Metabolic engineering of Gluconobacter oxydans 621H for increased biomass yield.

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources regio- and stereoselectively in the periplasm and therefore is used industrially for oxidative biotransformations, e. g., in vitamin C production. However, it has a very low biomass yield as the oxidized products largely remain in the medium and cannot be used for anabolism. Cytoplasmic carbon metabolism occurs via the pentose phosphate pathway and the Entner-Doudoroff pathway, whereas glycolysis and the tricarboxylic acid cycle are incomplete. Acetate is formed as an end product via pyruvate decarboxylase and acetaldehyde dehydrogenase. In order to increase the biomass yield from glucose, we sequentially replaced (i) gdhS encoding the cytoplasmic NADP-dependent glucose dehydrogenase by the Acetobacter pasteurianus sdhCDABE genes for succinate dehydrogenase and the flavinylation factor SdhE (strain IK001), (ii) pdc encoding pyruvate decarboxylase by a second ndh gene encoding a type II NADH dehydrogenase (strain IK002.1), and (iii) gdhM encoding the membrane-bound PQQ-dependent glucose dehydrogenase by sucCD from Gluconacetobacter diazotrophicus encoding succinyl-CoA synthetase (strain IK003.1). Analysis of the strains under controlled cultivation conditions in bioreactors revealed for IK003.1 that neither gluconate nor 2-ketogluconate was formed, but some 5-ketogluconate. Acetate formation was eliminated, and comparable amounts of pyruvate were formed instead. CO2 formation by IK003.1 was more than doubled compared to the reference strain. Growth of IK003.1 was retarded, but the biomass yield of this strain was raised by 60%. IK003.1 serves as suitable host for oxidative biotransformations and for further metabolic engineering.

SdhE-dependent formation of a functional Acetobacter pasteurianus succinate dehydrogenase in Gluconobacter oxydans--a first step toward a complete tricarboxylic acid cycle.

The obligatory aerobic α-proteobacterium Gluconobacter oxydans 621H possesses an unusual metabolism in which the majority of the carbohydrate substrates are incompletely oxidized in the periplasm and only a small fraction is metabolized in the cytoplasm. The cytoplasmic oxidation capabilities are limited due to an incomplete tricarboxylic acid (TCA) cycle caused by the lack of succinate dehydrogenase (Sdh) and succinyl-CoA synthetase. As a first step to test the consequences of a functional TCA cycle for growth, metabolism, and bioenergetics of G. oxydans, we attempted to establish a heterologous Sdh in this species. Expression of Acetobacter pasteurianus sdhCDAB in G. oxydans did not yield an active succinate dehydrogenase. Co-expression of a putative sdhE gene from A. pasteurianus, which was assumed to encode an assembly factor for covalent attachment of flavin adenine dinucleotide (FAD) to SdhA, stimulated Sdh activity up to 400-fold to 4.0 ± 0.4 U (mg membrane protein)(‒1). The succinate/oxygen reductase activity of membranes was 0.68 ± 0.04 U (mg membrane protein)(‒1), indicating the formation of functional Sdh complex capable of transferring electrons from succinate to ubiquinone. A. pasteurianus SdhE could be functionally replaced by SdhE from the γ-proteobacterium Serratia sp. According to these results, the accessory protein SdhE was necessary and sufficient for heterologous synthesis of an active A. pasteurianus Sdh in G. oxydans. Studies with the Sdh-positive G. oxydans strain provided evidence for a limited functionality of the TCA cycle despite the absence of succinyl-CoA synthetase.

High precision genome sequencing of engineered Gluconobacter oxydans 621H by combining long nanopore and short accurate Illumina reads.

State of the art and novel high-throughput DNA sequencing technologies enable fascinating opportunities and applications in the life sciences including microbial genomics. Short high-quality read data already enable not only microbial genome sequencing, yet can be inadequately to solve problems in genome assemblies and for the analysis of structural variants, especially in engineered microbial cell factories. Single-molecule real-time sequencing technologies generating long reads promise to solve such assembly problems. In our study, we wanted to increase the average read length of long nanopore reads with R9 chemistry and conducted a hybrid approach for the analysis of structural variants to check the genome stability of a recombinant Gluconobacter oxydans 621H strain (IK003.1) engineered for improved growth. Therefore we combined accurate Illumina sequencing technology and low-cost single-molecule nanopore sequencing using the MinION® device from Oxford Nanopore. In our hybrid approach with a modified library protocol we could increase the average size of nanopore 2D reads to about 18.9kb. Combining the long MinION nanopore reads with the high quality short Illumina reads enabled the assembly of the engineered chromosome into a single contig and comprehensive detection and clarification of 7 structural variants including all three known genetically engineered modifications. We found the genome of IK003.1 was stable over 70 generations of strain handling including 28h of process time in a bioreactor. The long read data revealed a novel 1420 bp transposon-flanked and ORF-containing sequence which was hitherto unknown in the G. oxydans 621H reference. Further analysis and genome sequencing showed that this region is already present in G. oxydans 621H wild-type strains. Our data of G. oxydans 621H wild-type DNA from different resources also revealed in 73 annotated coding sequences about 91 uniform nucleotide differences including InDels. Together, our results contribute to an improved high quality genome reference for G. oxydans 621H which is available via ENA accession PRJEB18739.