Patent Application
20080118943
The present invention relates to methods for selecting mutant bacteria producing increased folate levels. In these methods the antifolate methotrexate (MTX) is used as selection agent. Provided are also food and food supplement compositions comprising the mutant bacteria or derivatives or extracts of the bacteria.
Folate analogues and in particular inhibitors of dihydrofolate reductase have been the topic of research for many years because of their potential role in cancer chemotherapy and as agents against the malaria parasite. One of the first folate analogues described in literature is methotrexate from Lederle Laboratories. This compound has already been used in 1948 for the treatment of acute leukemia (Hitchings, G. H., Jr., 1989, In Vitro Cell Dev Biol 25:303-10).
The activity of methotrexate (MTX) is based on competitive inhibition of the target enzyme dihydrofolate reductase. This enzyme is needed for the production and recycling of tetrahydrofolate (THF) using dihydrofolate (DHF) as a substrate. The specificity of the methotrexate can be explained by small structural differences of dihydrofolate reductases in different species (Chang et al., 1978, Nature 275:617-24). Methotrexate prevents the cell from producing sufficient tetrahydrofolate for the biosynthesis of methionine, DNA and RNA. However in the presence of purines, thymidine, glycine, methionine and pantothenic acid (all metabolites that need tetrahydrofolate for their own biosynthesis) the demand of cells to synthesize tetrahydrofolate can be decreased (Harvey, R. J., 1973, J Bacteriol 114:309-22). Nevertheless cells will always be in some way hampered in growth by MTX, because THF is essential for formation of methionyl-tRNAfmet, a compound that is indispensable for the initiation of protein synthesis, regardless of the presence of folate dependent metabolites (Baumstark et al., 1977, J Bacteriol 129:457-71).
Lactic acid bacteria such as Lactococcus lactis, Lactobacillus plantarum and Lactobacillus casei show a high degree of variability in their response to methotrexate and another folate analogue, trimethoprim. L. lactis is known be insensitive to both antifolates (Leszczynska et al., 1995, Appl Environ Microbiol 61:561-6). It has been shown for numerous organisms that prolonged exposure to antifolates results in selection of resistant cell types. Several underlying mechanisms have been described to explain the resistance mechanism of both eukaryote cell lines as well as bacteria to methotrexate (MTX). Tamura et al. (1997, Microbiology 143: 2639-46) investigated the mechanisms leading MTX resistance in Enterococcus hirae. They found several phenotypic effects including (i) increased folic acid reductase (FAR) activity (ii) increased dihydrofolate reductase (DHFR) activity, (iii) decreased synthesis and intracellular retention of MTX containing two glutamyl residues, (iv) decreased uptake of MTX accompanied by decreased uptake of folates; and (v) reduction of folate-binding capacity. The form of folate present in the media during the development of resistance affected DHFR and FAR activities and the transport of folates.
However, although it is known that exposure to MTX can be used to select cells having increased resistance to MTX, no link between MTX resistance and folate levels of the cells has been disclosed in the prior art. In contrast to the MTX resistance mechanisms described above, the present inventors have surprisingly found that bacterial cells have another way of generating MTX resistance. This mechanism is the production of high levels of folate. This finding can be exploited in the screening and selection of (spontaneous) mutant bacteria producing high folate levels, by using methotrexate as selection agent. Such bacteria, especially food grade bacteria, can be used to fortify food products with folate. Folate is a soluble B vitamin, required for proper cell growth and functioning. Many food products, such as bread, cereals, dairy products, etc. are supplemented with folic acid to ensure adequate intake of folate. The mutant bacteria of the present invention enable fortification of food products with natural folate by for example fermentation using the mutant bacteria.
While testing whether recombinant folate overproducing bacteria have modified susceptibility to MTX, it was found that a number of wild type strains of L. plantarum, which were incubated for more than about 40 hours on inhibitory amounts of MTX (1,25 mg/L) were not inhibited by MTX, but had a growth rate similar to that on medium lacking MTX. When analysing the folate levels of these strains, approximately 5% of these colonies displayed a significant higher folate production compared to the (non mutated) wild type strain. The total folate pools were 4% to 63% higher compared to (average) wild-type levels. This observation demonstrated that MTX can be used to efficiently select natural (non-GMO) folate overproducing bacteria. The resistance to MTX was maintained, even when the strains were grown for more than 50 generations on medium lacking MTX, which indicated that the resistance is caused by (spontaneous) stable mutations in the bacterial genome.
