Kynurenine responsive and degrading bacteria

Abstract

Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive and degrading bacteria (e.g., such that the bacteria reduces or eliminates the tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences encoding: i) a kynurenine transcriptional regulator protein, ii) a kynurenine transporter protein, iii) a kynureninase (KynU) protein, and iv) at least one protein important or essential for growth of the bacteria expression of which is linked to a kynurenine responsive promoter (e.g., where the corresponding gene(s) has been deactivated or knocked out in the genome of the bacteria).

Description

[0001]Attorney Docket Number: CCF-44551.601 KYNURENINE RESPONSIVE AND DEGRADING BACTERIA The present application claims priority to U.S. Provisional application serial number 63 / 707,975, filed October 16, 2024, which is herein incorporated by reference in its entirety. This invention was made with government support under GM156762, CA267711 and CA043703 awarded by the National Institutes of Health and HT9425-24-1-0880 by the Department of Defense. The government has certain rights in the invention. SEQUENCE LISTING PARAGRAPH The text of the computer readable sequence listing filed herewith, titled “CCF_44551_601_SequenceListing.xml”, created October 16, 2025, having a file size of 28,432 bytes, is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive and degrading bacteria (e.g., such that the bacteria reduces or eliminates the tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences encoding: i) a kynurenine transcriptional regulator protein, ii) a kynurenine transporter protein, iii) a kynureninase (KynU) protein, and iv) at least one protein important or essential for growth of the bacteria expression of which is linked to a kynurenine responsive promoter (e.g., where the corresponding gene(s) has been deactivated or knocked out in the genome of the bacteria). BACKGROUND OF THE INVENTION Bacterial-based cancer therapy could be a promising treatment option for solid tumors especially for those cases in which the traditional approaches are inadequate and ineffective. This therapy is based on the high natural tendency of some bacteria like Salmonella enterica, Escherichia coli, and Clostridium novyi to grow specifically within the tumor compared to normal tissues causing both direct toxic effect on the tumor cells and activation of the immune system to better eradicate the tumor1-3. This preference for the tumor tissues has been attributed to multiple factors including nutrient availability, more protection from immune surveillance and the hypoxic environment within the solid tumors in case of anaerobic bacteria4. With the latest advances in bacterial synthetic biology, the bacterial- based cancer therapy became closer to reality with several clinical trials were completed or Attorney Docket Number: CCF-44551.601 are going using genetically modified bacterial strains as either monotherapy or in combination with other therapies including immune checkpoint inhibitors (ICIs) (e.g. NCT00004988, NCT01675765, NCT01924689, NCT03435952, etc.). A main limitation, however, for the wide-spread application of the bacterial-based cancer therapy is the need to improve the specificity of the bacteria to the cancer site especially if these bacteria were engineered to carry cytotoxic payload4. With regard to S. enterica, one of the most promising and widely tested bacteria as cancer therapeutic, several previous approaches aimed at attenuating it to reduce its toxicity and improve its tumor targeting5-7. Of note, S. enterica VNP20009 strain which has mutations in purI and msbB genes making it auxotrophic to alanine and less stimulatory of tumor necrosis factor-α production, was among the most promising strains when tested in murine models in terms of specificity to the tumor8. Initial attempts, however, testing VNP20009 in clinical trials found it ineffective in causing tumor regression and that we still need to improve the tumor localization of S. enterica9. This in turn highlights the need to find alternative ways to engineer S. enterica so that we can enhance its safety profile (and specificity to cancer) without reducing its efficacy. SUMMARY OF THE INVENTION Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive and degrading bacteria (e.g., such that the bacteria reduces or eliminates the tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences encoding: i) a kynurenine transcriptional regulator protein, ii) a kynurenine transporter protein, iii) a kynureninase (KynU) protein, and iv) at least one protein important or essential for growth of the bacteria expression of which is linked to a kynurenine responsive promoter (e.g., where the corresponding gene(s) has been deactivated or knocked out in the genome of the bacteria). Also provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive bacteria (e.g., such that the bacteria reduce or eliminate said tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences comprising: i) a first nucleotide sequence (e.g., not endogenous to said bacteria) encoding a kynurenine transcriptional regulator protein, and / or ii) a second nucleotide sequence (e.g., not endogenous to said bacteria or overexpressing an endogenous equivalent protein) encoding a kynurenine transporter protein. In particular embodiments, the kynurenine responsive bacteria further comprise at least one of the following: i) a third nucleotide sequence comprising a kynurenine responsive promoter Attorney Docket Number: CCF-44551.601 upstream of a plasmid replication gene encoding a plasmid replication protein, ii) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein, and iii) a fifth or more nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene essential for bacterial growth, or a gene codes a for therapeutic payload. In some embodiments, provided herein are composition comprising: a bacterial cell (e.g., engineered bacterial cell), wherein the bacterial cell comprises: A) at least one bacterial chromosome, wherein the at least one bacterial chromosome has at least one endogenous gene important or essential for growth of the bacterial cell: i) deactivated, or ii) knocked-out; B) optionally one or more expression vectors (e.g., 1, 2, 3, or 4) which optionally comprise one or more plasmids; and C) one or more nucleic acid sequences which are present in the bacterial genome and / or are present in the one or more expression vectors, wherein the one or more nucleic acid sequences comprise: i) a first nucleotide sequence encoding a kynurenine transcriptional regulator protein; ii) a second nucleotide sequence encoding a kynurenine transporter protein; iii) a third nucleotide sequence encoding a kynureninase (KynU) protein; iv) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of at least one gene that encodes at least one protein that compensates for (e.g., supplies the missing protein or missing protein function, so the bacteria can grow) the at least one endogenous gene that is deactivated or knocked-out; and v) optionally a fifth nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein. In particular embodiments, provided herein are methods comprising: administering the bacteria as described above or herein to a patient with a tumor or a disease in which kynurenine, and optionally a polyamine, which is optionally spermidine, is accumulating in a localized lesion. In particular embodiments, the patient is a human. In other embodiments, the administration is such that the bacteria localizes to the tumor, or the localized lesions, of the patient. In particular embodiments, the administration is such that the bacteria degrades the kynurenine (e.g., at least 50%, 60%, 70%, 80%, or 90% of the kynurenine present) or such that most or all of the tumor is gone after treatment. In other embodiments, the bacteria further degrades the polyamine, which is optionally spermidine. In further embodiments, the tumor or the localized lesion produces kynurenine and the bacteria preferentially localizes, and / or proliferates in, the tumor or lesion at least in part based on the kynurenine expression. In other embodiments, the bacteria are administered orally, systemically or through direct intratumor, or intra-lesion, injection. Attorney Docket Number: CCF-44551.601 In certain embodiments, provided herein are kits and systems comprising: a) the bacteria above and as described herein; and b) a delivery device or composition for administering the bacteria to a patient with a tumor or lesion, and / or a shipping container. In certain embodiments, the patient is a human. In further embodiments, delivery device or composition is selected from: syringes, oral tablets, capsules, enema, and IV solution. In particular embodiments, the bacteria herein (e.g., engineered bacteria) further comprise: vi) a sixth nucleotide sequence comprising a kynurenine responsive promoter upstream of at least one gene that codes for a therapeutic payload, wherein the at least one gene encodes Interferon gamma (INFy), IFN-alpha, IFN-beta, a type 1 interferon, IL-12, IL- 15, IL-18, IL-2, TNF-alpha, anti-CD40, ICAM-1, an immune checkpoint inhibitor, or a TLR agonist, or other