Novel in VIVO expression system for gene expression control and applications thereof

Abstract

The present invention relates to a novel in vivo expression system for biocontainment and gene expression control in microbes including modified and / or unmodified for application in processes of altering cell multiplication, activating cell lysis, altering metabolism and product formation, etc. The said gene expression control system can be switched on (activated) and off (deactivated) depending on presence and absence of externally and / or internally active inducer or product of gene activity, etc. The said biocontainment and gene expression control system are useful for in vitro applications (production in Bioreactors) and also for in situ applications including but not limited to application on plant, soil, water treatment, aquaculture, cattle feed, gut applications, etc.

Description

[0001] “NOVEL IN VIVO EXPRESSION SYSTEM FOR GENE EXPRESSION CONTROL AND APPLICATIONS THEREOF”

[0002] TECHNICAL FIELD OF THE INVENTION:

[0003] The present invention relates to a novel in vivo expression system for biocontainment and gene expression control in microbes including modified and / or unmodified for application in processes of altering cell multiplication, activating cell lysis, altering metabolism and product formation, etc. The said gene expression control system can be switched on (activated) and off (deactivated) depending on presence and absence of externally and / or internally active inducer or product of gene activity, etc. The said biocontainment and gene expression control system are useful for in vitro applications (production in Bioreactors) and also for in situ applications including but not limited to application on plant, soil, water treatment, aquaculture, cattle feed, gut applications, etc.

[0004] BACKGROUND AND PRIOR ART OF THE INVENTION:

[0005] An important aspect of bacterial control is the capacity of engineered strain to survive in a particular or specified environment. This prevents the escape of engineered strain to the unintended environment and avoids the mix up of genetic biodiversity. Small molecule-based kill switches have been developed to ensure the survival in specific environmental conditions, by allowing the expression of essential genes, by toxic gene regulation, non- natural amino acid based auxotrophic conditions.

[0006] US10760065B2 discloses the processes of tuing the microbial populations with programmable Nucleases, wherein methods and compositions are disclosed for reversing antibiotic resistance or virulence in destroying pathogenic microbial cells which include programmed expression of Toxin-Antitoxin (TA method) for controlling the microbial population. EP3478834B1 discloses the use of a gene oscillator or other cellular machinery that creates a periodic signal is used to trigger the insertion of polynucleotide sequences that record the passage of time. Making the DSB and integrating the HDR templates in response to signals creates a record in the polynucleotide of events experienced by the cell and their timing.

[0007] Stirling et al., (2020) developed a pH sensitive based biocontainment genetic circuit which enables the toxin expression in an unfavourable environment toxin thereby creating an effective bacterial containment system. Chan et al., 2016 developed a biocontainment system with unbalanced reciprocal transcriptional repression circuit-based control (Deadman and Passcode) of the cell viability system to prevent the escape of genetically engineered microbes into the environment. With the use of cold-inducible promoter, Stirling et al., (2017) developed “Essentializer and cryodeath” based genetic circuits which controls the bacterial death by a loss of a bi-stable cl / Cro memory switch. Cold inducible cryodeath switch employs toxin-antitoxin titration based evolutionarily stable construct to control the populations in 140 generations.

[0008] With the discovery of CRISPR-Cas based genome editing tools, it has been a forefront tool in strain engineering to produce the industry-relevant entities in the recent past. CRISPR-Cas based genetic circuits to control the cell population also have been developed. Rottinghaus et al., (2022) developed CRISPR based genetically stable kill switch to control the probiotic bacteria proliferation using a chemical and temperature responsive switch.

[0009] The previous methods of controlling gene expression in microbes have several limitations. These include the utilization of toxin-based kill-switches or the establishment of unfavourable conditions as a kill-switch. These methods are primarily employed in research and under in-vitro conditions. Additionally, they are specific to particular microbes. Consequently, there is a need for methodologies suitable for commercial applications in diverse microorganisms intended for a wide range of uses.

