Patent Application
20230263864
The present invention relates to engineered microbes that are suitable for in vivo therapeutic production of L-DOPA and Dopamine in a subject. The invention also relates to pharmaceutical formulations and uses of the same for the treatment or management of diseases and disorders that can be treated or managed with L-DOPA and Dopamine produced by the engineered microbes in vivo in the digestive track, such as the gut.
A number of bioactive molecules can be derived from L-tyrosine using different enzymes and enzymatic pathways as shown in
L-DOPA is a prodrug of dopamine that is administered to patients with Parkinson's due to its ability to cross the blood-brain barrier. Currently L-DOPA is administered as a pharmaceutical. However, maintaining a stable level of the compound in the blood is problematic.
Dopamine which is produced by decarboxylation of L-DOPA, modulates blood pressure, and also has a role in immune modulation, adipose tissue metabolism, nutrient absorption, and modulation of gut-brain axis functions.
The present application addresses the need for these molecules by providing engineered microbes which can produce L-DOPA and dopamine in the gut.
In vivo production of compounds in the gut by engineered microbes is complicated by the nature of enzymatic processes and potential for a variety of by-products that complicates therapeutic in vivo use of the engineered microbes.
However, the present inventors have engineered microbes which produce L-DOPA and dopamine in sufficient amounts to have in vivo efficacy. The by-product profiles of the constructs are also suitable for therapeutic use.
In a first aspect, there is provided a microbial cell adapted to produce L-DOPA, the cell comprising: a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase, for use as a medicament.
The microbial cell may for use in a method of treating Parkinson's disease or in a method of treating a dopamine-related disorder.
In a further aspect there is provided a microbial cell comprising a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase, wherein the microbial cell is a therapeutic microbial cell, optionally E. coli Nissle.
In a further aspect there is provided a pharmaceutical formulation comprising a microbial cell wherein the microbial cell comprises a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase.
The microbial cell may additionally comprise a nucleic acid encoding a compound which inhibits an L-DOPA metabolizing bacteria; or may be co-administered with:
i) a compound which inhibits an L-DOPA-metabolizing bacteria; or
ii) a further microbial cell which produces a compound which inhibits an L-DOPA-metabolizing bacteria.
The microbial cell may also additionally comprise:
a) a recombinant nucleic acid encoding a 4a-hydroxytetrahydrobiopterin dehydratase; and/or
b) an σ70 promoter.
In a second aspect of the invention, there is provided a microbial cell adapted to produce dopamine, the cell comprising:
a) a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase; and
b) a recombinant nucleic acid encoding an enzyme having L-DOPA decarboxylase activity.
The tyrosine hydroxylase be a mutant enzyme wherein:
a) the mutant tyrosine hydroxylase does not comprise a functional regulatory domain; and/or
b) the mutant tyrosine hydroxylase comprises a mutation in the catalytic domain.
The mutation may correspond to any one of amino acids 177-198 of SEQ ID NO. 2, optionally wherein the mutation is at an amino acid corresponding to amino acid 196 of SEQ ID NO. 2, optionally wherein the mutation is Ser196Glu or Ser196Leu; or any one of amino acids 22-43 of SEQ ID NO. 4, optionally wherein the mutation is at an amino acid corresponding to amino acid 41 of SEQ ID NO. 4, optionally wherein the mutation is Ser41Glu (SEQ ID NO. 6) or Ser41Leu (SEQ ID NO. 8).
The L-DOPA decarboxylase enzyme may belong to any one of the following:
a) EC:4.1.1.28, optionally wherein the enzyme has at least 70% sequence identity to SEQ ID NO.s 18, 20 or 22;
b) EC:4.1.1.105, optionally wherein the enzyme has at least 70% sequence identity to SEQ ID NO.s 20 or 22;
c) EC:4.1.1.25 optionally wherein the enzyme has at least 70% sequence identity to SEQ ID NO. 25.
Also provided is a pharmaceutical formulation comprising any of the microbial cells above which are adapted to produce dopamine.
Also provided is any of the microbial cells above adapted to produce dopamine for use as a medicament, for example in a method of treating a dopamine-related disorder.
The following may apply to any of the aspects above:
The tyrosine hydroxylase may belong to EC 1.14.16.2.
The tyrosine hydroxylase may not comprise the regulatory domain. For example, the tyrosine hydroxylase may comprise the catalytic domain and the tetramerization domain of the eukaryotic tyrosine hydroxylase enzyme, optionally wherein the tyrosine hydroxylase has at least 70% sequence identity to SEQ ID NO. 4.
The microbial cell may additionally comprise a nucleic acid encoding a mutant GTP cyclohydrolase I, the mutant GTP cyclohydrolase I having at least 70% sequence identity to SEQ ID NO. 10, and comprising one or more mutations wherein the mutant provides for an increased hydroxylation activity of the tyrosine hydroxylase. For example, the GTP cyclohydrolase I mutation may be at a position corresponding to amino acid 198 of SEQ ID NO. 10.
The microbial cell may further comprise a nucleic acid encoding a 4a-hydroxytetrahydrobiopterin dehydratase (phhB), optionally wherein the phhB belongs to EC 4.2.1.96 and/or has at least 70% sequence identity to SEQ ID NO. 14; and/or a nucleic acid encoding a dihydromonapterin reductase (FolM), optionally wherein the FolM has at least 70% sequence identity to SEQ ID NO. 12.
The nucleic acid(s) may be integrated into the genome of the microbial cell.
In a further aspect, there is provided a recombinant expression plasmid comprising:
a) a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase; and any one or more of the following:
b) i) a recombinant nucleic acid encoding a 4a-hydroxytetrahydrobiopterin dehydratase; and/or
In a further aspect, there is provided a recombinant expression plasmid comprising:
a) a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase; and
b) a recombinant nucleic acid encoding an enzyme having L-DOPA decarboxylase activity.