therapeutic, which is optionally human. In some embodiments, the therapeutic is selected from: an immunomodulatory agent, a bacterial toxin, and a drug- activating enzyme. In further embodiments, the bacterial further comprise: vi) a sixth nucleotide sequence encoding aromatic amino acid transaminase (TyrB) protein, and / or a seventh nucleotide sequence encoding aspartate aminotransferase (aspC) protein. In other embodiments, the bacterial further comprise: vi) one or more sixth nucleic acid sequences that alone or together encode: PauA protein (PauA (also called SpuB and PuuA)), PauB protein (PauB (also called PuuB)), PauC protein (PauC (also called KauB and PuuC)), and PauD protein (PauD (also called SpuA and PuuD)). In other embodiments, the bacterial further comprise: vi) a sixth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein. In certain embodiments, the reporter protein is selected from mCherry, luciferase, GFP, tdTomato, CFP, RFP, YFP, β- galactosidase and β-galactosidase. In other embodiments, the bacteria comprises all of the "sixth nucleotide sequences" described above. In other embodiments, the one or more expression vectors (e.g., adenoviral vector) are present in the bacterial cell, and wherein the first, second, third, and fourth nucleic acid sequences (and optionally others mentioned above or herein) are present in the one or more expression vectors. In certain embodiments, the one or more expression vectors comprise one or more plasmids, and wherein the fifth nucleotide sequence is present and is in the one or more plasmids. In certain embodiments, at least one of the first, second, third, and fourth nucleic acid sequences are in the at least one bacterial chromosome (e.g., 1 or 2 are present in the at least one bacterial chromosome). In other embodiments, the one or more expression vectors comprise one or more plasmids which comprise first and second plasmids, and wherein the first plasmid contains the first and second nucleotide sequences, and wherein the Attorney Docket Number: CCF-44551.601 second plasmid contains the fourth and / or fifth nucleotide sequences. In other embodiments, the one or more plasmids comprise three or four plasmids. In some embodiments, the third nucleotide sequence is: i) present in the at least one bacterial chromosome (e.g., as a knock-in), ii) present on the first or second plasmids, or iii) present on a third plasmid. In particular embodiments, the kynurenine transcriptional regulator protein comprises KynR protein or homologs thereof. In additional embodiments, the KynU and / or the TyrB are from Pseudomonas aeruginosa or other equivalent proteins from Gram-negative and Gram-positive bacteria. In further embodiments, the kynurenine transporter protein comprises an MTR protein, a Tna protein, or an AroP protein. In certain embodiments, the kynurenine responsive promoter comprises a kynurenine- responsive promoter DNA sequence (Pkyn), such as: PPakynB, PcnkynB, and PpfkynU. In other embodiments, the plasmid replication protein is present and comprises repL. In other embodiments, the bacteria is from the following genera which naturally harbor KynR: Pseudomonas, Cupriavidus, other proteobacteria, and Bacillus. In further embodiments, the kynurenine transporter protein comprises an MTR protein, a Tna protein, or an AroP protein, or their homologues other aromatic amino acid transporters which may transport kynurenine such as TyrP, and PheP. In other embodiments, the at least one, or at least two, or at least three of the first, second, third, fourth, or fifth nucleic acid sequences are non-endogenous to the bacteria. In further embodiments, the bacteria is E. coli or S. enterica. In further embodiments, the bacteria further comprise a nucleic acid sequence comprising a quorum sensing system upstream of the third nucleotide sequence encoding the KynU protein. In other embodiments, the quorum sensing system comprises a PluxI promoter upstream of a sequence encoding LuxI protein, and optionally further encoding LuxR protein. In further embodiments, the quorum sensing system comprises at least one of the following systems: i) a Plas promoter upstream of a sequence encoding lasI and / or lasR; ii) a Prhl promoter upstream of a sequence encoding rh1I and / or rh1R; iii) a Ptra promoter upstream of a sequence encoding traI and / or traR; iv) a sequence encoding ArgA, ArgB, ArgC, and ArgD; v) a sequence encoding: luxS, luxP, and luxQ; vi) a sequence encoding: qseC and qseB; and vii) a sequence encoding HHQ, PQS, HQNO, and NHQ. DESCRIPTION OF THE FIGURES Figure 1. KynR regulator with its cognate promoter (Pkyn) were cloned on a plasmid system and optimized in E. coli then moved to S. enterica. A series of constructs in which KynR with its cognate promoter (Pkyn) were cloned from P. aeruginosa on reporter Attorney Docket Number: CCF-44551.601 constructs and screened. A) Early constructs showed only modest performance in E. coli. B) Subsequent modification through optimizing the ribosomal binding site (RBS) sequence for kynR and mCherry genes to make pPaKynR2. C) Further modification through overexpressing the transporter, Mtr, to make pmtr-PaKynR2. D) Expression of the cloned Mtr transporter was tuned to further enhance the sensitivity to kynurenine in the construct designated p113mtr-PaKynR2. E) Comparing the performance of the different kynurenine- responsive promotors in E. coli when cloned upstream of mCherry on a plasmid that harbors KynR. F) A schematic diagram of the final optimized two-plasmid system (pKynR5) is shown. The kynurenine-sensing system was further improved using the dual -plasmid system, pKynR5 which employs kynR gene from P. aeruginosa together with the Mtr transporter on plasmid A while harboring kynurenine-inducible promoters PCnkyn and PPfkyn from P. fluorescens and C. nectar, respectively upstream of plasmid replication protein, RepL, and the reporter mCherry in plasmid B. G-H) Testing this system in both E. coli and Salmonella proved it responds to kynurenine with high ON / OFF ratio. Figure 2. S. enterica ATCC 14028 naturally harbors the promiscuous aspartate transaminase (AspC) in addition to other related transaminases including aromatic amino acid transaminase (TyrB). To first test if S. enterica can naturally metabolize kynurenine into kynurenic acid, and if AspC enzyme is responsible for this conversion, we generated S. enterica ∆aspC knockout. In parallel, we also transformed the wild-type S. enterica with a plasmid harboring kynurenine transporter (Tra). Analyzing the LB culture spent media of each of these strains through LC-MS / MS confirmed that S. enterica naturally metabolizes kynurenine into kynurenic acid albeit modestly. The expression of kynurenine transporter gene resulted in higher kynurenic acid. As expected, wild-type (Wt) S. enterica was unable to convert kynurenine to anthranilic acid as it does not harbor KynU enzyme. Knocking out aspC reduced the amount of kynurenic acid produced but did not abolish it suggesting that AspC is not the major player in this conversion. We anticipated this activity may be due to TyrB and therefore, we generated and tested ∆tyrB, and ∆aspC∆tyrB double knockout. The results confirmed that TyrB but not AspC is the major contributor for kynurenine metabolism into kynurenic acid. Next, we focused on improving the capability of S. enterica to metabolize kynurenine. To achieve this, we transformed it with a plasmid harboring the kynurenine transporter alone or in combination with either kynU gene (from P. aeruginosae), tyrB or aspC gene (from E. coli). We found that expressing KynU together with the kynurenine transporter allowed S. enterica to degrade kynurenine to anthranilic acid and abolished its native kynurenic acid production. Likewise, overexpressing tryB and to less extent aspC significantly enhanced the natural ability of S. enetrica to degrade kynurenine to Attorney Docket Number: CCF-44551.601 kynurenic acid. A) Structure of kynurenine and its two metabolites; anthranilic, and kynurenic acids. B) Wild-type S. enterica (Wt) and mutants were cultured in LB media supplemented with 100 µM kynurenine. Samples were analyzed after 24 h of incubation through LC-MS / MS. n=4. Plotted are means ± SE. Figure 3. Combining kynurenine targeting with kynurenine degradation in one S. enterica strain. A) To combine kynurenine targeting with kynurenine degradation in one strain, we modified the previously engineered S. enterica AD95+ strain (Santos et al. Science Advances, 2025). AD95+ strain has two genes essential for its growth (asd and murI genes) which were knocked out from its chromosome. These two genes were supplied (in trans) on two separate plasmids downstream kynurenine-responsive promotors. Consequently, AD95+ grows only in presence of kynurenine. This in turn makes it target kynurenine-rich tumors while unable to survive in other tissues which lack kynurenine. Here we modified AD95+ strain through inserting the kynureninase enzyme (KynU) within its chromosome. KynU enzyme was inserted either downstream of a constitutive promotor (PJ23114) or the quorum- sensing induced Plux promotor to generate strains AD31 and AD51, respectively. B) Testing the growth of each strain in