Gene GMS 01

#The present invention relates to microorganisms genetically engineered to increase yield and/or efficiency of biomass production from a carbon source, such as e.g. glucose. Processes for generating such microorganisms are also provided by the present invention. The invention also relates to polynucleotide sequences comprising genes that encode proteins that are involved in the bioconversion of a carbon source such as e.g. glucose into biomass. The invention also features polynucleotides comprising the full-length polynucleotide sequences of the novel genes and fragments thereof, the novel polypeptides encoded by the polynucleotides and fragments thereof, as well as their functional equivalents. Also included are processes of using the polynucleotides and modified polynucleotide sequences to transform host microorganisms leading to a microorganism with reduced carbon source diversion, i.e. higher yield and/or efficiency of biomass production from a carbon source such as e.g. glucose.

#The bioconversion of a carbon source may involve many different metabolic routes, and involve several enzymatic steps to generate biomass, wherein the enzymes may be located in the cytosol, on the membrane or in the periplasmic space of a host cell. Furthermore, transporters may also play an important role in the efficient conversion of a carbon source into biomass.

#For instance, in the case of acetic acid bacteria, which are obligate aerobe, gram-negative microorganisms belonging to the genus Acetobacter, Gluconobacter, and Gluconacetobacter, these microorganisms are able to oxidize D-glucose at the periplasmic membrane level to D-gluconate by means of a membrane-bound D-glucose dehydrogenase, and transport D-gluconate into the cytosol. D-gluconate can then be phosphorylated by gluconokinase to D-gluconate-6-phosphate. In addition to that, it is believed that D-glucose can be directly transported into the cytosol and then converted into D-gluconate by means of a cytosolic NAD(P)-dependent D-glucose dehydrogenase. Furthermore, D-glucose transported into the cytosol can also be first phosphorylated to D-glucose-6-phosphate by glucokinase and then be dehydrogenated to D-gluconate-6-phosphate. D-gluconate-6-phosphate may enter the pentose phosphate pathway, being further metabolized to produce reducing power in the form of NAD(P)H and tricarboxylic compounds necessary for growth and maintenance.

#For determination of biomass production, cells of G. oxydans were grown at 27° C. for 3 days on MB agar, containing 25 g/l mannitol, 5.0 g/l yeast extract (Difco), 3.0 g/l Bactopeptone (Difco) and 15 g/l agar. Cells were resuspended in 10% glycerol (10 ml per plate) and 1 ml aliquots were stored at −80° C.

#A baffled 500 ml shake flask with 100 ml medium No. 5 containing 50 g/l D-glucose, 0.5 g/l glycerol, 15 g/l yeast extract (Difco), 2.5 g/l MgSO4.7H2O, 0.3 g/l KH2PO4, 15 g/l CaCO3 and 1 drop antifoam was inoculated with a 1 ml aliquot and incubated at 30° C. and 180 rpm in a shaker for 48 h. The optical density at 600 nm (OD600) was determined using a Spectronic 4001/N spectrophotometer and an identical medium No. 5 flask was then inoculated with this culture, such that the start OD600 in the second flask was 0.25. This flask was then further incubated at 30° C., 180 rpm for another 60 h with samples of 3 ml taken every 12 h for analysis of biomass and glucose concentrations as follows:

#Production of biomass was determined via measurement of the dry weight of the sample. Reaction tubes were pre-dried at 40° C. under vacuum for 48 h and the weight of the empty tubes measured [w1]. The sample was added and the weight of the full tube determined [w2]. 0.1 ml 18.5% HCl was added to dissolve the CaCO3. The tubes were subsequently centrifuged for 3 min at 13000 rpm. The supernatant was discarded, 0.5 ml of water was added and the pellet was resuspended. The tube was centrifuged again for 3 min at 13000 rpm and the supernatant was discarded. The pellet was dried at 40° C. under vacuum for 48 h and the tube containing the pellet again weighted [w3]. The concentration of biomass [B] in the sample was calculated as follows: (w3−w1)/(w2−w1)×1000=concentration of biomass [g/kg]