In a further aspect there is provided a mutant eukaryotic tyrosine hydroxylase wherein the mutation is at any one of amino acids 177-198 of SEQ ID NO. 2, optionally wherein the mutation is at an amino acid corresponding to amino acid 196 of SEQ ID NO. 2, optionally wherein the mutation is Ser196Glu or Ser196Leu. The tyrosine hydroxylase enzyme may also be the truncated form lacking the regulatory domain therefore, optionally the mutation is at any one of amino acids 22-43 of SEQ ID NO. 4, optionally wherein the mutation is at an amino acid corresponding to amino acid 41 of SEQ ID NO. 4, optionally wherein the mutation is Ser41Glu (SEQ ID NO. 6) or Ser41Leu (SEQ ID NO. 8).
Microbial Cell
By microbial cell is meant a bacteria and/or yeast cell.
The microbial cell may be a therapeutic cell meaning a cell suitable for use in medical treatment. These cells are nonpathogenic and may be commensal, i.e. part of the normal flora of the gut. The microbial cell may be an aerobic organism. Alternatively, the microbial cell may be an anaerobe which can survive and optionally grow in the presence of oxygen. That is, the microbial cell is not an obligate anaerobe. The microbial cell may be a probiotic microbial cell.
The microbial cell must be able to tolerate oxygen. That is, they can survive in the presence of oxygen. To test if a cell can survive in the presence of oxygen, this can be done for instance using the thioglycolate test. Fluid thioglycolate media is made such that an oxygen gradient concentrates high oxygen at the top of the broth and low oxygen at the bottom of the broth. Organisms that tolerate oxygen will cluster near the top and organisms that cannot tolerate oxygen will cluster near the bottom.
Microbial cells which are anaerobes and can survive in the presence of oxygen are as follows: The microbial cell may be a facultative anaerobe. A facultative anaerobe can grow without oxygen but can use oxygen if present. Alternatively, the microbial cell may be an aerotolerant anaerobe which cannot use oxygen for growth but will tolerate it's presence.
The microbial cell may be able to colonize where there is oxygen in the small and/or large intestine, for example an oxygen gradient. For example, the mucous layer of the small and/or large intestine, for example the inner and/or outer layer of mucous. For example the inner or outer layer of mucous of the large intestine.
Suitable therapeutic cells include Escherichia coli, for example E. coli Nissle. Other examples of suitable therapeutic cells include lactic acid bacteria for example Lactobacillus and/or Lactococcus. Other examples of therapeutic cells include Akkermansia, for example Akkermansia muciniphila, Bifidobacterium, Bacteroides, Salmonella or Listeria.
Other examples include Saccharomyces boulardii.
The cell may alternatively be a synthetic microbial cell.
Where the microbial cell is a combination of cells, the yeast may for example produce tyrosine hydroxylase and optionally any 1 or more of the co-factors: FoIE, FolM, FoIX or phhB; and the bacterial cell may produce any 1 or more of the co-factors: FoIE, FolM, FoIX or phhB. For example, the yeast cell may produce tyrosine hydroylase and the bacterial cell may produce FolE and FolM.
The microbial cell may be a combination of bacterial cells also where one type of bacterial cell produces tyrosine hydroxylase and optionally 1 or more of the co-factors, and another type of bacterial cell produces one or more of the co-factors.
The resulting combination of microbial cells may be described as a composition of microbial cells.
Mutant
By mutant is meant an enzyme which differs from the full length wild-type form.
By corresponding to is meant the equivalent amino acid in any sequence for that enzyme. For example Ser 196 in a tyrosine hydroxylase other than rat. The corresponding or equivalent amino acid in a tyrosine hydroxylase from another species can be found using sequence alignment software such as the BLAST sequence alignment tool described below.
Nucleic Acids
The nucleic acids may have 70, 75, 80, 85, 90, 95 or 100% sequence identity with those listed in Table 3.
Pharmaceutical Formulation
A pharmaceutical formulation includes excipients to preserve the activity or to deliver the cell to the gut. Preferably the formulation is an oral formulation.
The microbial cell may be formulated to preserve its activity and/or for delivery to the gut via an oral tablet or capsule or the like.
For example, the microbial cell may be lyophilized and include a lyoprotectant. The formulation may alternatively or additionally include any other excipient required to preserve the activity of the cell.
The formulation may be in an oral dosage form with a coating which allows delivery to the gut, for example an enteric coating.
Plasmid
The enzymes for expression in the microbial cell may be cloned into one of the native plasmids of a therapeutic bacteria.
For example, E. coli contains 2 native plasmids which are maintained stably in the strain. Cloning the enzymes into these plasmids ensures stability of the plasmid and enzymes at a controlled, low copy number. Additionally, this minimizes the amount of foreign DNA introduced to the strain, and it is non-transferrable to other bacteria, ensuring safety.
Alternatively, the enzymes may be expressed in a plasmid which is not native to the bacteria.
A yeast plasmid may also be used when yeast is the or one of the microbial cell(s). The plasmid may comprise any of the enzymes and/or promoters listed below in combination for expression of L-DOPA or dopamine in the microbial cell.
Integrated into the Genome
Alternatively, the genes encoding the enzymes may be integrated into the genome of the therapeutic microbial cell. This can be done using the CRISPR technique. Alternatively this can be done by various other methods including clonetegration (Shearwin et al (2013), ACS Synthetic Biology, Vol 2, pp 537-541).
Promoters
A promoter is a nucleotide sequence capable of controlling the expression of a gene. The promoter may be a σ70 promoter or a modified version of such a promoter where the nucleotide composition has been optimized for in vivo expression levels.
The promoters claimed have been tested for predictability and robustness in the mammalian GI tract. They have been selected from a large library of promoters, causing the most stable gene expression under any conditions (e.g. +/−oxygen, in exponential or stationary growth phase, in the upper and lower part of the GI tract, in the lumen vs. in the mucus layer), which are important for making robust therapeutic bacteria.
The tyrosine hydroxylase and/or L-DOPA decarboxylase genes may be under the control of the promoter. Additionally one or more of the other enzymes for L-DOPA or dopamine production listed may also be under the control of the promoter. Therefore, the microbial cell or recombinant plasmid may comprise one or more of the following promoters.