The same effect, that increased folate production leads to high resistance against MTX, was seen in recombinant L. plantarum strains, which were transformed with the complete folate biosynthesis gene cluster under the control of a strong promoter. These recombinant strains produced high levels of folate and were resistant to MTX when grown in a medium lacking folate dependent metabolites. Without limiting the scope of the invention, it is speculated that the intracellular dihydrofolate levels compete with the accumulated MTX for the active site on the enzyme dihydrofolate reductase (DHFR).
This finding was applied to develop a rapid screening method, based on growth in the presence of MTX, to select folate overproducing bacterial strains. The folate overproducers that were identified in this study displayed a high increase in folate levels compared to the wild type folate producers. The total folate levels ranged from 20 to 60 times the level of the wild type. The intracellular folate pools found in the folate overproducers were 10 to 20 times higher compared to the levels found in the wild type strain. These increased intracellular folate levels cause a decreased sensitivity (i.e. resistance) towards MTX. This explained why growth of the folate overproducing strains was not significantly affected by MTX. Sensitivity of the wild-type towards MTX was only observed when all the metabolites which are associated with folate were omitted from the medium.
Therefore, in one embodiment of the invention mutant bacteria are provided which comprises an increased amount of intracellular and/or extracellular folate compared to the wild type and which have a growth rate (μ) of at least 0.1 h−1 when grown on medium comprising (at least) 1.25 mg/l methotrexate and lacking folate dependent metabolites (i.e. they are said to be “resistant” to MTX). Preferably under these growth conditions, the growth rate of the mutant bacterium is not significantly different than that of the wild type strain when the wild type strain is grown on the same medium lacking MTX (i.e. “normal growth”). Most importantly, the mutant bacterium shows significant growth at an MTX concentration which is inhibitory to the wild type strain. An “Inhibitory concentration” is the concentration of MTX which results in substantially no growth of the wild type strain.
Growth rate can be measured by various means. For example, at certain time points following inoculation of the medium with the bacterial strain or isolate spectrophotometric readings can be taken and the growth rate over time calculated.
The mutant bacterium preferably comprises an increased amount of intracellular and/or extracellular folate compared to the wild type strain. Preferably, the amount of total folate is at least about 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, more preferably at least about 60%, 63% (or more) higher than the average amount of intracellular folate of the wild type strain(s). As in some bacterial species the wild type strains show significant variation in intracellular folate levels, it is preferred that a number of different wild type strains (e.g. 2, 3, 4 or more strains) are analysed for intracellular folate levels and the average folate level is calculated.
Preferably it is a food grade lactic acid bacterium. It may be a species selected from the group consisting of Lactobacillus reuteri, L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, L. casei, L. plantarum, L. paracasei, L. murinus, L. jensenii, L. salivarius, L. minutis, L. brevis, L. gallinarum, L. amylovorus, Lactococcus lactis, Streptococcus thermophilus, Leuconostoc mesenteroides, Lc. lactis, Pediococcus damnosus, P. acidilactici, P. parvulus, Bifidobacterium bhifidum, B. Iongum, B. infantis, B. breve, B. adolescente, B. animalis, B. gallinarum, B. magnum, and B. thermophilum.
In an alternative embodiment the mutant bacterium is an induced mutant having increased folate and being resistant to MTX. Such a mutant may be obtained using the same method as used in the selection of natural mutants (described herein below), except that prior to the screening method a mutagenesis step is included. A mutagenesis step may include one or more known mutagenesis methods, such as exposure to a chemical and/or physical mutagen (e.g. N-methyl-N′-nitro-N-nitrosoguanidine; UV radiation, gamma-radiation, etc.). Following mutagenesis, the bacteria are grown in/on medium comprising inhibitory amounts of MTX and isolates which do grow are selected and analysed for folate content.
In yet another embodiment, the mutant bacteria according to the invention can be characterized by their gene expression pattern. This is illustrated by the finding that methotrexate resistant L. plantarum strains producing at least 50% more folate compared to the wild-type show a 2-fold (or more) overexpression of one or more of thefolC genes in the genome. The genefoiC codes for the enzyme dihydrofolatesynthase. In one embodiment the bacteria according to the invention show overexpression compared to the wild-type of one or more genes coding for enzymes of the folate biosynthesis pathway. In addition to the gene directly involved in folate biosynthesis, also higher transcription levels were found for the following genes: FK (coding for fructokinase), POX (pyruvate oxidase), LOX (lactate oxidase) and PDH (pyruvate dehydrogenase). Compared to the wild-type, a significantly lower transcript level was found for: DAK (dihydroxyacetone phosphotransferase or glycerone kinase). Thus, in one embodiment of the invention, the bacterium/bacteria according to the invention comprise significantly enhanced mRNA transcript levels of one or more genes ofthe group encoding dihydrofolate synthase (EC 6.3.2.17), fructokinase (EC 2.7.1.4), pyruvate oxidase (EC 1.2.3.3), and 6-phospho-β-glucosidase (EC 3.2.1.86). Additionally or alternatively, the bacteria comprise significantly reduced mRNA transcript levels of the gene encoding for dihydroxyacetone phosphotransferase (=glycerine kinase) (EC. 2.7.1.29).