minimal media containing different kynurenine concentrations found that AD51 but not AD31 could grow in a kynurenine-controlled manner similar to the original AD95 strain. Figure 4. S. enterica VNP20009 engineered to express KynU enzyme together with kynurenine transporter (pTra-KynU), reduces tumor growth. A) Experiment scheme. B, C) KPCA.A tumors were developed subcutaneously in C57BL / 6 mice. When tumors were evident, S. enterica mutants were intraperitoneally injected at a dose of ~ 2×106CFU weekly. This dose was chosen to be lower than the dose used in other experiments to reduce systemic toxicity of VNP20009. Mice were euthanized 31 days after the tumor injection (when the tumors in the PBS group reached the endpoint). Tumor size was measured weakly while tumor weight was determined at the endpoint. D, E) Tumor kynurenine and anthranilic acid. Plotted are means ± SE. n=9~19 per group for each experiment. Data in C, D, and E are combined from 2 experiments. Figure 5. Tumor targeting by engineered AD51 compared to the parent AD95+. The modified AD51 strain (capable of both targeting and degrading kynurenine) still retains similar tumor specificity as the parent AD95+ strain when tested in subcutaneous KPCA.A ovarian murine tumor models. A) Subcutaneous KPCA.A tumors were injected in C57BL / 6 mice. When tumors reached a medium size, S. enterica mutants were i.v. injected at a dose of ~2×106colony forming units (CFU). B-D) Mice were euthanized 7 days later, and organs Attorney Docket Number: CCF-44551.601 harvested for CFU counting and to determine the ratio of the bacterial numbers in tumors to their numbers in liver and spleen. Plotted are means ± SE. Figure 6. KPCA.A Tumor suppression caused by the engineered AD51 compared to the parent 95+. S. enterica AD51 engineered to express KynU enzyme reduces tumor growth more efficiently than the parent AD95+ strain. KPCA.A tumors were developed subcutaneously in C57BL / 6 mice. When tumors were evident, S. enterica mutants were intravenously injected at a dose of ~ 2×106CFU weekly. Mice were euthanized when the tumors in the PBS group reached the endpoint. A, B) Tumor size was measured weakly while tumor weight was determined at the endpoint. C, D) Tumor kynurenine and anthranilic acid. Plotted are means ± SE. Figure 7. KPCA.A Tumor suppression caused by the engineered AD51 compared to the parent AD95+ with and without IFNg. S. enterica AD51 engineered to express KynU enzyme reduces KPCA.A ovarian tumor growth more efficiently than the parent AD95+ strain and the effect is more significant when combined with IFNγ. KPCA.A tumors were developed subcutaneously in C57BL / 6 mice. When tumors were evident, S. enterica mutants were intravenously injected at a dose of ~ 2×106CFU weekly while IFNγ was injected twice weekly intratumorally at a dose of 5 µg. Mice were euthanized when the tumors in the PBS group reached the endpoint. Tumor size (7A) was measured weakly while tumor weight (7B) was determined at the endpoint. Plotted are means ± SE. The data shows that combining the kynurenine-degrading AD51 with IFNγ caused the most significant reduction in tumor growth. Figure 8. B16 / F10 Tumor suppression caused by the engineered AD51 compared to the parent AD95 with and without IFNg. S. enterica AD51 engineered to express KynU enzyme reduces B16 / F10 melanoma tumor growth more efficiently than the parent AD95+ strain and the effect is more significant when combined with IFNγ. B16 / F10 tumors were developed subcutaneously in C57BL / 6 mice. When tumors were evident, S. enterica mutants were intravenously injected at a dose of ~ 2×106CFU weekly while IFNγ was injected twice weekly intratumorally at a dose of 5 µg. Mice were euthanized when the tumors in the PBS group reached the endpoint. Tumor size (8A) was measured weakly while tumor weight (8B) was determined at the endpoint. Plotted are means ± SE. The data shows that combining the kynurenine-degrading AD51 with IFNγ caused the most significant reduction in tumor growth. Figure 9. In order to enhance the dependance of our engineered S. enterica strain on kynurenine and improve tumor kynurenine degradation, we aimed at deleting the native trpE gene in S. enterica AD51 to create the strain AD51E. This is expected to disrupt the native Attorney Docket Number: CCF-44551.601 ability of Salmonella to synthesize tryptophan from chorismic acid and will force Salmonella to utilize anthranilic acid produced from kynurenine degradation as a substrate to synthesize its own tryptophan (see pathway at 9A). The data shown proved that this hypothesis is true with stricter kynurenine-controlled growth for AD51E when compared to AD51 in absence of additional tryptophan in the media (B, C). When tryptophan is supplied in the media, the growth of AD51E becomes less dependent on kynurenine (D, E). Figure 10. Sequences and gene neighborhood of the cloned kynurenine-responsive promoters. A) Kynurenine is sensed in P. aeruginosa as intermediate metabolite in tryptophan to anthranilic acid metabolic pathway. Kynurenine binding the transcriptional regulator KynR activates transcription from the cognate PkynB promoter. B-C) Structure of the kynurenine metabolizing operons in selected Gram-negative and Gram positive bacteria. The dotted red square shows the sequence in (C) cloned upstream of mCherry and in combination with P. aeruginosa KynR in Figure 2E to make plasmids pKynR-Cn-cherry, pKynR-Pf-cherry, pKynR-Pa-cherry, pKynR-Bt-cherry, pKynR-Bc- cherry, pKynR-Bp-cherry, pKynR-Rs-cherry, pCerKynR1-cherry, respectively. For pCerKynR1-cherry, the original B. cereus kynR was incorporated in the plasmid rather than P. aeruginosa KynR as KynR in B. cereus belongs to tetR family regulators unlike KynR in Gram negative strains which is Lrp / AsnC family type regulator. D) Sequences highlighted in yellow in (C) were analyzed at Berkley Weblogo to find the consensus sequence among the highly responsive Pkyn promoters in Gram-negative bacteria. B. pertussis PkynB was excluded due to poor activity as shown in Figure 2. B. cereus PkynU was also excluded. The sequence in blue in P. aeruginosa PkynB depicts the predicted -35, -10 and transcriptional start site based on the online BPROM promoter finding tool. For P. aeruginosa PkynB, there was originally an extra 5’ sequence (not shown) which was trimmed in later experiments when found unnecessary for the activity. Figure 11A, B. The two plasmids of the pkynR7 system in AD95+ strain. Plasmid A harbors KynR, murI gene under kynurenine-responsive promotor and the mtr transporter gene for kynurenine uptake. Plasmid B harbors asd gene downstream of another kynurenine- responsive promotor. The replication protein of this plasmid is also placed downstream of a third kynurenine-responsive promotor. Figure 12 shows the nucleic acid sequence of S. enterica asd knockout (SEQ ID NO:1). Figure 13 shows the nucleic acid sequence of S. enterica murI knockout (SEQ ID NO:2). Attorney Docket Number: CCF-44551.601 Figure 14A shows map for p113m-mur-114KynU plasmid. This plasmid was used to to generate VNP20009 / ptra-kynU which is used in Figure 2 and 4. It harbors the KynU gene together with the mtr transporter. It also harbors murI gene which is essential for bacterial growth. murI was knocked out from VNP20009 chromosome and is supplied on the plasmid to enhance the plasmid stability in absence of antibiotics. Figure 14B shows p113m-mur- 114aspC which serves the same function as p113m-mur-114KynU except that KynU is replaced with aspC to degrade kynurenine into kynurenic acid rather than anthranilic acid. Another version of the same plasmid backbone was also generated through replacing aspC with tyrB gene to do the experiments in Figure 2. Figure 14C shows the map for the suicide plasmid used to insert LuxR-pluxI-KynU in AD95+ chromosome to generate strain AD51. Figure 14D shows the map for the suicide plasmid used to knockout trpE gene from AD51 chromosome to generate AD51E. Figure 15 shows metabolism in S. enterica of Spermidine to N-acetyl-Spermidine and metabolism in Pseudomonas aeruginosa of Spermidine to putreanine or Isoputreanine via the PauABCD operon. Figure 16. Cloning PauABCD genes from Pseudomonas aeruginosa into S. enterica enabled degrading spermidine in the culture rather than converting it into N-acetyl- spermidine. Figure 17. The pau operon (for spermidine degradation) was combined with either KynU or TyrB expression to degrade both kynurenine and spermidine. Figure 18A shows the map for plasmid p113mur-114pau1 which was transformed into Salmonella enterica in Figure 16 to render it capable of degrading spermidine and N- acetyl-spermidine. This plasmid harbors the pau operon from Pseudomonas aeruginosa under the constitutive promotor PJ23114. It also harbors murI gene which is essential for bacterial growth. murI was knocked out from Salmonella chromosome and is supplied on the plasmid to enhance the plasmid stability in absence of antibiotics. Figures 18B and 18C show the maps for p113mur-115pau1-114kynU and p113mur-115pau1-114tyrB. These two plasmids are used to transform Salmonella to perform the experiments shown in figure 17. They have similar backbone as in p113mur-114pau1 except