Microorganisms having enhanced sucrose mutase activity

The invention relates to the biotechnological production of isomaltulose and isomaltulose-containing compositions and improved means, therefore particularly microbial cells.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Phase of PCT/EP2010/006487, filed Oct. 23, 2010, which claims benefit of German Application Number 10 2009 053 566.7, filed Nov. 11, 2009, the contents of each are incorporated by reference herein in their entirety.

The sequence protocol includes:

SEQ ID No:1: DNA-sequence upstream in reference to the smuA-gene, comprising the native smuA-promoter/operator, which can be substituted entirely or partially by the substitute promoter according to the invention or its functional substitute promoter fragment;

SEQ ID No:2 to 21: Sequences of functional substitute promoters, which can regulate the expression of the smuA-gene in the cell instead of the native smuA-promoters.

In order to substitute the identified strong promoters for the native smuA-promoter, according to the invention preferably a substitution occurs, which is performed without the introduction of novel, non-homologue sequences into the organism. For this purpose, according to the invention preferably the following process occurs essentially: A promoter substitute plasmid is created, which preferably comprises approx. 1000 bp of a DNA-region upstream and downstream of the smuA-promoters. Subsequently a non-scarring substitution of the native smuA-promoter occurs, located in the plasmid and showing a size of approx. 400 bp, for another homologue promoter. The base-equivalent substitution may be confirmed, for example by DNA-sequencing. The promoter substitute plasmid obtained in this way is transferred into the organism and the plasmid is chromosomally integrated via homologue recombination into the smuA-unit. The substitution of promoters preferably occurs by the simultaneous elimination of the substitute plasmid by a targeted selection of the second recombination event.

If applicable, the correct, non-scarring promoter substitution and the absence of any external sequences in the organism can be verified by PCR-methods and/or sequencing methods in a manner known per se. Additional securing is possible by southern-hybridization.

In order to substitute the native smuA-promoter, preferably the inter-genetic region between a potential regulator of the expression of the region coding the sucrose mutase and a region coding the sucrose mutase SmuA is selected on the chromosome of the bacteria. Particularly preferred, the interim fragment is removed by way of hydrolysis at the regions Munl and Pmel. Preferably the targeted insertion of the homologue substitute promoter, preferred according to the invention, occurs as a Munl or EcoRI/Pmel-fragment.

Substitution of the smuA-Promoters by Self-Cloning inP. rubrum

All cloning and DNA-modifications are performed as described in Sambrook et al., 1989 (Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). PCR-charges, kits for the isolation of nucleic acids, detection and selection methods, and cultivation were performed in a manner known per se, unless stipulated otherwise, according to the specifications of the respective manufacturers.

Chromosomal DNA of theP. rubrumwild type is isolated and partially metabolized with Alul. The fragments are cloned into the Stul interfaces by two different promoter probe-vectors shown inFIG. 2, which essentially differ in their number of copies: While the vector pUCTT-gusA shows a pUC-derivative with a high number of copies and carrying the chloroamphenicol-resistant gene cat of pBR328 (DSMZ, Brunswick), the vector pSCTT-gusA is a vector with a low number of copies, deducted from the known plasmid pSC101 (DSMZ, Brunswick) and carrying the canamycin-resistant gene aphll. In both vectors the gusA-gene is used as the promoter-reporting gene, which is coded for a β-glucuronidase and expressed after the insertion of a functional promoter fragment.

After the addition of the substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) a successful expression can be detected by blue coloration (execution according to Platteeuw C. et al., 1994, Appl Environ Microbiol. 60:587-93). The three terminators (Ter) can prevent any undesired expression of the reporter gene by potential, plasmid-internal promoter elements. Both vector types replicate inE. colias well as inP. rubrum.

In order to control the promoter function of the host cell-internal substitute promoter fragments, the chromosomal β-glucuronidase gene uidA, naturally present inE. Coli, can be deactivated by one trained in the art using methods known to him/her, in order to create anE. coligusA-test stemE. coliDH10BΔuidA.

The transformation of pUCTT-gusA or pSCTT-gusA-gene bank of the substitute promoters inE. coliDH10BΔuidA leads to blue colonies, in which the promoter-less gusA-reporter gene is expressed by the substitute promoter fragment cloned in.

The restriction analysis of plasmid-DNA, isolated from blue colonies, shows that differently sized chromosomalP. rubrumfragments also have promoter functions.

Using the electroporation (preparation of electro-competent cells and execution according to Dower et al., 1988, Nucleic Acids Res., 16:6127-6145) the clones can be transferred toP. rubrumand the promoter activity, already detected inE. coli, can be confirmed.