The term “upregulated” or “downregulated” refers to an increased mRNA transcription level or a decreased mRNA transcription level compared to the gene in the wild type strain.
A “significantly enhanced transcript level” or a “significantly enhanced transcription level” refer to an amount of mRNA transcript of at least about 2 fold, preferably at least about 3 fold, or even about 4 fold or more compared to the transcript level found in the wild type. Likewise, a “significantly reduced transcript level” or “transcription level” refers to an amount of mRNA transcript of at least about 2 fold, 3 fold, etc. less than found in the wild type.
Optionally, the transcription pattern (i.e. the enhanced or reduced transcript levels of one or more of the genes mentioned above) may be used as a selection criterion. For example, bacteria may first be screened for altered expression levels in a first screen, and subsequently analysed for methotrexate resistance and/or folate levels (as described below). Likewise, it may be used as an additional screening/selection step in the method described below (for example between steps (b) and (c) or after step (d), or it may even replace steps (a) and (b).
The transcription profile of one of the methotrexate resistant strains described herein (Lactobacillus plantarum NIZO B2550) has been compared with that of the parental strain (Lactobacillus plantarum WCFS1 (the full sequence of L. plantarum WCFS1 has been deposited in the EMBL database under accession no. AL93.5263)). This was done by using whole genome DNA micro-arrays of Lactobacillus plantarum WCFS1. Total mRNA samples of the parental strain and the methotrexate resistant mutant were isolated and subsequently treated with reverse transcriptase to synthesize fluorescently labelled cDNA. The samples of parental strain and mutant were differentially labelled. Subsequently, a mixture of the labelled cDNA samples was hybridized on the DNA-microarray to determine the relative abundance of the mRNA of all genes in the mutant relative to the parental strain of L. plantarum.
In one embodiment the mutant bacteria are the bacterial strains (or any derivatives thereof) deposited by NIZO Food Research, P.O. Box 20, 6710 BA Ede, the Netherlands at the CBS (Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under the Budapest Treaty under Accession number CBS 117120.
A further aspect of the invention relates compositions comprising mutant bacteria as described herein above or derivatives or extracts thereof, as well as to methods for the production of such compositions. The bacteria of the invention are cultured under appropriate conditions, optionally recovered from the culture medium and optionally formulated into a composition suitable for the intended use. Methods for the preparation of such compositions are known per se.
Preferably, the mutant bacteria, derivatives or extracts thereof, are used to make food or food supplement compositions comprising natural folate.
A preferred composition according to the invention is suitable for consumption by a subject, preferably a human or an animal. Such compositions may be in the form of a food supplement or a whole food or food composition, which besides the bacteria of the invention also contains a suitable food base. A food or food composition is herein understood to include not only solid and semi-solid compositions, but also liquids for human or animal consumption, i.e. a drink or beverage. The food or food composition may be a solid, semi-solid and/or liquid food or food composition, and in particular may be a dairy product, such as a fermented dairy product, including but not limited to a yoghurt, a yoghurt-based drink or buttermilk. Such foods or food compositions may be prepared in a manner known per se, e.g. by adding one or more mutant bacteria of the invention to a suitable food or food base, in a suitable amount.
Also food supplements can be made, comprising suitable amounts of one or more mutant bacterial strains, or extracts therefrom (e.g. partially or substantially purified natural folate). Food supplements include for example vitamin tablets, pills, capsules or powders, comprising recommended daily amounts of various vitamins, including folate. The synthetic folic acid normally used in such preparations may be replaced with the natural folate according to the invention.
In a further preferred embodiment, the live bacteria are used in or for the preparation of a food or food composition, e.g. by fermentation. In doing so, the bacteria of the invention may be used in a manner known per se for the preparation of such fermented foods or food compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid bacteria. In such methods, the bacterial cells of the invention may be used in addition to the micro-organism usually used, and/or may replace one or more or part of the micro-organism usually used. For example, in the preparation of fermented dairy products such as yoghurt or yoghurt-based drinks, a food grade mutant bacterium of the invention may be added to or used as part of a starter culture or may be suitably added during such a fermentation. The “growth medium” of the bacteria in this case is a food-grade medium, such as milk-based.