that pau operon is under the PJ23115 promotor and they harbor kynU and TyrB genes, respectively. Figure 19A shows the amino acid sequence of KynR from P. aeruginosa species: (SEQ ID NO:3). Other homologues of KynR can be also used (e.g. UniRef id # UPI0027D38BD6, UPI000D14D06C, UPI000D7418B9). Figure 19B shows the amino acid sequence of exemplary kynurenine transport proteins: i) MTR protein from E. coli species (SEQ ID NO:4); ii. TnaB protein from E. coli species (SEQ ID NO:5); and iii) AroP protein Attorney Docket Number: CCF-44551.601 from E. coli species (SEQ ID NO: 6). Figure 19C shows the amino Acid sequence of KynU from P. aeruginosa species (SEQ ID NO:7). Other homologues of kynU (kynureninase) enzyme from either prokaryotic or eukaryotic origin can be also used instead of P. aeruginosa KynU (e.g. UniprotKB accession # Q16719, Q9CXF0, P70712, P83788, Q54Q04, Q05979, A8XKT0, A9VHP9, Q59QC4, Q6CDM0, Q62M98). Figure 19D shows the amino acid sequence of murl from Escherichia fergusonii species (SEQ ID NO: 8). Figure 19E shows the amino acid sequence of asd from Salmonella enterica species (SEQ ID NO: 9). Figure 19F shows the amino acid sequence of LuxR from Vibrio fischeri (aka Aliivibrio fischeri) species (SEQ ID NO:10). Figure 19G shows the amino acid sequence of LuxI from Vibrio fischeri (Aliivibrio fischeri) species (SEQ ID NO 11). Figure 19G shows the nucleic acid sequence from PluxI from Vibrio fischeri (Aliivibrio fischeri) species (SEQ ID NO: 12). DESCRIPTION OF THE INVENTION Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive and degrading bacteria (e.g., such that the bacteria reduces or eliminates the tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences encoding: i) a kynurenine transcriptional regulator protein, ii) a kynurenine transporter protein, iii) a kynureninase (KynU) protein, and iv) at least one protein important or essential for growth of the bacteria expression of which is linked to a kynurenine responsive promoter (e.g., where the corresponding gene has been deactivated or knocked out in the genome of the bacteria). In certain embodiments, none of the one or more nucleic acid sequences are endogenous to the bacteria, or only some are endogenous. Also provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive bacteria (e.g., such that the bacteria reduce or eliminate said tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences comprising: i) a first nucleotide sequence (e.g., not endogenous to said bacteria) encoding a kynurenine transcriptional regulator protein, and / or ii) a second nucleotide sequence (e.g., not endogenous to said bacteria or overexpressing an endogenous equivalent protein) encoding a kynurenine transporter protein. In particular embodiments, the kynurenine responsive bacteria further comprise at least one of the following: i) a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein, ii) a Attorney Docket Number: CCF-44551.601 fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein, and iii) a fifth or more nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene essential for bacterial growth, or a gene codes a for therapeutic payload. In some embodiments, expression of the kynureninase (KynU) protein is under the control of a constitutive promoter. However, in other embodiments, expression of the kynureninase (KynU) protein is under the control of a quorum sensing system. Such regulated expression of KynU is important as kynurenine is generally needed for the bacteria to grow with the important or essential proteins needing kynurenine presence to grow (and the ability of KynU to degrade kynurenine that is present). In general, in such embodiments with a quorum sensing system, the KynU enzyme will not be expressed when the bacteria is present at low numbers (e.g., at or near a tumor or lesion). For example, using the Lux quorum sensing system for illustrative purposes, the Plux promotor (upstream of KynU) is activated only when the bacteria reaches a certain threshold biomass. In this system in the Example below, we knocked in the LuxR gene (the regulator) and the LuxI gene with its native promotor (LuxI makes the quorum sensing molecule AHL). KynU was then knocked in downstream of LuxI so that it is expressed as one operon together with LuxI. In this system, luxI encodes LuxI, an autoinducer synthase enzyme that produces an acyl- homoserine lactone (AHL) signaling molecule. Next, luxR encodes LuxR, a transcriptional activator protein that binds AHL. The LuxR–AHL complex then binds to a promoter region to activate transcription. The PluxI promoter (sometimes called Plux1) is the promoter that drives transcription of the luxI gene. It is activated by the LuxR–AHL complex, creating a positive feedback loop where: i) LuxI makes AHL, ii) AHL accumulates as the bacterial population grows (e.g., in or near tumor or lesion), iii) AHL binds LuxR, and iv) LuxR–AHL activates PluxI, increasing luxI (and often luxR) expression, and thereby increasing KynU expression. Other quorum sensing systems are known in the art and can be employed, see e.g., Chakraborty et al., Heliyon, Volume 9, Issue 5, May 2023, e16205; Sunny Cui & Esther Kim, Communicative & Integrative Biol., 2024, VOL.17, NO.1, 2415598; Patel et al., The Microbe, Volume 6, March 2025, 100224; Zeng et al., Microbiological Research Volume 273, August 2023, 127414; and Boo et al., Current Opinion in Systems Biology Volume 28, December 2021, 100378; all of which are herein incorporated by refernece in their entirities and particularly for the components of Quorum sensing systems. Exemplary quorum sensing systems include, but are not limited to, the following: i) a Plas promoter Attorney Docket Number: CCF-44551.601 upstream of a sequence encoding lasI and / or lasR; ii) a Prhl promoter upstream of a sequence encoding rh1I and / or rh1R; iii) a Ptra promoter upstream of a sequence encoding traI and / or traR; iv) a sequence encoding ArgA, ArgB, ArgC, and ArgD; v) a sequence encoding: luxS, luxP, and luxQ; vi) a sequence encoding: qseC and qseB; and vii) a sequence encoding HHQ, PQS, HQNO, and NHQ. In certain embodiments, the bacteria herein express a therapeutic payload, or a therapeutic is separately provided to the patient along with the bacteria. In particular embodiments, the therapeutic payload is IFNg (e.g., human or otherwise mammalian) expressed by the bacteria, or separately administered to the patient at or about the same time as the bacteria. IFNg is a cytokine that has a potent proinflammatory and antitumor effect. IFNg anti-tumor effect is enhanced when it is combined with microbial products. However, the normal downside of providing IFNg to cancer patients is that this results in increased kynurenine. However, the bacteria described herein degrade kynurenine, and therefore combining IFNg (expression in bacteria or separate administration) with the KynU degrading bacteria herein allows for the antitumor proinflammatory benefits of IFNg without increasing kynurenine concentration in the tumor. In some embodiments, the bacteria herein are obligate anaerobic bacteria. In some embodiments, the bacteria herein are facultative anaerobic bacteria. In some embodiments, the bacteria herein are aerobic bacteria. In some embodiments, the bacteria herein are gram- positive bacteria. In some embodiments, the bacteria herein are gram-negative bacteria. In some embodiments, the bacteria herein are non-pathogenic bacteria. In some embodiments, the bacteria herein are commensal bacteria. In some embodiments, the bacteria herein are probiotic bacteria. In some embodiments, the bacteria herein are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria herein include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria, Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55, Clostridium butyricum miyairi, Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT, Clostridium paraputrificum, Clostridium pasteureanum, Clostridium pectinovorum, Attorney Docket Number: CCF-44551.601 Clostridium perfringens, Clostridium roseum, Clostridium sporo genes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, and Vibrio cholera. The bacteria herein may be administered as part of a composition, which may be a pharmaceutical composition (e.g., comprising a therapeutically effective amount of the bacteria herein in and a pharmaceutically acceptable carrier. The compositions may include a carrier, which refers to a diluent, adjuvant, excipient, or vehicle with which the bacteria is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a useful carrier when the pharmaceutical composition is administered, for example, intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The pharmaceutical compositions herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation generally depends on the route of administration. The bacteria herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, Attorney Docket Number: CCF-44551.601 injectable, intravenous, sub-cutaneous, intratumoral, peritumor, immediate-release, pulsatile- release, delayed-release, or sustained release). Suitable dosage amounts for the bacteria herein may range, for example, from about 104 to 1012 bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal. The