The σ70 promoter may have at least 70, 75, 80, 85, 90, 95 or 100% sequence identity to SEQ ID NO. 32 or 33.
For example, the promoter for the tyrosine hydroxylase may have a consensus sequence as follows:
Where the sequence is in accordance with the IUPAC code below.
For example, the promoter may be SEQ ID NO. 32 or 33 or a sequence comprising 90, 95 or 98% sequence identity with either SEQ ID NO. 32 or 33. The promoter may consist of consensus sequence SEQ ID NO. 55.
The promoter for any or all of FoIE, FolM and/or FoIX may be an Anderson promoter. The promoter for any or all of FoIE, FolM and/or FoIX may have a consensus sequence as follows (again with reference to the IUPAC code above):
For example, the promoter may be SEQ ID NO. 38-50 or a sequence comprising 70, 75, 80, 85, 90, 95 or 98% sequence identity with either SEQ ID NO. 38-50. The promoter may consist of consensus sequence SEQ ID NO. 56.
Functional variants with different degrees of sequence identity can be checked for retention of activity by comparing expression of a suitable reporter under the control of the variant promoter and compare this activity with the reporter under the control of the non-variant promoter. It is generally preferred that a promoter with less that 100% sequence identity retains at least 25, 50, 75, 80, 85, 90, 95 or 100% activity of the reference promoter. In addition to sequence identity, the promoters may be shortened at 1 or both ends of the sequence. This shortening may be by 1 or 2 nucleotides at 1 or both ends. These shortened variants can be checked for retention of activity as explained above.
Recombinant
By recombinant is meant an exogenous nucleic acid sequence which is not native to the cell in which the nucleic acid is being expressed.
The cell may contain 1 copy of the enzyme(s) or more than 1. For example, there may be more than 1 copy of the nucleic acid encoding the tyrosine hydrolase present in the cell, either in a plasmid or integrated into the genome.
Sequence Identity
Sequence identity may be calculated using any suitable software such as BLAST (Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410.)
The enzymes claimed may have at least 70%, 75%, 80%, 85%, 90%, 95% or 90% sequence identity to any of the enzymes listed in Table 3. The enzymes may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
Features for L-DOPA Production
L-DOPA
L-DOPA, L-3,4-dihydroxyphenylalanine, is made from the amino acid tyrosine. This is shown in
It is the precursor to the neurotransmitter dopamine. Conversion to dopamine occurs in the CNS (after L-DOPA crosses the blood brain barrier) and in the peripheral nervous system.
Tyrosine Hydroxylase
The eukaryotic tyrosine hydroxylase (TyrOH) is a member of the biopterin-dependent aromatic amino acid hydroxylase family of non-heme, iron(II)-dependent enzymes. TyrOH catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA) as shown in
The tyrosine hydroxylase of the invention may belong to EC 1.14.16.2. The enzyme may be an animal enzyme, for example a mammalian enzyme.
The sequence of the full length rat tyrosine hydroxylase is as follows:
The above sequence is SEQ ID NO. 2. The tyrosine hydroxylase may have at least 70, 75, 80, 85, 90, 95, 97 or 100% sequence identity with SEQ ID NO. 2.
The enzyme may be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
GTP Cyclohydrolase I (folE)
In humans, the production of L-DOPA requires synthesis and regeneration of the co-factor tetrahydrobiopterin. Bacteria and yeast do not produce this co-factor. Therefore, the native cofactor tetrahydromonapterin pathway is exploited instead. The synthesis pathway for this native cofactor is shown in
The GTP cyclohydrolase I may belong to E.C. 3.5.4.16.
The GTP cyclohydrolase I may have at least 70, 75, 80, 85, 90, 95 or 100% sequence identity with SEQ ID NO. 10. The enzyme may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
The mutation may increase hydroxylation of the tyrosine hydroxylase by at least 120% as compared to the native or wild-type unmutated enzyme (under the same conditions). The mutation may be at any one of the following positions in SEQ ID NO. 10: D97-E112, K121-D130, N170-H180, S193-L200 and S207-N222. For example, D97, M99, T101, V102, A125, K129, N170, V179, T196, T198 (excluding T198P), S199, L200, S207, H212, E213, F214, L215 and H221.
The mutation may be selected from: D97V, D97L, D97A, D97T, M99C, M99T, M99V, M99L, M991, T101I, T101V, T101L, V102M, N170K, N170D, N170L, V179A, V179M, T1961, T196V, T196L, T198I, T198V, T1983, T198L, 3199Y, 3199F, L200P, L200C, L2003, L200A, S207R, S207K, S207M, H212R, H212K, E213K, E213R, F214A, F214G, F2143, L215P, L215Q, L215N, L215D, L215T, L215S, L215G, L215A, L215C, L215F, L215M, H221R and H221K.
The mutant may also comprise any combination of these mutations.
For example, the GTP cyclohydrolase I mutant may have at least 70% sequence identity with SEQ ID NO. 10, and comprise any one or more of the above mutations.
The GTP mutant may be the endogenous, native GTP cyclohydrolase which is mutated i.e. not an additional recombinant copy.
Additional Enzymes which Aid Tyrosine Hydroxylation Activity
In addition or as an alternative to the FolE mutation to increase co-factor production which in turn increases tyrosine hydroxylation, other enzymes in the pathway of
For example, the microbial cell may over-express (compared to the wild-type under the same conditions) any nucleic acid encoding:
The nucleic acid may also be any encoding enzymes with these activities and having at least 70, 75, 80, 85, 90, 95 or 100% sequence identity with the above SEQ ID NO.s. The enzymes may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the constructs.
Upregulating expression may be via a recombinant nucleic acid, for example an additional copy of the gene on a plasmid or integrated into the genome, or alternatively via upregulating the endogenous sequence.
