Abstract
Background5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid-based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated.ResultsFor plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF production rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression.ConclusionWe identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization.
Highlights
The strictly aerobic acetic acid bacterium Gluconobacter oxydans contains at least eight membrane-bound dehydrogenases catalyzing the rapid chemo, regio, and stereoselective oxidation of sugars, alcohols, and polyols in Battling et al Microb Cell Fact (2020) 19:54 involves G. oxydans include dihydroxyacetone [11] or the anti-diabetic drug miglitol [12, 13].Due to the vigorous incomplete periplasmic substrate oxidation of G. oxydans, only a small fraction of the carbon source is taken up into the cytoplasm and enters the central carbon metabolism, which is characterized by an incomplete glycolysis and an incomplete tricarboxylic acid (TCA) cycle [14]
Selection of genomic integration sites for the fdhSCL genes and design of integration constructs In this study, we wanted to generate plasmid-free G. oxydans strains for the production of 5-KF by genomic integration of the fructose dehydrogenase (FDH) genes fdhSCL from G. japonicus
The intergenic regions (IGRs) were chosen based on the following criteria: (i) the genomic positions should be close to the origin of replication to profit from a positive gene dosage effect; (ii) the positions should be located between the 3′-ends of two convergent genes to avoid an interference with the regulation of neighboring genes; (iii) the expression levels of the adjacent genes should vary for the three sites to test the influence of the genomic vicinity on fdhSCL expression; (iv) the chromosomal gene upstream of the fdhSCL genes should possess a terminator to enable comparison of the three loci without readthrough from an upstream promoter
Summary
The strictly aerobic acetic acid bacterium Gluconobacter oxydans contains at least eight membrane-bound dehydrogenases catalyzing the rapid chemo-, regio-, and stereoselective oxidation of sugars, alcohols, and polyols in Battling et al Microb Cell Fact (2020) 19:54 involves G. oxydans include dihydroxyacetone [11] or the anti-diabetic drug miglitol [12, 13].Due to the vigorous incomplete periplasmic substrate oxidation of G. oxydans, only a small fraction of the carbon source is taken up into the cytoplasm and enters the central carbon metabolism, which is characterized by an incomplete glycolysis and an incomplete tricarboxylic acid (TCA) cycle [14]. As a consequence of the small fraction of substrate metabolized within the cell, the biomass yield of G. oxydans is quite low (about 0.1 g cell dry weight/g glucose). One of the resulting strains, IK003.1, which is derived from the parent strain 621H, lacks both the membrane-bound and the soluble glucose dehydrogenase as well as pyruvate decarboxylase. It has a 60% increased biomass yield on glucose and accumulates pyruvate instead of acetate [17]
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