bacteria herein may be administered intravenously, e.g., by infusion or injection. Alternatively, the bacteria may be administered intratumorally and / or peritumorally. In other embodiments, the bacteria may be administered intra-arterially, intramuscularly, or intraperitoneally. In some embodiments, the bacteria colonize about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the tumor. The bacteria may be administered via intratumoral injection, resulting in bacteria that is directly deposited within the target tumor. Depending on the location, tumor type, and tumor size, different administration techniques may be used, including but not limited to, cutaneous, subcutaneous, and percutaneous injection, therapeutic endoscopic ultrasonography, or endobronchial intratumor delivery. In some embodiment, other techniques, such as laparoscopic or open surgical techniques are used to access the target tumor. For some tumors, percutaneous injection can be employed, which is one of the least invasive administration methods. Ultrasound, computed tomography (CT) or fluoroscopy can be used as guidance to introduce and position the needle. Single insertion points or multiple insertion points can be used in percutaneous injection protocols. Using a single insertion point, the solution may be injected percutaneously along multiple tracks, as far as the radial reach of the needle allows. In other embodiments, multiple injection points may be used if the tumor is larger than the radial reach of the needle. In certain embodiments, intratumoral administration for lung cancer, such as non-small cell lung cancer, can be achieved through endobronchial intratumor delivery methods. The dose to be injected is derived from the type and size of the tumor. The dose of the bacteria herein is typically lower, e.g., orders of magnitude lower, than a dose for systemic intravenous administration. The volume injected into each lesion is based on the size of the tumor. To obtain the tumor volume, a measurement of the largest plane can be conducted. The estimated tumor volume can then inform the determination of the injection Attorney Docket Number: CCF-44551.601 volume as a percentage of the total volume. For example, an injection volume of approximately 20-40% of the total tumor volume can be used. In some embodiments, the treatment regimen will include one or more intratumoral administrations. In some embodiments, a treatment regimen will include an initial dose, which followed by at least one subsequent dose. One or more doses can be administered sequentially in two or more cycles. For example, a first dose may be administered at day 1, and a second dose may be administered after 1, 2, 3, 4, 5, 6, days or 1, 2, 3, or 4 weeks or after a longer interval. Additional doses may be administered after 1, 2, 3, 4, 5, 6, days or after 1, 2, 3, or 4 weeks or longer intervals. In some embodiments, the first and subsequent administrations have the same dosage. In other embodiments, different doses are administered. In some embodiments, more than one dose is administered per day, for example, two, three or more doses can be administered per day. In certain embodiments, the bacteria herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. In some embodiments, the bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. In another embodiments, the pharmaceutical composition comprising the bacteria may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In some embodiments, the compositions with the bacteria are formulated for intra- intestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as Attorney Docket Number: CCF-44551.601 suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and / or dispersing agents. The bacteria may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant. The programmable bacteria may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. The bacteria herein may be used to treat cancer (e.g., in a human or other mammal). Examples of cancer include but are not limited to, carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include melanoma, lung cancer, head and neck cancer, renal cell cancer, colon cancer, colorectal cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, endometrial carcinoma, myeloma (such as multiple myeloma), salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, and esophageal cancer. The present disclosure is not limited to the particular sequences provided herein, such as those shown in Figure 19A-G. For example, it is now well known in the art that one can find homologous sequences in other species through relatively simple sequence searches (e.g., NCBI BLASTp, DELTA-BLAST, OrthoFinder, OrthoDB, eggNOG, Ensembl Compara, Ensembl Genomes, InterProScan, HHsearch, HHpred, KEGG, EFI-EST, InterProScan, OrthoFinder + IQ-TREE, FastTree, etc.). For example, one could provide any of the genes in Figure 19 (e.g., KynR, MTR, TnaB, AroP, KynU, murl, asd, etc.) in a sequence search (e.g., in the programs above) to locate similar sequence (e.g., homologues) in other species. One can also use the name of the gene and / or a description of the role of the protein to find homologous that perform the same function, but might only have limited sequence similarity. This is well known in the art at this time. Also, provided below, in Table 1, are various homologs of the genes and proteins recited herein along with their accession numbers (e.g., as examples of alternatives that one of skill in the art could use, or use to screen for further homologs). Attorney Docket Number: CCF-44551.601 TABLE 1 Gene Accession (UniProt) Species tyrB P74861 Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) / C2A) Attorney Docket Number: CCF-44551.601 Streptomyces griseus Q59228 Geobacillus stearothermophilus (Bacillus stearothermophilus) / Attorney Docket Number: CCF-44551.601 Klebsiella pneumoniae subsp. pneumoniae (strain ATCC 700721 / MGH 78578) P9WHH3 Attorney Docket Number: CCF-44551.601 EXAMPLES EXAMPLE 1 Kynurenine responsive and degrading Bacteria for Tumor Treatment Kynurenine is immunosuppressive metabolite produced by almost all solid tumors including GBM10,11, breast cancer12-15, ovarian cancer16-18, colorectal cancer19-21, head and neck squamous cell carcinoma (HNSCC)22and others23creating an immunosuppressive microenvironment that allows the tumor to escape immune surveillance24,25. The level of kynurenine production differs between tumors and is induced by the T-cell infiltration and the associated inflammatory cytokines especially IFNɣ within the tumor microenvironment26. The immunosuppressive and protumorgenic action of kynurenine is mainly attributed to its binding and activation of the arylhydrocarbon receptor (AhR) in the T-cells leading to their conversion into Treg cells among other actions15,27. Several IDO inhibitors have entered different phases of oncology clinical trials in combination with other immunotherapy approaches (e.g., NCT01792050, NCT02077881, and NCT02471846) with the hypothesis that countering the kynurenine pathway will lead to immune stimulation. Nevertheless, recent studies focusing on ovarian cancer found IDO1 blockade to be ineffective in the clinical trials due to the tumor metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This results in elevated nicotinamide adenine dinucleotide (NAD+) and finally reduction of T cell proliferation and activity28. This in turn highlights the need to find alternative ways to overcome the kynurenine accumulation in the tumors. Some bacteria such as Pseudomonas aeruginosae harbor kynureninase (KynU) enzyme which metabolizes kynurneine into anthranilic acid (Figure 10A)29-32. Unlike kynurneine, anthranilic acid is not known to be a ligand for AhR. Previous studies showed that intra-tumor administration of purified bacterial PEGylated KynU enzyme successfully degraded the intratumor kynurenine and inhibited tumor growth in melanoma, breast, and colon carcinoma models27. That study provided proof of principle that IDO1 protumorgenic effect is in large part due to kynurenine production rather than tryptophan depletion. In that Attorney Docket Number: CCF-44551.601 study, however, the authors had to PEGylate KynU to improve its stability in the tumor microenvironment27. Genetically-engineered bacteria including Salmonella enterica and other strains are emerging as cancer therapeutics and may be a future potential treatment option for solid tumors1-3including GBM33with several clinical trials being performed or are still going using commensal and genetically modified bacterial strains (e.g., NCT00004988, NCT01675765, NCT01924689, NCT03435952, etc.). Recent advances in bacterial synthetic biology have enabled engineering of the highly attenuated and tumor-specific S. enterica VPN20009 strain8,34. These bacteria both directly kill the cancer cells, and more importantly, owing to their natural immunogenic properties, stimulate the immune cells within the tumor microenvironment to better attack and eradicate the tumor. Widespread application of this approach, however, is limited and there is still a need to improve the therapeutic efficacy of these bacteria in stimulating the immune system4. In this Example, we