The microbial cell may have increased activity of FolE and/or FolM. Therefore, the microbial cell comprises a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase (for example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 4) and upregulated FolE and/or FolM. This may be by additional recombinant FolE and/or FolM being added to the cell. The FolE enzyme may be mutated as described above.
Alternatively, the microbial cell comprises a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase (for example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 4) and utilizes the endogenous FolE and FolM cofactors. The FolE enzyme may be mutated as described above.
Expression of the tyrosine hydroxylase (or example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 4) may be under a promoter comprising or consisting of consensus SEQ ID NO. 55. Expression of one or more of the co-factors (for example, FolE and/or FolM) may be under the control of a promoter comprising or consisting of SEQ ID NO. 56. The enzymes (and optionally the promoters described above) are preferably integrated into the genome of the cell.
Compound which Inhibits L-DOPA-Metabolizing Bacteria
Bacteria such as E. faecalis metabolize L-DOPA in the gut (see
The enzyme in E. faecalis responsible for metabolizing L-DOPA is TyrDC. Therefore, the compound may inhibit any bacteria which express TyrDC, for example, any bacteria comprising a nucleic acid encoding an enzyme with at least 70% sequence identity to SEQ ID No. 25.
Such a compound may be a bacteriocin. For example: Ubericin A, Hiracin, JM79 or Enterocin A (for example SEQ ID NO.s 29, 27 or 31). Alternatively the bacteriocin may be any of the below.
Bacillus subtilis
Micrococcus varians
Lactobacillus curvatus
Lactobacillus sakei
Lactobacillus sakei
Enterococcus faecium
Lactobacillus johnsonii
Bacillus subtilis
Enterococcus faecium
Lactobacillus johnsonii
Lactobacillus plantarum
Bacillus halodurans
Lactobacillus plantarum
Pediococcus pentosaceus
Enterococcus faecalis
Staphylococcus aureus
Enterococcus hirae DCH5
Enterococcus faecalis
Streptococcus uberis
Carnobacterium maltaromaticum
Enterococcus faecalis
Streptococcus uberis
Streptococcus bovis HJ50
Weissella cibaria 110
Enterococcus durans QU 49
Streptococcus uberis
Enterococcus faecium T8
Lactococcus lactis QU 5
Leuconostoc pseudomesenteroides
Leuconostoc pseudomesenteroides
Enterococcus avium
Enterococcus faecium
Enterococcus faecium
Lactococcus sp. QU 12
Lactococcus garvieae
Weissella
paramesenteroides DX
Bacillus thuringiensis
Enterococcus faecalis
Leuconostoc
mesenteroides TK41401
Staphylococcus
epidermidis 224
Enterococcus faecalis
Enterococcus faecalis
Bacillus thuringiensis DPC
Bacillus thuringiensis DPC
Lactococcus garvieae
Enterococcus faecalis
Enterococcus faecium
The bacteriocin may also be any which has at least 70, 75, 80, 85, 90 or 95% sequence identity to any of the above bacteriocins. For example any of SEQ ID NO.s 27, 29 or 31.
Parkinson's Disease
Parkinson's disease causes impairment in both motor and non-motor functions. Current treatment is with L-DOPA in the form of tablet or inhalable powder.
Features Specific to Dopamine Production
Dopamine
Dopamine is a hormone and a neurotransmitter that plays several important roles in the brain and body. It is an organic chemical of the catecholamine and phenethylamine families. It is an amine synthesized by removing a carboxyl group from a molecule of its precursor chemical L-DOPA. The structure of dopamine and the pathway from L-tyrosine is shown in
Mutant Tyrosine Hydroxylase
The tyrosine hydroxylase may be a mutant, i.e. the enzyme differs from the full length wild type enzyme sequence.
The wild type full length rat enzyme comprises:
The mutant may not comprise the regulatory domain. The entire regulatory domain may be deleted or only part of the regulatory domain may be deleted.
Truncation may be at any point in the regulatory domain to reduce the complexity of the protein for expression in a microbial cell and/or to decrease negative feedback by dopamine for the dopamine-producing microbial cell. The skilled person would be aware of suitable points to truncate the regulatory domain whilst maintaining the activity of the enzyme guided by the crystal structure (Goodwill, K., Sabatier, C., Marks, C. et al. Crystal structure of tyrosine hydroxylase at 2.3 Å and its implications for inherited neurodegenerative diseases. Nat Struct Mol Biol 4, 578-585 (1997).
The tyrosine hydroxylase may comprise the catalytic domain (and not the regulatory domain or tetramer domain); or the catalytic domain and the tetramer domain (and not the regulatory domain). These domains may comprise the above amino acids sequences or have at least 70, 75, 80, 85, 90, 95, 99 or 100% sequence identity with the above amino acid sequences, and optionally be further truncated to the core secondary structure elements to provide function, for example by removing 1-20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids from the N and/or C termini of the constructs.
For example, the truncated enzyme may comprise the catalytic and tetramer domains, amino acids:
Optionally the truncated enzyme may be SEQ ID NO. 4.
Alternatively, the truncated enzyme may comprise the catalytic domain only:
SAREDKVPWFPRKVSELDKCHHLVTKFDPDLDLDHPGFSDQVYRQRRKLIAEIAFQYKHGE PIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFL KERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLA DRTFAQFSQDIGLASLGASDEEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHS LSEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPF (amino acids 158-456 of SEQ ID NO. 2). Optionally the truncated enzyme may be amino acids 1-301 of SEQ ID NO. 4.
The tyrosine hydroxylase may be any sequence having at least 70, 75, 80, 85, 90 or 95% sequence identity to the above truncated forms. The enzyme may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
The truncated forms described above may be used for L-DOPA as well as dopamine production.
The following mutants are particularly adapted for dopamine production.
The tyrosine hydroxylase may alternatively or additionally be mutated to increase flux through the pathway and/or to prevent dopamine inhibition of tyrosine hydroxylase.
The tyrosine hydroxylase may not comprise an active regulatory domain meaning the regulatory domain is mutated to prevent feedback inhibition by dopamine.