In a further embodiment the bacteria used may be dead (e.g. lysed) or non-viable, or lyophilized.
The compositions manufactured using one or more mutant bacterial strains according to the invention or supplemented with a suitable amount of one or more of the strains, comprise an enhanced amount of natural folate, produced by the bacteria. One or more strains which are high overproducers of folate may be mixed or may be added sequentially to the composition or during its production. The total amount of folate present in the final product can be analysed using for example HPLC analysis.
Methotrexate is used for the selection of bacteria having increased intracellular and increased total folate levels.
In one embodiment a method for selecting bacteria having increased intracellular and/or total folate levels is provided. The method comprising the steps of:
“Growing” in step (a) may also mean contacting the bacteria with such a medium. The medium may comprise various concentrations of MTX. For example a first round of selection may be carried out on a relatively low concentration, such as 1.25, 1.5, 2.0, 2.5 mg/l medium. Isolates growing on one or more of these concentrations may then be screened directly for folate content or may be subjected to one or more further rounds of MTX selection, using for example a higher concentration of MTX, such as 4, 5, 10 or more mg/liter. The stringency of the selection process can be varied by selecting more stringently, e.g. only isolates with a high growth rate at high concentration, or less stringently, e.g. isolates showing moderate growth at relatively low MTX concentrations. Preferably, in each MTX selection step, the isolates are grown on or in the medium for at least 40 hours, more preferably at least 50 hours or more. Suitable controls, e.g. wild type isolates, should always be included, as the mutants are selected based on the difference in growth between the mutant and the control (e.g. the wild type). The control need not necessarily be a wild type, but can also be a previously selected strain. Optimum incubation temperature, pH of the medium, and other parameters can be easily determined by the skilled person. The exact conditions depend on the bacterial species used.
Preferably, the method is set up in such a way that large numbers of isolates can be screened simultaneously, e.g. in 96-well microtitre plates or the like.
The method can be applied on all bacteria which show a natural sensitivity for methotrexate. Using the folate analogue methotrexate, one can select and isolate efficiently natural folate overproducing bacteria which can be used for the production of fermented food products with increased folate concentration (natural fortification or fermentation fortification) as described above.
Any bacterium obtainable according to the method and having significantly increased folate levels is comprised herein.
Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y.; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and TI of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.), all incorporated herein by reference.
Wild type L. plantarum WCFS1 strains and its derivatives were grown at 37° C. on Chemically Defined Media (CDM), containing 19 g/L β-phosphoglycerate (6) or modified CDM. In this modified CDM, glycine, inosine, orotic acid, thymidine, guanine, adenine, uracil and xantine were omitted from the normal CDM. Additionally 8 ng/ml chloroamphenicol was added to all the media used. Methotrexate (Sigma, aminopterin) was added to both types of CDM, in concentrations ranging from 0 to 10 mg/l.
The plasmids used in this study are listed in Table 1.
The vector pNZ8148 is an empty vector containing the nisin inducible promoter, a multiple cloning site, and a chloramphenicol resistance marker. The pNZ8148 derivatives and the L. plantarum genomic DNA was isolated using established procedures. PCR was performed using Pfx DNA polymerase (Invitrogen, Paisley, UK) in a Mastercycler PCR apparatus (Eppendorf, Hamburg, Germany) using the following cycles: denaturation at 94° C. for 30 s (3 minutes the first cycle), annealing at 45° C. for 25 s, and elongation at 68° C. for 1 minute per 1000 bps for a total of 30 cycles.
The folate levels of the three genetically engineered L. plantarum strains were analyzed using the Lactobacillus casei microbiological assay as described previously (Sybesma et al., 2003. Appl. Environ. Microbiol. 69:3069-76).
Microtiter plates were used to determine the growth rate of the three L. plantarum strains harbouring the different vectors. The growth of the three strains was monitored in a 96 well microtiter plate for 35 hours using a spectrophotometer (SPECTRAmax®, Molecular Devices, Sunnyvale, Calif., USA). The growth was tested on the two types of media, and each construct was analyzed in triplicate on both media for the following MTX concentrations: 0, 0.3125, 0.625, 1.25 and 2.5 mg/L.