enhanced the efficacy of bacterial-based cancer therapy in modulating the tumor immune microenvironment through engineering S. enterica to degrade the pro-tumorigenic immunosuppressive kynurenine secreted by the tumor cells as well as other pro-tumorgenic metabolites secreted in the tumor such as spermidine. In addition, we combined kynurenine-controlled growth with kynurenine degradation. In this regard, the aim is, once a certain density of bacteria reached (controlled synthetically via quorum sensing systems), the enzymes cloned to degrade the metabolite of interest such as kynurenine or spermidine can be expressed. RESULTS Investigating natural kynurenine metabolism in S. enterica Some bacteria including E. coli metabolize kynurenine into kynurenic acid through transaminases such as aspartate transaminase (AspC)35,36. S. enterica ATCC 14028 naturally harbors the promiscuous aspartate transaminase (AspC) in addition to other related transaminases including aromatic amino acid transaminase (TyrB). In previous work,37it was found that S. enterica also metabolizes kynurenine into kynurenic acid (Figure 2). To first test if S. enterica metabolize kynurenine into kynurenic acid through AspC enzyme, we generated S. enterica ∆aspC knockout. In parallel, we also transformed the wild-type S. enterica with a plasmid harboring the aromatic amino acid transporter (Mtr). This transporter was found in the previous study to enhance kynurenine importing37. Analyzing Attorney Docket Number: CCF-44551.601 the LB culture spent media of each of these strains through LC-MS / MS confirmed that S. enterica naturally metabolizes kynurenine into kynurenic acid albeit modestly (Figure 1B). The overexpression of the Mtr transporter resulted in higher kynurenic acid. Wild-type (Wt) S. enterica was unable to convert kynurenine to anthranilic acid as it does not harbor KynU enzyme. Knocking out aspC reduced the amount of kynurenic acid produced but did not abolish it suggesting that AspC is not the major player in this conversion. We anticipated this activity may be due to TyrB and therefore, we generated and tested ∆tyrB, and ∆aspC∆tyrB double knockout. The results confirmed that TyrB but not AspC is the major contributor for kynurenine metabolism into kynurenic acid (Figure 2). Engineering Kynurenine to Anthranilic acid pathway in S. enterica Next, we focused on improving the capability of S. enterica to metabolize kynurenine. To achieve this, we transformed it with a plasmid harboring the kynurenine transporter alone or in combination with either kynU gene (from P. aeruginosae), tyrB or aspC gene (from E. coli). We found that expressing KynU together with the kynurenine transporter allowed S. enterica to degrade kynurenine to anthranilic acid and abolished its native kynurenic acid production (Figure 2). Likewise, overexpressing tryB and to less extent aspC significantly enhanced the natural ability of S. enetrica to degrade kynurenine to kynurenic acid. The two kynurenine degrading plasmids (pMtr-KynU and pMtr-TyrB) were then transformed into S. enterica VNP200009. As pointed earlier, this strain is a derivative of the wildtype S. enterica ATCC 14028 which is attenuated to reduce its toxicity and improve its tumor targeting5-7. To ensure plasmid stability, the transformed VNP200009 were rendered d-glutamate auxotrophic through knocking out murI gene (needed for d-glutamate synthesis and subsequent synthesis of the peptidoglycan cell wall)38. A copy of the murI gene was then supplied on the kynurenine-degrading plasmids. Testing these VNP20009 mutants confirmed the enhanced capability to degrade kynurenine to either kynurenic or anthranilic acid similar to what we observed with the wild-type S. enterica . To confirm ability of the KynU-harboring S. enterica VNP20009 to degrade tumor kynurenine to anthranilic acid in vivo, we developed subcutaneous KPCA.A tumors in C57Bl / 6J mice. The mice were i.p. injected weekly with either PBS, parent VNP20009 or the KynU-harboring mutant (Figure 4). When the tumors in the PBS group reached the endpoint, the mice were sacrificed, and the selective bacterial colonization in tumors was confirmed through plating on LB agar plates. Tumor kynurenine and anthranilic acid concentrations were analyzed through LC-MS-MS. The results confirmed the capability of Attorney Docket Number: CCF-44551.601 KynU-harboring S. enterica to degrade tumor kynurenine into anthranilic acid (Figure 4D, E). Further, the tumors in VNP20009 / pMtr-kynU group exhibited significantly reduced growth compared to control (PBS) treated mice. The reduction in tumor growth was more evident in VNP20009 / pMtr-kynU group compared to the parent VNP20009 treated-mice suggesting a therapeutic benefit of the kynureninase-expressing S. enterica treatment (Figure 4B, C). Combining kynurenine targeting and degradation in the same S. enterica strain. In the previously mentioned study, kynurenine-targeting S. enterica AD95+ strain were developed. In contrast to VNP20009 which is auxotrophic to purines, AD95+ is dependent for its growth on kynurenine37. This dependency was attained through deleting murI and asd genes needed for the synthesis of d-glutamate and diaminopimelic acid (DAP), respectively. These two genes were then supplied in trans under kynurenine-controlled genetic circuits. It was previously demonstrated that AD95+ has significantly higher tumor specificity and less toxicity compared to VNP2000937. We therefore aimed at moving our kynurenine degradation cassettes to the better tumor-specific AD95+. Since AD95+ is dependent on kynurenine for growth, we thought this could be challenging. For our first engineered AD95+ derivative (Hereafter named AD31), KynU enzyme was expressed chromosomally downstream the constitutive synthetic promotor PJ23114 (Figure 3A). Unlike the parent AD95+, AD31 was unable to grow in M9 defined media even when supplemented with kynurenine. Next, we developed methods for controlling KynU gene expression through Acyl- homoserine lactone (AHL) – based quorum sensing. AHL-quorum sensing (QS) was first described in the bacterium Vibrio fischeri (Aliivibrio fischeri) where it controls the transcription of the luminescence (lux) operon39,40(both of which are herein incorporated by reference for this system). In this system, the luxI gene codes for AHL synthase, while LuxR acts as transcriptional regulator which upon binding to the autoinducer AHL, activates the transcription of the promotor upstream of the lux operon leading to upregulation of the luxI gene together with the other lux genes downstream of luxI. When the bacteria are present at low number in the medium, the concentration of the produced AHL molecules is very low. However, when the bacteria grow to sufficient numbers, the concentration of the produced AHL in the medium reaches a certain threshold which allows it to bind to the LuxR and activate the expression of the lux operon in a positive feedback loop. Previous studies by Attorney Docket Number: CCF-44551.601 Hasty’s and Danino’s groups successfully employed this luxR-luxI quorum-sensing (QS) based system to induce periodic controlled self-lysis of the anti-tumor bacteria upon reaching a certain threshold number in the tumor. This is basically achieved through placing a bacteriophage-derived lysin gene under the control of the luxI promotor41-43. When the bacteria grow to a sufficient density in the tumor, the expression of this lysin gene is induced, leading to the lysis of the majority of the bacterial cells and the release of their intracellular components. The remaining bacteria which escape this lysis, start to grow again and the cycle is repeated in a self-controlled manner. Expressing KynU enzyme under control of the LuxI-LuxR QS system will therefore ensures that KynU is produced only after the bacteria grow and accumulate in the tumor. Indeed, when we replaced PJ23114 with the LuxR-PLuxI casstte (Figure 3B), the resultant strain (Hereafter named AD51), could grow in M9 media when supplemented with kynurenine in a pattern akin to the original AD95+ strain. AD51, however showed maximum growth at kynurenine concentrations of 25 µM While the original AD95+ showed maximum growth at 10 µM concentration (Figure 3B). S. enterica AD51 successfully target as well as degrade tumor kynurenine in vivo. To first assess the ability of AD51 to retain tumor specificity, we developed KPCA.A subcutaneous tumors. The mice were then i.v. injected (via retroorbital injection) with either the original kynurenine-targeting AD95+ strain or the modified kynurenine-targeting and degrading AD51 strain. 