The tyrosine hydroxylase may alternatively or additionally comprise a mutation in the catalytic domain which increases dopamine production, for example by 3-fold compared to the wild type. The mutation may be in amino acids 177-198 of SEQ ID NO. 2. These amino acids form a loop as shown by the crystal structure of the enzyme. The inventors have surprisingly found that mutating an amino acid in this loop increases dopamine production. The amino acid mutated in this loop may be at position 196. The mutant may be Ser 196Glu or Ser196Leu. These are shown below in the rat full length enzyme, and truncated enzyme. The mutation in the truncated form corresponds to position 41, optionally to Glu/Leu (Ser 40 without the start codon, and as referred to in
Full Length Mutant (Loop 177-198 is Underlined; Mutation 196 is in Brackets)
Truncated Mutant without the Regulatory Domain (Loop 22-43 is Underlined; Mutation 41 is in Brackets
This mutation at position 196 in the full length or 41 in the truncated form may also be applied to any of the truncated mutants above, for example the truncated form comprising only the catalytic domain.
Therefore, the tyrosine hydroxylase may comprise any of the truncated forms above and additionally comprise a mutation in the loop: CHHLVTKFDPDLDLDHPGFSDQ, optionally at the underlined serine position.
For example, the mutant may be SEQ ID NO. 6 or 8, or a mutant with at least 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO. 6 or 8.
The tyrosine hydroxylase may have at least 70, 75, 80, 85, 90, 95 or 100% sequence identity with any of the above mutant forms. Additionally, the mutant may be further truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) from the N and/or C termini of the constructs.
The inventors have surprisingly found that the above mutants (with the mutation at position 196 in the full length sequence and position 41 in the truncated sequence without the regulatory domain) produced less L-DOPA, for example 5, 10, 15 or 20% less L-DOPA compared to the wild-type, but at least 1.5 fold, 2 fold, 2.5 fold or 3 fold higher dopamine. This is set out in the table below and
Also see
L-DOPA Decarboxylase Activity
To produce dopamine from L-DOPA, the L-DOPA is decarboxylated.
The L-DOPA decarboxylase used may be any of the following:
The decarboxylase may also be any with at least 70, 75, 80, 85, 90, 95, 97 or 99% sequence identity with the above enzymes. The enzyme may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
Dopamine-Related Disorders
Peripheric dopamine can affect browning of adipocytes, energy expenditure, levels of glucose in blood and contribute to insulin signaling. Therefore, the microbial cell expressing dopamine may help treat diabetes, obesity and/or other metabolic diseases.
Furthermore, Dopamine modulates the immune system. Therefore, the microbial cell expressing dopamine could be to regulate the immune response in the gut. For example, the microbial cell could be used to treat Irritable bowel disease, ulcerative colitis, Chrohn's disease, Intestinal cancers.
The microbial cell may also be used to treat other immune-mediated inflammatory diseases. For example, the microbial cell may be used to treat ankylosing spondylitis, psoriasis, psoriatic arthritis, Behcet's disease, arthritis and allergy.
Dopamine can also regulate blood pressure. Therefore, the microbial cell may be used as a blood pressure modulators. For example, the microbial cell may be used to treat high or low blood pressure.
As L-DOPA can be converted into dopamine peripherally, the L-DOPA producing microbial cells can also be used to deliver dopamine and hence treat any of the dopamine-related disorders above.
Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the method or kit includes a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.
Aspects of the present invention will now be illustrated by way of example only and with reference to the following experimentation.
Strains and Cultivation Conditions
For general lab procedures strains were grown using LB media at 37° C., unless otherwise stated. Strains generated were stored at −80° C., glycerol stocks (glycerol 25%). Proper antibiotics were used accordingly to the resistance markers of the different strains.
L-DOPA Production Cultures
L-DOPA production cultures were carried out in 96 deep well plates and 350 μl media. Biological triplicates of each strain were used to inoculate precultures in M9 media with 0.4% glucose with or without 0.2% CAS amino acids and L-Tyrosine. Precultures were grown for 24 hours at 37° C. in a shaking incubator at 250 RPM. Production cultures were carried by inoculating the preculture with 1:100 ratio of the total volume and incubated at 37° C. in a shaking incubator at 250 RPM for 22 hours. After 22 hours the cultures were centrifuged at 4700 RPM and the supernatant was collected and frozen until further analysis.
Plasmids
Plasmid Construction and Purification
L-DOPA and dopamine producing plasmids were constructed using USER cloning. pMUT plasmid, truncated tyrosine hydroxylase, decarboxylases and other genes were amplified using Phusion U polymerase and uracil containing primers. These fragments were later purified using Thermofisher PCR purification kit and were subsequently cloned together using the USER enzyme. Top10 chemically competent cells were transformed by heat-shock with 5 μl of USER reaction and plated in LB plates supplemented with kanamycin. Plates were incubated at 37° C. overnight. Correct constructs were screen by colony PCR and confirmed by sequencing. Correct colonies were incubated in 2×YT supplemented with kanamycin at 37° C. overnight. Plasmids were later extracted from the cultures using MACHEREY-NAGEL plasmid purification kit.
The plasmids are shown in
The truncated TH, phhB and all DDCs except for EF have been codon optimized. folE, folM and folX are native sequences from E. coli.
E. coli Nissle
Transformation of E. coli Nissle
1 colony of E. coli Nissle was grown overnight at 37° C. in a shaking incubator. Next day, 1:100 dilution was inoculated in 10 ml of 2×YT for 3-4 hours. At OD600=0.4-0.5 cells were harvested, washing 3 times with cold 10% glycerol in MQ water, and were later electroporated using Bio-RAD MicroPulser electroporator and 0.1 mm electroporation cuvettes. Transformants cells were recovered in 1 ml of SOC media at 37° C. for 1 hour before plating them in LB plates supplemented with kanamycin and incubated at 37° C. overnight.