[0010] Hence, there exists a need in the art to devise an effective procedure for controlling gene expression in genetically engineered microbes. This procedure should inhibit alterations in gene biodiversity by preventing the intermingling of genetically engineered / enhanced microbes with the natural environment. There is also a need for a biological safety mechanism for the genetically engineered microbes to maintain in a contained environment which allows the modified microbe to survive only in the specific environment, and for timely activation and / or inactivation and / reduction of processes involved in cell metabolism, product formation process, cell lysis, etc.

[0011] OBJECT OF THE INVENTION:

[0012] The objective of the present invention is to establish a novel in vivo expression system for genetic biocontainment and gene expression control of genetically enhanced / edited / engineered microbes, in controlling cell metabolism, product formation, cell lysis process of the microbes, and also limiting the spread of the microbe to unintended environment and reducing the risk of genetic biodiversity mix up. The proposed method includes kill-switch processes such as and not limited to controlling gene expression in genetically engineered microbes based on the anticipated results, aimed at delaying / halting / repressing cellular metabolism, growth, and gene expression subsequent to gene manipulations. The said gene expression control system are for application including but not limited to application on plant, soil, water treatment, aquaculture, cattle feed, gut applications, etc.

[0013] SUMMARY OF THE INVENTION:

[0014] In a main aspect, the present invention provides a unique in vivo expression system for gene expression control and biocontainment of genetically engineered microbes, based on naturally occurring gaseous and / or non-gaseous molecules present in in vivo environments in plants, aquatic species, mammalian species, wherein the expression results in activation or suppression of cell multiplication, metabolism, product formation, cell lysis, etc.

[0015] In another aspect, the present invention provides processes for genetic biocontainment of microbes / genetically enhanced / edited / engineered microorganisms, wherein the microbe is manipulated to alter and timely tune the cell metabolism and / or gene expression and / or prevent its spread to unintended environments. The said manipulations in the microbe are also for preventing the mix up of engineered microbes.

[0016] In another aspect, the expression elements are regulated based on sensing the presence and / or absence of the naturally occurring gaseous and / or non-gaseous compounds, wherein the gaseous compounds that are sensed by the expression system includes but not limited to CO, CO2, CH4, NO, N2O, ethylene, acetylene, H2S, NH3, TMA-Trimethyl amine, etc., and the non-gaseous compounds that are sensed by the expression system includes but not limited to sugars, sugar isomers, organic acids, amino acids, fatty acids, peptides, proteins, phenolic acids, hormones, urea, guanidine, methanol, formic acid, formaldehyde, acetaldehyde, etc.

[0017] In another aspect, the system for gene expression control in genetically engineered microbes includes and not limited to the following process either in combination or alone:

[0018] 1. Activation of gene expression for product formation,

[0019] 2. Inactivation / Repression of gene expression and product formation

[0020] 3. Enhancing cell multiplication

[0021] 4. Arresting cell multiplication

[0022] 5. Activating cell lysis (self-destruction)

[0023] 6. Altering / delaying cell metabolism In yet another aspect, the present process for gene expression control in genetically engineered microbes uses induction compounds such as, and not limited to, sugars & sugar analogs like lactose, Isopropyl thiogalactoside (IPTG), Arabinose, Nitrogen compounds, amino acids, Ions, Nucleic acids, for controlled inducible gene expression.

[0024] In another aspect, the establishment of Gene control switch on & off occurs by the induced expression of compounds leading to the arrest of microbe growth or metabolism or gene expression.

[0025] In yet another aspect, the metabolism or growth-arresting compounds include but are not limited to, cell lytic enzymes, repressors for gene expression, toxins, essential gene transcription and / or translation repressors, gene silencing elements etc.

[0026] In another aspect, the present process for gene expression control in genetically engineered microbes includes the incorporation of CRISPR-Cas, such as but not limited to, variants of Cas9, Casl2, Casl3, based gene control elements for arresting microbe metabolism or growth or gene expression.

[0027] In yet another aspect, the present invention provides the establishment of controlled expression process, where in the presence and absence of the inducer compound, determines the metabolism and growth of the microbe.