1 week later, the mice were sacrificed and bacterial counts in tumors and other organs were determined through plating (Figure 5). The results found AD51 still keeps high specificity to the tumor as the original AD95+ strain (Figure 5B). Surprisingly, there was slight enhancement of tumor colonization compared to AD95+. However, colonization of other organs was also slightly increased resulting in almost similar tumor / liver and tumor / spleen ratios as AD18 (Figure 5C, D). To assess the tumor-mitigating effects of AD51 compared to AD95+, we injected KPCA.A subcutaneous tumors in a different set of mice. The bacteria or control PBS solution were then i.v. injected weekly after the tumors were evident. Tumor growth was assessed weekly for each group and all mice were sacrificed ~ 1 month later. The results found both strains significantly reduced both tumor growth over time and the tumor weight at the endpoint (Figure 6A-B). AD51, however, was more effective in reducing tumor burden. This was consistent with the reduction in tumor kynurenine seen in AD51 group but not in AD95+ group (Figure 6C-D). Attorney Docket Number: CCF-44551.601 Combining the treatment with intertumoral injection of IFNγ resulted in a more significant reduction in tumor growth (Figure 7). Similar results were also obtained when the treatment was tested in B16F10 melanoma tumor model (Figure 8) demonstrating that this approach is applicable nearly all solid tumors as kynurenine accumulation was reported in almost all kinds of solid tumors. In order to enhance the dependance of the engineered S. enterica strain on kynurenine and improve tumor kynurenine degradation, we aimed at deleting the native trpE gene in S. enterica AD51 to create the strain AD51E. This is expected to disrupt the native ability of Salmonella to synthesize tryptophan from chorismic acid and will force Salmonella to utilize anthranilic acid produced from kynurenine degradation as a substrate to synthesize its own tryptophan. The data shown (Figure 9) proved that this hypothesis is true with stricter kynurenine-controlled growth for AD51E growth in absence of additional tryptophan in the media. When tryptophan is supplied in the media, the growth of AD51E becomes less dependent on kynurenine. Similar to kynurenine, spermidine is another metabolite which accumulates in tumors leading to immunosuppressive effects. Spermidine is a polyamine originates from arginine and ornithine metabolism, and is overproduced in various solid tumors creating a pro-tumorigenic environment that contribute to tumor growth and proliferation44-46. Similar to its close relative, E. coli, S. enterica naturally converts excess spermidine in the media into N-acetyl-spermidine as metabolic end product which cannot be metabolized further47,48. In contrast, some bacteria such as P. aeruginosa are capable of degrading spermidine and use it as carbon source through a series of oxidation reactions catalyzed by specialized spu (spermidine utilization) gene clusters49-52. Consequently, we cloned the five main spu genes needed for spermidine degradation in a single operon on a plasmid and tested the capability to degrade spermidine when this plasmid was expressed in S. enterica. Our results (Figures 15-16) indicated that expressing this spu-plasmid enabled S. enterica to degrade spermidine in the medium, abolished N-acetyl spermidine formation, and therefore, reduced the total concentration of spermidine and N-acetyl spermidine. Combining KynU or TyrB enzymes with the Pau operon in one S. enterica strain enabled degrading both kynurenine and spermidine (Figure 17). In summary, through a successive series of engineering approaches, we constructed bacterial kynurenine-responsive genetic circuits which allowed controlling gene expression and growth in harboring bacteria (including E. coli and S. enterica) in response to kynurenine secreted by solid tumors. Subsequent engineering further improved the efficacy of these Attorney Docket Number: CCF-44551.601 bacteria through enabling them to also degrade kynurenine. The engineered bacteria are useful as next generation drug delivery chasses for cancer therapy. Furthermore, applications of these strains both as therapeutics and as diagnostics for kynurenine accumulation could extend beyond cancer to other inflammatory diseases in which kynurenine is also overproduced including inflammatory bowel diseases. Combining kynurenine degradation with the capability to degrade other immunosuppressive metabolites such as spermidine as shown here can result in better anti-tumor efficacy. REFERENCES: 1. Chein et al., Advances in bacterial cancer therapies using synthetic biology. Current opinion in systems biology 5, 8 (2017). 2. Daschner et al., Bugs as Cancer Drugs: Challenges and Opportunities. Mol Cell Biol 39(2019). 3. Salicrup et al., A. Bugs as Drugs, potential self-regenerated innovative cancer therapeutics approach for global health. J Glob Health 10, 010311 (2020). 4. Duong, M.T., Qin, Y., You, S.H. & Min, J.J. Bacteria-cancer interactions: bacteria-based cancer therapy. Exp Mol Med 51, 1-15 (2019). 5. Pawelek, J.M., Low, K.B. & Bermudes, D. 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Attorney Docket Number: CCF-44551.601 39. Doublet, P., van Heijenoort, J., Bohin, J.P. & Mengin-Lecreulx, D. The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity. J Bacteriol 175, 2970-2979 (1993). 40. Clairmont, C., et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 181, 1996- 2002 (2000). 41. Ait-Belkacem, R., et al. Microenvironment Tumor Metabolic Interactions Highlighted by qMSI: Application to the Tryptophan-Kynurenine Pathway in Immuno-Oncology. SLAS Discov 22, 1182-1192 (2017). 42. Almansour, N.M. Triple-Negative Breast Cancer: A Brief Review About Epidemiology, Risk Factors, Signaling Pathways, Treatment and Role of Artificial Intelligence. Front Mol Biosci 9, 836417 (2022). 43. Bianchini, G., Balko, J.M., Mayer, I.A., Sanders, M.E. & Gianni, L. 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Methods Mol Biol 2021, 77-84 (2019). 50. Adams, S., et al. Involvement of the kynurenine pathway in human glioma pathophysiology. PloS one 9, e112945 (2014). 51. Riess, C., et al. Activation of the Kynurenine Pathway in Human Malignancies Can Be Suppressed by the Cyclin-Dependent Kinase Inhibitor Dinaciclib. Frontiers in immunology 11, 55 (2020). Attorney Docket Number: CCF-44551.601 52. Triplett, T.A., et al. Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme. Nat Biotechnol 36, 758-764 (2018). 53. Odunsi, K., et al. Metabolic adaptation of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses. Sci Transl Med 14, eabg8402 (2022). 54..Mehta, N., et al. Bacterial Carriers for Glioblastoma Therapy. Mol Ther Oncolytics 4, 1- 17 (2017). 55. Han, Q., Fang, J. & Li, J. Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase. Biochem J 360, 617-623 (2001). 56. Jansen, R.S., et al. Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis. Nat Commun 11, 1960 (2020). 57. Santos, A., et al. Leveraging dysregulated tumor metabolism for targeting anticancer bacteria. Sci Adv 11, eads1630 (2025). 58. Waters, C.M. & Bassler, B.L. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21, 319-346 (2005). 59. Smith, D., et al. Variations on a theme: diverse N-acyl homoserine lactone-mediated quorum sensing mechanisms in gram-negative bacteria. Sci Prog 89, 167-211 (2006). 60. Din, M.O., et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81-85 (2016). 61. Harimoto, T., et al. Rapid screening of engineered microbial therapies in a 3D multicellular model. Proc Natl Acad Sci U S A 116, 9002-9007 (2019). 62. Gurbatri, C.R., et al. Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies. Sci Transl Med 12(2020). 63. Akinyele, O. & Wallace, H.M. Characterising the Response of Human Breast Cancer Cells to Polyamine Modulation. Biomolecules 11(2021). 64. Soda, K. The mechanisms by which polyamines accelerate tumor spread. J Exp Clin Cancer Res 30, 95 (2011). 65. Miska, J., et al. Polyamines drive myeloid cell survival by buffering intracellular pH to promote immunosuppression in glioblastoma. Sci Adv 7(2021). 66. Fang, S.B., et al. speG Is Required for Intracellular Replication of Salmonella in Various Human Cells and Affects Its Polyamine Metabolism and Global Transcriptomes. Front Microbiol 8, 2245 (2017). Attorney Docket Number: CCF-44551.601 67. Fukuchi, J., Kashiwagi, K., Yamagishi, M., Ishihama, A. & Igarashi, K. Decrease in cell viability due to the accumulation of spermidine in spermidine acetyltransferase-deficient mutant of Escherichia coli. The Journal of biological chemistry 270, 18831-18835 (1995). 68. Yao, X., He, W. & Lu, C.D. Functional characterization of seven gamma- Glutamylpolyamine synthetase genes and the bauRABCD locus for polyamine and beta- Alanine utilization in Pseudomonas aeruginosa PAO1. J Bacteriol 193, 3923-3930 (2011). 69. Lu, C.D., Itoh, Y., Nakada, Y. & Jiang, Y. Functional analysis and regulation of the divergent spuABCDEFGH-spuI operons for polyamine uptake and utilization in Pseudomonas aeruginosa PAO1. J Bacteriol 184, 3765-3773 (2002). 70. Yao, X., Li, C., Zhang, J. & Lu, C.D. gamma-glutamyl Spermine Synthetase PauA2 as a potential target of antibiotic development against Pseudomonas aeruginosa. Antimicrob Agents Chemother 56, 5309-5314 (2012). 71. Krysenko, S. & Wohlleben, W. Polyamine and Ethanolamine Metabolism in Bacteria as an Important Component of Nitrogen Assimilation for Survival and Pathogenicity. Med Sci (Basel) 10(2022). 72. Dey, G., et al. LCK facilitates DNA damage repair by stabilizing RAD51 and BRCA1 in the nucleus of chemoresistant ovarian cancer. J Ovarian Res 16, 122 (2023). All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, medicine, and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