E. coli Nissle strains were inoculated in biological triplicates and grown for 24 hours in M9 media with 0.4% glucose (Preculture). Production culture was inoculated in 1:100 ratio from the preculture and grown for 22 hours in M9 media with 0.4% glucose and supplemented with 100 mg/L of L-Tyrosine. Production cultures were centrifuged at 4500 RPMs and supernatant was collected for H PLC analysis.
HPLC analysis was carried out as follows:
Quantitative analysis of L-DOPA, L-Tyrosine and dopamine in cell-free supernatant was performed by High-Performance Liquid Chromatography (HPLC) on an UltiMate 3000 UHPLC system (ThermoScientific). The system consisted of an LPG-3400RS quaternary pump and a WPS-3000RS autosampler with a TCC-3000 column oven and a DAD-3000 diode array detector. Samples were run at a pressure of 600 bar through a CORTECS column (1.6 μm, 2.1×150 mm) at 30° C. with an injection volume of 1 μl and a flowrate of 0.350 ml/min in 10 mM ammonium formate as mobile phase.
Constructing Promoter Variants
pHM181 (=pDOPA_1) was used as the starting point from which all other variants tested in
Production of L-DOPA
Biological triplicates of E. coli Nissle strains harboring the different promoter constructs were grown, using 96 deep-well plates, in 350 μl of M9 minimal media with 0.4% glucose for 24 hours in a shaking incubator at 37° C. and 250 RPM. Production culture was inoculated with 1:100 inoculum from the preculture in fresh M9 minimal media with 0.4% glucose. The plate was incubated for 22 hours at 37° C. and 250 RPM. The production culture was then centrifuged at 4500 RPM and supernatants were transferred into a 96 well microtiter plate and stored at −20° C. until HPLC analysis.
Results Summary
The results are shown in
The FolE mutant shows increased production as seen in
Promoter MSKL7 and MSKL8 with the tyrR KO produce the most L-DOPA. Promoter 7 was chosen for further experiments as it showed an increase of L-DOPA production in both genotypes with and without tyrR KO compared to promoter 8.
The same cultivation method as previously described was used to test the effect of changes to the co-factor production on the amount of L-DOPA produced.
The following differences in plasmids were tested:
pMUT-DOPA_5 is shown in
Variations made to this plasmid were as follows:
5.4=changed promoter of phhB for the trc promoter
5.5=added folE(T198I) gene from E. coli Nissle
5.6=added folE(T198I) and folM from Nissle (also shown in
Additionally, a further experiment was carried out to probe the effect of over-expression of phhB.
Results Summary
The results are shown in
In addition to folE, the addition of folM also increase L-DOPA production.
With regards to phhB, it was found that in a background strain containing the genomic folE(T198I) mutation, co-expression of phhB increases L-DOPA production in a small but statistically significant manner (
Bacteriocin Assay (
Three colonies of each E. coli Nissle strain were inoculated in 5 ml of BHI broth and grown overnight at 37° C. in a shaking incubator. The following day, 1 ml of the culture was washed with PBS buffer once and 10 μl were used to spot the strains on top of a BHI agar plate. After drying, top BHI agar was mixed with 500 μl of previously grown E. faecalis, and was placed on top of the BHI agar containing the dried spots of the E. coli strains. Plates were dried for 10 minutes and then incubated at 37° C. overnight.
Competition Experiments (
E. faecalis and L-DOPA EcN strains expressing different bacteriocins were grown overnight in Brain Heart Infusion (BHI) broth (NutriSelect™), without supplementation of antibiotics. The next day, EcN cultures were washed once and resuspended in PBS. Cultures were diluted accordingly to have a concentration of 10{circumflex over ( )}7 and 10{circumflex over ( )}6 CFU/ml of EcN and E. faecalis respectively in 10 ml of BHI. Throughout the experiment 200 μl were taken periodically for CFU plating and 1 ml for future HPLC quantification. Samples for HPLC quantification were centrifuged at 10 000 g for 3 min, supernatant was transferred into a 96-well plate. Before HPLC quantification the supernatants were filtered using a 96-well filter plate (AcroPrep™) Culture dynamics were followed by transferring 200 μl of the competition culture into a 96-well microtiter plate and running a kinetic experiment measuring OD and GFP in a fluorescent microtiter plate reader (Synergy H1). Competition experiment was performed for 48 hours.
Results Summary
The results are shown in
6D-A: L-DOPA producing EcN strains, which co-express bacteriocins outcompete E. faecalis compared to an L-DOPA producing strain, which does not produce bacteriocins.
6D-B these strains also are able to maintain higher levels of L-DOPA in the supernatant and for longer time than the EcN that does not produce bacteriocins.
6D-C,D These strains inhibit the metabolism of tyrosine into tyramine by E. faecalis. The enzyme tyrDC, responsible for this is also the one that turns L-DOPA into dopamine and contributes to the degradation of L-DOPA and a poor therapeutic response in PD patients.
These results show the strain can not only express L-DOPA but also inhibit E. faecalis in the vicinity of the L-DOPA producing strain meaning higher levels of L-DOPA can be maintained instead of being metabolised to dopamine.
Oral Gavage of Engineered E. coli Nissle:
Female mice (NMRI, supplied by Taconic Biosciences, 6 weeks of age) were group-housed on a 12-h light:dark cycle at constant temperature with ad libitum access to food and water in a Specific Pathogen Free (SPF) facility. Upon delivery, mice were given 5 days to adjust to new location. Cohort size was 8 animals, and 4 different cohorts were tested, see below. All animals received Streptomycin (5 g/L) in the drinking water to ensure colonization, 3 days before being gavaged and throughout the experiment. A single oral gavage of 108 cells was administered of either L-DOPA-producing (called ‘EcN_DOPA’) or a control E. coli Nissle (‘EcN_CTRL’) strain without expression of the tyrosine hydroxylase gene. Samples were taken for the following 7 days, after which animals were euthanized and final blood samples and gut content samples were collected. 2 of the 4 cohorts were also treated with the TDC inhibitor Carbidopa via intraperitoneal injection (10 mg/kg body weight) every 24 h. Fresh fecal samples were collected daily for 7 days to quantify colonization and metabolite levels. Plasma samples were taken on day 2 (submandibular sampling) and day 7 (vena cava) after gavage, and urine samples were taken on day 3 and 6.