[0028] DESCRIPTION OF FIGURES:

[0029] Figure 1 : Depicts the tuning of one-step biocontainment method of gene expression control and cell lysis

[0030] Figure 2: Depicts the tuning of two-step biocontainment method of gene expression control and cell lysis under in-vivo condition (in plant)

[0031] Figure 3: Depicts the general pathway of Lysine biosynthesis in yeast with emphasis on rate -limiting step. DETAILED DESCRIPTION OF THE INVENTION DETAILS OF BIOLOGICAL ORGANISMS USED IN THE INVENTION

[0032] The present invention provides a unique in vivo expression system based on naturally occurring gaseous and / or non-gaseous molecules present in in vivo environments in plants, aquatic species, mammalian species, wherein the expression results in activation or suppression of cell multiplication, metabolism, product formation, cell lysis, etc.

[0033] The present invention also provides processes for Genetic biocontainment of microbes / genetically enhanced / edited / engineered microorganisms, wherein the microbe is manipulated to alter and timely tune the cell metabolism and / or gene expression and / or prevent its spread to unintended environments. The said manipulations in the microbe are also for preventing the mix up of engineered microbes. In a preferred embodiment, the present invention provides a unique in vivo expression system for gene expression control and biocontainment of genetically engineered microbes based on naturally occurring gaseous and / or non-gaseous molecules present in in vivo environments in plants, aquatic species, mammalian species, wherein the expression results in activation or suppression of cell multiplication, metabolism, product formation, cell lysis, and the like.

[0034] In another embodiment, the expression system of the present invention can be an inducible and / or constitutive system and involves the expression elements such as and not limited to promoter, operator, Riboswitch elements and Translation regulators like UTR (UnTranslated regions), RBS (Ribosome binding site), enhancers of transcription / translation, and the like.

[0035] In an embodiment, the expression elements of the expression system are regulated based on sensing the presence and / or absence of the naturally occurring gaseous and / or non-gaseous compounds, wherein the gaseous compounds that are sensed by the expression system includes but not limited to CO, CO2, CH4, NO, N2O, ethylene, acetylene, H2S, NH3, TMA-Trimethyl amine, etc., and the non-gaseous compounds that are sensed by the expression system includes but not limited to sugars, sugar isomers, organic acids, amino acids, fatty acids, peptides, proteins, phenolic acids, hormones, urea, guanidine, methanol, formic acid, formaldehyde, acetaldehyde, etc.

[0036] In an embodiment, the present invention provides novel process of genetic biocontainment and gene expression control in microbes including modified and / or unmodified microbes. The said biocontainment and gene expression control process are for application under in vitro applications (production in Bioreactors) and also for in situ applications including but not limited to application on plant, soil, water treatment, aquaculture, cattle feed, gut applications, etc. In another embodiment, the process for gene expression control in genetically engineered microbes includes and not limited to the following process either in combination or alone.

[0037] 1. Activation of gene expression for product formation,

[0038] 2. Inactivation / Repression of gene expression and product formation

[0039] 3. Enhancing cell multiplication

[0040] 4. Arresting cell multiplication

[0041] 5. Activating cell lysis (self-destruction)

[0042] 6. Altering / delaying cell metabolism

[0043] In another embodiment, the gene expression control enables biocontainment of the microbe in a specific environment and prevents the microbe survival in non-specific environment.

[0044] In another embodiment, the gene expression controls comprises in vitro handling of engineered microbes for enhancing product formation, enhancing microbial multiplication, in bioreactors, shake flasks, Fermenters, and the like.

[0045] In another embodiment, the gene expression controls can also be applied for in situ / in vivo applications including but not limited to soil environment, plant foliar and rhizosphere environment, animal gut, aquaculture, water treatment plant, etc., for enhancing cell multiplication, timely activation of product formation, activation of cell metabolism, timely repression of cell multiplication, kill-switch for cell lysis, etcand the like.

