Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans

Abstract

Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome

“…The hewl gene is substantiated to be active against Bacillus species recently and can be used as a powerful marker for counter-selection [22]. The upp gene has been widely used in markerless modifications, such as gene disruption, point mutation, and large-scale deletion [23], [25]–[28]. Although it is feasible as a counter-selectable marker, the proportion of cells which undergo pop-out is expected to be of the order of 10 −6 [23] and actually needs to be enhanced to facilitate the application of markerless system.…”

Utilization of d -Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

“…The generated fusion construct and pK18mobsacB (20), a mobilizable plasmid that does not replicate in G. oxydans, were digested with EcoRI and HindIII, respectively, and were then linked by using T 4 DNA ligase to form pK18mobsacB-⌬GOX1253. The plasmid was transferred into G. oxydans via a triparental mating method as described previously (21). E. coli DH5␣ harboring pK18mobsacB-⌬GOX1253 was used as the donor strain, E. coli HB101 harboring the plasmid pRK2013 as the helper strain, and G. oxydans 621H as the recipient strain.…”

A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette

“…To date, several homology-directed repair (HDR)-based genetic modification technologies have been developed in the model microorganism, Escherichia coli , and two-step strategies including a negative selection step make it possible to perform markerless genome editing (Datsenko and Wanner, 2000; Tischer et al, 2006; Peters et al, 2013; Wang et al, 2014). A large number of toxic genes have been verified to work well as negative selection markers in E. coli , including sacB, ccdB and codAB (Yu et al, 2008; Kostner et al, 2013; Wang et al, 2014).…”