In vivo sample analysis: Plasma, tissue samples, gut content and fecal samples were analyzed for DOPA-derived and related serotonin metabolites using LC-MS. For plasma: blood samples were collected using BD microtainer tubes with Li-Heparin coating, and plasma was prepared according to the manufacturer's instructions and frozen at −80 C. Urine samples were collected within 30 minutes of urination and immediately frozen at −80 C. Both sample types were then thawed, mixed with an internal standard buffer (IS buffer) containing 0.9% NaCl, 0.2% Ascorbic acid and 20 mg/L C13, N15-labelled Tryptophan, and then methanol-precipitated. After drying samples using a vacuum centrifuge, they were reconstituted in 50 ul ddH2O for LC-MS/MS analysis. Gut content and fecal samples were weighed, then homogenized in ice-cold IS buffer, centrifuged for 1 min at 500 g and the supernatant was immediately stored at −80 C for analysis. Gut and brain tissue was also frozen for real-time quantitative PCR (RT-qPCR) and metabolite analysis. Quantification of metabolites from in vivo samples was performed as described above (‘LC-MS analysis’).
Results Summary
The results are shown in
Oral delivery of the genetically modified E. coli Nissle strains of the invention and their effect on host physiology was demonstrated in mice. The L-DOPA producing strain was shown to affect metabolite levels in urine and plasma, compared to a non-producing control strain (
General Protocol for Production of Dopamine
Biological triplicates of E. coli Nissle strains harboring the different promoter constructs were grown, using 96 deep-well plates, in 350 μl of M9 minimal media with 0.4% glucose and 0.2% Cas amino acids for 24 hours in a shaking incubator at 37° C. and 250 RPM. Production culture was inoculated with 1:100 inoculum from the preculture in fresh M9 minimal media with 0.4% glucose and 0.2% Cas amino acids. The plate was incubated for 22 hours at 37° C. and 250 RPM. The production culture was then centrifuged at 4500 RPM and supernatants were transferred into a 96 well microtiter plate and stored at −20° C. until HPLC analysis.
A panel of L-DOPA decarboxylases was tested in combination with tyrosine hydroxylase. The DDCs were on a different plasmid, called pMK-DDC (
The same culture conditions were used as described above, with the only difference that 100 mg/L L-tyrosine was supplemented in the medium. This information is also in the table above.
Draconibacterium
orientale
Nisaea denitrificans
Verrucosispora
Candidatus
Koribacter versatilis
Sus scrofa
Catharanthus
roseus
Homo sapiens
Capsicum annuum
Oryza sativa
Japonica
Camptotheca
acuminata
Ophiorrhiza pumila
Enterococcus
faecalis
Enterococcus
faecalis
Detection and quantification of L-DOPA, dopamine, tyrosine, tyramine, phenethylamine, serotonin, tryptamine, tryptophan, and 5-HTP were conducted by liquid chromatography mass spectrometry (LC-MS) measurements on a Dionex UltiMate 3000 UHPLC (Fisher Scientific, San Jose, Calif.) connected to an Orbitrap Fusion Mass Spectrometer (Thermo Fisher Scientific, San Jose, Calif.). The system used an Agilent Zorbax Eclipse Plus C18 2.1×100 mm, 1.8 μm column kept at 35° C. The flow rate was 0.350 mL/min with 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B) as mobile phase. The gradient started as 5% B and followed a linear gradient to 35% B over 1.5 min. This solvent composition was held for 3.5 min after which it was changed immediately to 95% B and held for 1 min. Finally, the gradient was changed to 5% B until 6 min. The sample (1 uL) was passed on to the MS equipped with a heated electrospray ionization source (HESI) in positive-ion mode with sheath gas set to 60 (a.u.), aux gas to 20 (a.u.) and sweep gas to 2 (a.u.). The cone and probe temperature were 380° C. and 380° C., respectively, and spray voltage was 3500 V. Scan range was 50 to 500 Da and time between scans was 100 ms. Quantification of the compounds was based on calculations from calibration standards analyzed before and after sets of 24 samples. All reagents used were of analytical grade.
Results Summary
The results are shown in
The best DDCs that produced measurable amounts of dopamine were: DRO, CK, SS, EF, EFop). These were selected for further testing with variants of the TyrH enzyme as described below in Example 7.
The truncated tyrosine hydroxylase was used as the background for testing mutations top optimize dopamine production.
A mutation at position 196 in the full length: position 40 in the truncated enzyme was made (position 41 including the start codon).
This is at the following sequence for the truncated enzyme:
The same methods as described above (Plasmid construction, Nissle transformation and Production of L-DOPA) were used to test for L-DOPA production.
Results Summary
The results are shown in
Variation of ser40 of tyrosine hydroxylase (Ser41 with the start codon included) to ser40glu and ser40leu affects production of L-DOPA. The characterized variations surprisingly decrease L-DOPA production yet increase dopamine production. These truncated mutants are SEQ ID NO.s 6 and 8.
Uracil primers containing the codon substitution for ser40 were used to amplify the plasmid containing the TH. The PCR product was purified and USER cloning protocol was followed (described above). The correct construct was later transformed into E. coli Nissle for further production characterization (also described previously).
The mutant was then tested with various decarboxylases to look for the strain which produced the most dopamine and the fewest side products.
The best DDCs were combined with the different versions of the TH (still using a 2 plasmid system, and feeding 100 mg/L L-tyrosine in the medium) and tested for production of dopamine under the same culture conditions, but in the absence of 100 mg/L supplemented Tyrosine. Therefore, all dopamine produced in
Results Summary
The results are shown in
Under these conditions, a construct was found (TyrH(Ser40Leu)+SS-DDC) which produces dopamine with a titer of approximately 25 mg/L without any detectable byproducts.
A further promoter (Anderson J23101) was tested for driving the expression of cofactor genes.