[0046] In yet another embodiment, the gene expression controls are suitable for wide class of microbes including but limited to Enterobacter, E.coli, Serratia, Azorhizobium, cyanobacteria, Gluconacetobacter, Acetobacter, Beijerinckia, Duganella, Delftia, Sinorhizobium, Bradyrhizobiun, Halomonas, Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Methylomonas, Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, Acetabacterium, Streptomyces, Rhodococcus, Frankia sp, Microbacterium, Curtobacterium, Bradyrhizobium japonicum, Ralstonia eutropha, Epichloe typhina, Rhodococcus, cyanobacteria, methylobacteria, methanogens, methylotrophs, Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azotobacter, Azorhizobium, Beijerinckia, Duganella, Bradyrhizobiun, Sinorhizobium, Methylomonas, Methylosarcina, Methylococcus, Nitrospira, Nitromonas, Nictrobacter, Bacillus, Paenibacillus, Streptomyces, Rhodobacter sphaeroides, Cupriavidus necator, Rhodobacter spp, Azospirillum lipoferum, Rhodopseudomonas palustris, Flavobacterium, Xanthobacter spp; algae such as chlorella, Chlamydomonas, and the like, fungi, yeasts such as Pichia, saccharomyces, Rhodosporidium, myceliopthora, Schizosaccharomyces, Priformospora indica, Aspergillus, Pichia stipitis, and the like.

[0047] In an embodiment, the gene expression control by cell lysis comprises gene manipulations in microbial genome for intracellular expression of killer compounds such as and not limited to lytic enzymes (such as lysozyme, lyticase, zymolyase, chitinase, protease, nucleases), dsRNA, RNA inhibitors, anti-sense RNA, and the like.

[0048] In another embodiment, the gene expression control can be under control of constitutive promoter such as and not limited to promoters of enzymes involved in Glycolysis, TCA cycle, etc., and / or inducible promoter such as and not limited to lac, tac, trc, lactose / arabinose / raffmose / galactose based promoter, and the like.

[0049] In another embodiment, the gene expression under inducible promoter requires induction compounds such as and not limited to sugars, sugar analogues, sugar isomers, proteins, peptides, nitrogen compounds like urea, guanidine, amino acids, and the like.

[0050] In yet another embodiment, the gene expression control comprises generating auxotrophism which requires the presence of essential key compounds for cell survival such as but not limited to amino acids, nucleotides and / or their precursors or derivatives, etc.

[0051] In another embodiment, the gene expression control comprises combination of more than one processes such as and not limited to presence and absence of inducer compounds, and / or Auxotrophism and / or in situ compounds and / or environmental factors like abiotic / biotic / pH / temperature stress, and the like.

[0052] In one major embodiment, the gene manipulations for gene expression control for in vivo / in situ application such as in plant is achieved by enabling the activation of microbial metabolism by sensing the plant intrinsic compound such as and not limited to plant sap sugars, phenolic compounds, plant hormones, plant pigments, compounds like Guanidine, chlorophyll breakdown products, etc.

[0053] In another embodiment, the gene manipulations for gene expression control for in vivo / in situ application such as in animal gut, especially in cattle gut is achieved by enabling the ingestion of engineered microbe and activation of microbial metabolism by sensing the gut intrinsic compounds

[0054] In another major embodiment, the present process for gene expression control in genetically engineered microbes uses compounds such as, and not limited to, sugars & sugar analogs like lactose, Isopropyl thiogalactoside (IPTG), Arabinose, Nitrogen compounds, amino acids, Ions, Nucleic acids, for controlled inducible gene expression.

[0055] In a further embodiment, the present invention provides controlled inducible gene expression by inclusion of gene expression elements such as and not limited to promoter, operator, Translation regulators like UTR (UnTranslated regions), RBS (Ribosome binding site), Riboswitch, etc. specific for the inducer compound. In one embodiment, the establishment of Gene control switch on & off occurs by the induced expression of compounds leading to the arrest of microbe growth or metabolism or gene expression.