The wild type L. plantarum WCFS1 containing pNZ8148 was again cultivated in modified CDM containing 2.5 mg/L MTX, but this growth medium was split-up into 96 wells of the microliter plates. This microliter plate was incubated for 3 days, and hereafter aliquots from each of the 96 wells were dispersed into two fresh 96 well microliter plate containing the modified CDM with this time 10 mg/L MTX. Through each step the original microliter plate was mixed with 60% glycerol and stored in a −80° C. freezer. The glycerol stored microliter plates were used to inoculate the mutated or adapted cells on fresh modified CDM medium. The folate levels of these 288 MTX resistance cultures were checked using a quick screening method. This method is based on the normal microbiological assay as described previously, however some changes were adopted. In this quick screening method the folate produced by the wild type is compared, to the folate produced by the mutant strains. Cultivation of the indicator strain on the folate produced by the wild type leads to a relative low optical density. The optical density of the indicator strain on folate overproducers will be higher then of the wild type. Strains producing an increased optical density of the indicator strain were reanalyzed in by using the extended Lb. casei microbiological assay.
The folate production levels of L. plantarum wild type carrying pNZ8148 and the two genetic constructs pNZ7019 and pNZ7021 were studied on normal CDM medium. Folate production of L. plantarum WCFS1 strain containing the empty vector pNZ8148 was found to be 100 ng folate/ml culture (
Next, the specific growth rates of the two folate overproducing strains and the wild type strain, were compared on CDM with and without folate dependent metabolites in the presence of a range of methotrexate (MTX) concentrations (
Prolonged incubation (>40 hours) of the wild type L. plantarum WCFS1 on modified CDM supplemented with 2.5 mg/L MTX, resulted in significant growth of the culture. If this culture was diluted in fresh modified CDM supplemented with 2.5 mg/L MTX, immediate growth of the culture was observed. Single colonies were isolated from this culture to test if natural mutants with increased MTX resistance were selected or if some kind of temporary adaptation takes place. Modified CDM containing 2.5 mg/l MTX was inoculated with a single colony derived from an MTX resistant culture. Next, the growth medium was split into two cultures; one culture was grown for 50 generation in the modified CDM with 2.5 mg/l MTX, the other culture was grown for 50 generations in the same medium in the absence of MTX. After the 50 generation both cultures where transferred again to modified CDM containing the 2.5 mg/l MTX. Both strains with a different history revealed a comparable growth rate on this medium indicating that a stable mutation causes the MTX resistance phenotype.
We isolated 288 colonies from cultures containing 2.5 mg/L or 10 mg/L MTX.
These colonies were analysed for folate overproduction using a rapid pre-screening method. Based of this rapid screen, 5 out of 288 colonies gave raise to cultures with higher folate levels compared to the wild type. These 5 cultures were subsequently plated on modified CDM supplemented with 2.5 mg/L MTX. From each plate 3 single colonies were isolated, stored and analyzed again for folate production in the following way. Folate levels in cultures derived from 15 positive colonies were monitored (
Using oligonucleotide microarray technology for genome-wide transcription profiling, we have identified genes in the methotrexate resistant mutant L. plantarum NIZO B2550 (derived from L. plantarum WCFS1) which are specifically up- or down regulated compared to the wild-type. Cultures of L. plantarum WCFS1 and NIZO B2550 were grown on modified CDM and at a turbidity (OD600) of 1, cells were harvested and mRNA was isolated. The mRNA samples were processed for analysis on DNA micro-arrays. The relative abundance of specific mRNA's was expressed as the fold change in expression of genes in the methotrexate resistant strain compared to the corresponding genes in the parental strain. Analysis of the transcriptome profile reveals that in comparison with the parental strain,folC1, the gene coding for a dihydrofolate synthase is specifically upregulated 4-fold (see Table 2) in the methotrexate resistant mutant compared to the wild-type. This enzyme couples L-glutamate to dihydropteroate to form dihydrofolate, which is a structural analogue of methotrexate and serves as the substrate for the enzyme dihydrofolate reductase. More dihydrofolate synthase through overexpression of folC1 is expected to lead to higher intracellular dihydrofolate pools, which in turn could compete with methotrexate for conversion by dihydrofolate reductase. This observation reveals the mechanism of methotrexate resistance in the isolated mutants. It also explains why these mutants produce higher levels of folate in the absence of methotrexate.
L. plantarum NIZO B2550 compared to L. plantarum WCFS1.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05101533.7 | Mar 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL06/50042 | 3/1/2006 | WO | 00 | 11/12/2007 |