Attorney Docket Number: CCF-44551.601 CLAIMS: We claim:

1. A composition comprising: a bacterial cell, wherein said bacterial cell comprises: A) at least one bacterial chromosome, wherein said at least one bacterial chromosome has at least one endogenous gene important or essential for growth of said bacterial cell: i) deactivated, or ii) knocked-out; B) optionally one or more expression vectors which optionally comprise one or more plasmids; and C) one or more nucleic acid sequences which are present in said bacterial genome and / or are present in said one or more expression vectors, wherein said one or more nucleic acid sequences comprise: i) a first nucleotide sequence encoding a kynurenine transcriptional regulator protein; ii) a second nucleotide sequence encoding a kynurenine transporter protein; iii) a third nucleotide sequence encoding a kynureninase (KynU) protein; iv) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of at least one gene that encodes at least one protein that compensates for said at least one endogenous gene that is deactivated or knocked-out; and v) optionally a fifth nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein.

2. The composition of claim 1, further comprising: vi) a sixth nucleotide sequence comprising a kynurenine responsive promoter upstream of at least one gene that codes for a therapeutic payload, wherein said at least one gene encodes Interferon gamma (INFy), which is optionally human INFy.

3. The composition of claim 1, further comprising: vi) a sixth nucleotide sequence encoding aromatic amino acid transaminase (TyrB) protein, and / or a seventh nucleotide sequence encoding aspartate aminotransferase (aspC) protein.Attorney Docket Number: CCF-44551.601 4. The composition of claim 1, further comprising: vi) one or more sixth nucleic acid sequences that alone or together encode: PauA protein (PauA (also called SpuB and PuuA)), PauB protein (PauB (also called PuuB)), PauC protein (PauC (also called KauB and PuuC)), and PauD protein (PauD (also called SpuA and PuuD)).

5. The composition of claim 1, further comprising: vi) a sixth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein.

6. The composition of claim 1, wherein said one or more expression vectors are present in said bacterial cell, and wherein said first, second, third, and fourth nucleic acid sequences are present in said one or more expression vectors.

7. The composition of claim 6, wherein said one or more expression vectors comprise one or more plasmids, and wherein said fifth nucleotide sequence is present and is in said one or more plasmids.

8. The composition of claim 1, wherein at least one of said first, second, third, and fourth nucleic acid sequences are in said at least one bacterial chromosome.

9. The composition of claim 1, wherein said one or more expression vectors comprise one or more plasmids which comprise first and second plasmids, and wherein said first plasmid contains said first and second nucleotide sequences, and wherein said second plasmid contains said fourth and / or fifth nucleotide sequences.

10. The composition of claim 9, wherein said third nucleotide sequence is: i) present in said at least one bacterial chromosome as a knock-in, ii) present on said first or second plasmids, or iii) present on a third plasmid.

11. The composition of claim 1, wherein said kynurenine transcriptional regulator protein comprises KynR protein.

12. The composition of claim 1, wherein said KynU and / or said TyrB are from Pseudomonas aeruginosa or other equivalent proteins from Gram-negative and Gram- positive bacteria.Attorney Docket Number: CCF-44551.601 13. The composition of of claim 1, wherein said kynurenine transporter protein comprises an MTR protein, a Tna protein, or an AroP protein.

14. The composition of claim 1, wherein said kynurenine responsive promoter comprises a kynurenine-responsive promoter DNA sequence (Pkyn).

15. The composition of claim 1, wherein said plasmid replication protein is present and comprises repL.

16. The composition of claim 1, wherein said bacteria is from the following genera which naturally harbor KynR: Salmonella, Escherichia, Shigella, Proteus, other Enterobacteriaceae, Pseudomonas, Cupriavidus, other Gamma proteobacteria, and Bacillus.

17. The composition of claim 16, wherein at least one, or at least two, or at least three of said first, second, third, fourth, or fifth nucleic acid sequences are non-endogenous to said bacteria.

18. The composition of claim 1, wherein said bacteria is E. coli or S. enterica.

19. The composition of claim 1, further comprising vi) a sixth nucleic acid sequence comprising a quorum sensing system upstream of said third nucleotide sequence encoding said KynU protein.

20. The composition of claim 19, wherein said quorum sensing system comprises a Plux1 promoter upstream of a sequence encoding Lux1 protein, and optionally further encoding LuxR protein.

21. The composition of claim 19, wherein said quorum sensing system comprises at least one of the following systems: i) a Plas promoter upstream of a sequence encoding lasI and / or lasR; ii) a Prhl promoter upstream of a sequence encoding rh1I and / or rh1R; iii) a Ptra promoter upstream of a sequence encoding traI and / or traR; iv) a sequence encoding ArgA, ArgB, ArgC, and ArgD; v) a sequence encoding: luxS, luxP, and luxQ;Attorney Docket Number: CCF-44551.601 vi) a sequence encoding: qseC and qseB; and vii) a sequence encoding HHQ, PQS, HQNO, and NHQ.

22. A method comprising: administering the bacteria of any one of claims 1-21 to a patient with a tumor or a disease in which kynurenine, and optionally a polyamine, which is optionally spermidine, is accumulating in a localized lesion.

23. The method of claim 22, wherein said patient is a human.

24. The method of claim 22, wherein said administration is such that said bacteria localizes to said tumor, or said localized lesions, of said patient.

25. The method of claim 22, wherein said administration is such that said bacteria degrades said kynurenine.

26. The method of claim 25, wherein said bacteria further degrades said polyamine, which is optionally spermidine.

27. The method of claim 22, wherein said tumor or said localized lesion produces kynurenine and said bacteria preferentially localizes, and / or proliferates in, said tumor or lesion at least in part based on said kynurenine expression.

28. The method of claim 22, wherein said bacteria are administered orally, systemically or through direct intratumor, or intra-lesion, injection.

29. A kit or system comprising: a) said bacteria of any one of claims 1-21; and b) a delivery device or composition for administering said bacteria to a patient with a tumor or lesion.

30. The kit or system of claim 29, wherein said patient is a human.

31. The kit or system of claim 29, wherein said delivery device composition is selected from: syringes, oral tablets, capsules, enema, and IV solution.

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