/pAK1 showed no growth under anaerobic conditions on M9 agar plates with glycerol as the sole carbon and energy source.…”