1.2 Non-scarring insertion of substitute promoters

The substitute promoters according to the invention (SEQ ID NO: 2 to 21) are each substituted in a non-scarring fashion for the native chromosomal smuA-promoter/operator range (cf. SEQ ID NO: 1 and FIG.3ABC), by way of homologue recombination.

This approach is based on the chromosomal integration of a promoter substitute plasmid by way of homologue recombination. A base vector is provided, not reproducing inP. rubrum, at least conditionally. As an example, pUT-derivatives are inserted based on a R6K replication origin (Herrero et al., 1990, J. Bacteriol., 172:6557-67). These vectors reproduce only when the coding pir-gene essential for the replication of π-protein is present. Such a stem isE. coli S17-1λpir (Herrero et al., 1990, J. Bacteriol., 172:6557-67); it is used to construct the respective promoter substitute plasmids.

All promoter substitute plasmids according to the invention based on pUT-vectors are constructed such that the new promoter fragment to be substituted is flanked in a non-scarring fashion upstream by a 925 bp sized DNA fragment, coding the gntR-regulator, and downstream by a 1066 by sized DNA fragment, coding the 5′ region of smuA.FIG. 3shows in detail the realization of the cloning strategy, as an example for the ribB-promoter being the substitute promoter fragment (3C1). An exemplary, final promoter substitute plasmid (pUT-GntR-Pr3C1-SmuA′), triggering the substitution of the fragment 3C1 for the smuA-promoter, is shown inFIG. 4.

1.3 Creating promoter reorganization stems

The transfer of the promoter substitute plasmids to formP. rubrumis preferably realized by an inter-generic conjugation betweenE. coliS17-1λpir andP. rubrum. The pUT-plasmids used carry an “Origin of Transfer” (oriT) of the RP4 plasmid and can be mobilized by the RP4 plasmid chromosomally integrated in theE. coliS17-λpir toP. rubrum(Herrero et al., 1990, J. Bacteriol., 172:6557-67).

The conditions of the inter-generic conjugation have been optimized as follows: The selection of potentialP. rubrumtransconjugants requires a plasmid marker that can be selected inP. rubrum(e.g., aphll, canamycin-resistant), and a possibility to selectively inhibit theE. colidonor. By way of plating on a rifamycin-containing medium (100 μg/ml), spontaneously rifamycin-resistantP. rubrumwild type colonies are generated (P. rubrumRif), which show no other differences from the wild type. The conjugation is performed as follows:

E. coliS17-λpir donor stems carrying the respective promoter substitute plasmid are drawn out over night in 5 ml dYT medium (per 1 liter: 16 g bacto trypton, 10 g bacto yeast extract, and 5 g NaCl) with canamycin (50 μg/ml) added at 37° C. The recipientP. rubrumwas also drawn out over night in 5 ml dYT under the addition of rifamycin (100 μg/ml) at 30° C. 1 ml each of the overnight culture was injected into a conical flask with 100 ml dYT-medium (for additions, see above) and incubated at 30° C. (P. rubrum) or 37° C. (E. coli) and 250 rpm up to OD (600 nm) from 0.4 to 0.8. The donor and the recipient are mixed at a ratio of 1:4, centrifuged, washed with 1 ml dYT, and finally accepted in 100 μl dYT-medium. The suspension is applied by pipettes on a nitro-cellulose filter (0.45 μm pore size) located on a dYT plate and incubated over night at 30°. The cells are then rinsed off the filter with 1 ml dYT, diluted, and plated on selection plates (dYT+canamycin 50 μg/ml and rifamycin 100 μg/ml) and incubated over night at 30° C.

By the canamycin selection, suchP. rubrumtransconjugants are yielded, in which the plasmids are preferably integrated in the chromosome via one of the two homologue regions (FIG. 5). The incubation of canamycin-free medium allows the selection of promoter substitute stems, in which the integrated plasmid is disintegrated from the chromosome by a second cross-over. In order to prove the successful disintegration, color markers xylE can be inserted on a substitute plasmid. The xylE-gene codes for a catechol-2,3 dioxygenase, converting the catechol to 2-hydroxymuconic acid semialdehyde, which can be detected phenotypically by a striking yellow coloring. The disintegration of the previously integrated substitute plasmid is a rare occasion (1 of 1000 colonies) and can be detected by these markers via the designed clones not showing the yellow coloration after the addition of catechol (spray reagent: 0.2 ml of a 0.5 mol/l aqueous solution). Due to the fact that the disintegration of the substitute plasmid can also lead to the reconstitution of the wild type, all generated promoter reorganization stems can be tested and verified by PCR-experiments, southern-blot analyses, and genomic sequencing. The results show that the new stems generated developed free from scarring by base-identical substitution (self-cloning) and show no external sequences.