This promoter is SEQ ID NO. 39 (tttacagctagctcagtcctaggtattatgctagc). Other Anderson promoters that could be used are in SEQ ID NO.s 38 and 40-50.
The strains tested were as follows:
Briefly, 514 is an empty control, 519 does not have overexpression of folE and folM and 838 is the new strain with the codon optimized folE and folM and the Anderson promoter.
Results Summary
The results are shown in
Although the 838 strain produced lower amounts of L-DOPA, the Anderson promoter still produces L-DOPA in large amounts. The Anderson promoter is therefore an option for in vivo expression. By varying this promoter sequence, the amount of L-DOPA can be modified further (either up or down).
Genome integration was carried out using the pOSIP clone integration approach (St-Pierre et al, “One-step Cloning and Chromosomal Integration of DNA”, ACS Synth. Biol. 2013, 2, 9, 537-541).
The integration site used was the att186 integration site.
Results Summary
The results are shown in
The constructs tested are listed on the x-axis:
Column 1=strain 514 (empty plasmid)
All the further columns tested constructs with the codon optimized folE and folM and the new Anderson promoter (J23101). Instead, the promoter driving tyrosine hydroxylase expression was varied along with the RBS.
The constructs on the x axis are listed in the table below.
For example MSKL 8-8117 means the promoter used was MSKL 8 with a RBS with TIR of 8117. The terms “big” and “small” refer to the colony size only. Both types produced L-DOPA.
Varying the RBS did not alter L-DOPA production. The best construct as denoted by the arrow in
Biological triplicates of E. coli Nissle strains harboring the different tyrosine hydroxylases or bacterial enzymes constructs were grown, using 96 deep-well plates, in 350 μl of M9 minimal media with 0.4% glucose for 24 hours in a shaking incubator at 37° C. and 250 RPM. Production culture was inoculated with 1:100 inoculum from the preculture in fresh M9 minimal media with 0.4% glucose. The plate was incubated for 22 hours at 37° C. and 250 RPM. The production culture was then centrifuged at 4500 RPM and supernatants were transferred into a 96 well microtiter plate and stored at −20° C. until HPLC analysis.
Results Summary
The results are shown in
The results show that in the same genetic background and under the same conditions, the 838 and 519 strains produced more L-DOPA compared to the bacterial enzyme pathway which is based on E. coli native enzymes (HpaBC). A further advantage of TyrH, is that it is highly specific towards tyrosine unlike the bacterial enzymes (monooxygenases like hpaBC) that tend to be promiscuous in their substrate preference.
Additionally, we show that expressing the full length tyrosine hydroxylase from rat (codon optimized) E. coli Nissle is able to produce L-DOPA.
Gavage Preparation:
A single colony of each bacterial strain was grown in 50 ml of 2×YT for at least 16 hours at 37° C. and 250 RPM in a shaking incubator. Cultures were then washed with PBS and adjusted to contain 0.5×1010 CFU/ml.
Animals and Experiments:
Male Sprague Dawley rats were acclimatized for 1 week before randomized grouping (4 per group). 5 g/L of Streptomycin in drinking water was started 3 days prior of the gavage regime and was maintained throughout the experiment. Animals were gavaged 2 ml of 1010 CFU daily for 3 days (days 0-2). On day 3, animals were given 25 mg/Kg (IP) of carbidopa 1 hour prior the gavage containing 4×1010 CFU/ml and tyrosine (50 mg/Kg). Animals were sacrificed on day 4 and jugular blood was collected after decapitation. CFUs were determined from fecal and gut content samples.
Extraction of Plasma L-DOPA:
L-DOPA from plasma was extracted using an Ostro Protein Precipitation & Phospholipid Removal Plate following manufacturer's guidelines (100 μl of plasma), samples were dried using a speedvac with no heating and resuspended in MQ water containing 0.1% Ascorbic acid and formic acid. C-13 L-DOPA internal standard was spiked before the extraction method to account for any loss throughout the procedure. Internal standard solution also contained 0.1% ascorbic acid.
Lc-Ms/Ms Quantification:
The LC-MS/MS analysis was performed on a Vanquish Duo UHPLC binary system (Thermo Fisher Scientific, USA) coupled to the IDX-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, USA). The analytes were separated using a Waters ACQUITY BEH C18 (10 cm×2.1 mm, 1.7 μm) column equipped with an ACQUITY BEH C18 guard column kept at 40 C. The mobile phases consisted of MilliQ© water+0.1% formic acid (A) and acetonitrile+0.1% formic acid (B). The initial composition was 2% B held for 0.8 min, followed by a linear gradient till 5% in 3.3 min, and to 100% B in 10 min held for 1 min before going back to initial conditions. Re-equilibration time was 2.7 min. The flow rate was set at 0.35 mL/min. The MS measurements were done in positive and negative-heated electrospray ionization (HESI) mode with a voltage of 3500 V and 2500 V respectively acquiring in full MS/MS spectra (Data dependent Acquisition-driven MS/MS) in the m/z range of 70-1000. The acquired data were processed using QuanBrowser from the Xcalibur software v 4.4 (Thermo Fisher Scientific, USA).
Results Summary
The results are shown in
These results show that L-DOPA plasma levels were substantially increased in rats that were treated with a EcN with L-DOPA production capabilities (a: 0.511) (strain 519) compared to an empty strain, which does not produce any L-DOPA (a: 0.034).
Methods:
The same cultivation methods as described above were used to test expression of L-DOPA from the following strains:
L-DOPA was quantified using HPLC as described above.
Results Summary
The results are shown in
The results indicate that an extra expression component (where the integrated strain ALSO has the plasmid) boosts L-DOPA production (P=0.0273).
The results support that multiple copies in the chromosome, plasmid or combined (chromosome and plasmid) should increase microbial L-DOPA production capabilities, allowing the titration of L-DOPA in vivo.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010928.6 | Jul 2020 | GB | national |
| 2107473.7 | May 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/069895 | 7/15/2021 | WO |