Product spectrum and synthesis performance

2.1 Analysis of the carbohydrate composition using HPLC

The measurement was performed under the following chromatographic conditions: injection volume: 10 μl; flow rate: 1.0 to 1.8 ml/min. The flow rate to be adjusted for optimal separation depends on the type and condition of the separating column as well as the composition of the eluent. For additional analysis parameters see Table 2.

The growth of stems according to the invention and wild type-control stems occurred in 30 ml LB-medium (Start-OD600of 0.05). The cultures are each incubated at 30° C., 200 rpm in a horizontal shaking flask. After initially 24 hours of fermentation, 5×OD cells are removed, centrifuged, and washed with 1 ml Ca-acetate buffer (0.01 mol/l, pH 5.5). The cell pellets (equivalent to 5×OD cells) are each re-suspended in 1.25 ml Ca-acetate buffer (0.01 mol/l, pH 5.5) with a sucrose solution of 0.584 mol/l (200 g/l). The charges were incubated for biotransformation in deep-well plates under slight shaking at room temperature for 90 min. The reaction was stopped by heat treatment (5 min 98° C.).

Table 3 shows the results of the HPLC-analysis of the residue after fermentation under sucrose growth (S) or under glucose growth (G) with two individually created promoter substitute stems (No. 3C1A and No. 3C1 B) in reference to the wild type (WT):

The isomaltose amount of the 3C1 stems (3C1A2S and 3C1B2S) created under sucrose growth (S) is elevated in reference to the respective wild type (WT2S) by approx. the factor of 3.2.

Under glucose growth (G) the difference is more distinct because under these conditions SmuA is expressed in the wild type (WT2G) only in small amounts. The stems according to the invention (3C1A2G, 3C1B2G) produce only approx. 10% less isomaltulose compared to the sucrose growth.

During the further progression of the fermentation (more than 24 hours) isomaltulose is yielded by 70 to 90% and perhaps more than 90%.

2.3 Whole-Cell biotransformation in the fermenter

Wild typeP. rubrumZ12 and the substitute stems ofP. rubrumcreated by way of self-cloning were cultivated in the same medium. The growth of the pre-culture occurred in the shaking flask at 30° C. under aerobic conditions.

The fermenter was injected with 5 ml of a pre-culture in an exponential growth phase. The fermentation in the 500 ml-fermenters occurs under the above-mentioned parameters at 30° C. for 15 hours. After the end of the fermentations the cells were centrifuged for 30 min at 17,600×g and the clear residue is discarded. Subsequently the cell yield (dry bio mass) and its sucrose mutase activity were determined.

The dry biomass was determined by way of filtration of 10 ml fermentation suspension with a 0.45 μm filter and dehydration of this filter at 105° C. Two fermentations per stem were performed each.

The wild type yielded, after fermentation for 15 hours, a dry biomass of 85.1±5.6 g/kg and exemplarily the promoter substitute stem No. 3C1 yielded 80.9±1.0 g/kg. Based on this data no significant difference could be observed in the yield of dry biomass.

In order to determine the sucrose mutase activity 1 g moist biomass (BFM) each of the wild type and the self-cloned stems according to the invention were suspended in 50 g 10 mmol/l Ca-acetate buffer, pH 5.5 each, comprising 40% [w/v] sucrose. The cell suspension was incubated for 24 hours at 25° C. under shaking and samples were taken at different times. They were examined via HPLC for residual sucrose, isomaltulose, trehalulose, glucose, and fructose.

FIG. 6shows analysis data of the formation of isomaltulose from a 40% conc. sucrose in 10 mmol/l Ca-acetate buffer, pH 5.5. Samples were taken at different times and examined for the composition of the carbohydrates. The figure shows the conversion of 40% conc. sucrose into isomaltulose by the sucrose mutase of the wild type (WT) in reference to the exemplarily selected promoter substitute stem No. 3C1A. After 5 hours of incubation, using the sucrose mutase of the wild type 150 g isomaltulose, with the promoter substitute stem No. 3C1A, 320 g isomaltulose could already be proven, though.

Based on the conversion kinetics, the following specific activities could be calculated: Activity of the wild type: 1020±17.8 units/g dry biomass; activity of the stems (No. 3C1A and No. 3C1B): 3118±86.2 units/g dry biomass. The sucrose mutase activity was elevated in the substitute stems according to the invention in reference to the wild type by a factor of approximately 3.