[0056] In another embodiment, the metabolism or growth-arresting compounds include but are not limited to, cell lytic enzymes, repressors for gene expression, toxins, essential gene transcription and / or translation repressors, gene silencing elements etc.

[0057] In one embodiment, the present process is also for activation of brekdown. Hydrolysis of external substrates / compounds such as and not limited to sugars (mono / di / tri / oligo / polysaccharides), sugar isomers, proteins, peptides, fattyacids, lipids, phenolic compounds, TMA, polymers, and the like.

[0058] In one embodiment, the present process for gene expression control in genetically engineered microbes includes the incorporation of CRISPR-Cas, such as but not limited to, variants of Cas9, Casl2, Casl3, based gene control elements for arresting microbe metabolism or growth or gene expression.

[0059] In another embodiment, the present invention provides the establishment of controlled expression process, where in the presence and absence of the inducer compound, determines the metabolism and growth of the microbe.

[0060] In an embodiment, the present invention provides a process for gene expression control in genetically engineered microbes by switching on & off either microbe metabolism or growth or gene expression in engineered microbes. The said biocontainment systems are based on self-destruction of the microbes after certain generations of growth and expression of destined function.

[0061] In another embodiment, the metabolism or growth arresting compounds include, and not limited to, o Cell lytic enzymes, including proteolytic enzymes or nucleic acid lytic enzymes o Repressor for gene expression, o Toxins o Essential gene transcription and / or translation repressors, o Gene silencing elements such as and not limited to RNAi, siRNA, dsRNA, Anti-sense RNA, etc. o Inhibitors of metabolism such as Antibotics. o Lack of essential compounds (Auxotrophism) such as Amino acid or nucleotides, precursors, etc.

[0062] In another embodiment, the invention describes the combination of containment process and gene expression control methods as two-stage containment process. For example, the presence of in vivo compound is for activation of the product formation and amino acid auxotrophism for cell lysis and cell containment.

[0063] EXAMPLES

[0064] Example 1: In vivo expression system with Guanidine as example for inducer compound:

[0065] Gene expression control was assessed under in vitro condition from E.coli Host cells. Guanidine was used as the inducer compound for activating gene expression. Gene expression control is depicted in figure 1.

[0066] Lysozyme protein was expressed as the gene product and effect of expression was assessed by the cell lysis profile.

[0067] Gene coding for Lysozyme (SEQ ID NO: 3) was cloned downstream of the promoter with Guanidine-specific 5’UTR region (represented by SEQ ID NO: 1), in E.coli. SEQ ID NO: 2 depicts the Promoter sequence and 5’UTR sequence together (as example, Tuf promoter sequence + UTR sequence).

[0068] Expression of lysozyme was assessed in different sets with addition of Guanidine in varying concentrations. The effect of lysozyme expression was observed by the cell density (absorbance at 600nm) measurements. The results are depicted in table 1 below:

[0069] Table 1

[0070] Result:

[0071] As the concentration of Guanidine increased from 100 to 200 mM concentration, the cell density decreased showing the linear tuning of lysozyme expressing in correlation with Guanidine concentration.

[0072] Example 2: I In vivo expression system with Guanidine as example for inducer compound- two-step control of cell lysis.

[0073] • in vivo expression system with two-step control of cell-lysis kill-switch mechanism, as shown in Figure 2., was developed for tight control of gene expression and lysis

[0074] • Gene coding for the cell lytic enzyme lysozyme was cloned under tac promoter which got inactivated by lac inhibitor. Inhibition was controlled by expression of lac repressor which was expressed under the Guanidine specific promoter. • In presence of Guanidine, lac repressor expression was activated and this inhibited the expression of lysozyme.

[0075] • , when guanidine concentration went low, lac repressor expression weas disabled which enabled the lysozyme expression.

[0076] Result:

[0077] Methylobacterium strain was enabled with two-step in vivo expression system, in the absence of the Guanidine in culture medium, the lysozyme expression was activated and lead to reduction in culture density.

[0078] EXAMPLE 3: In vivo expression system in tuning cell lysis in yeast cells - kill switch control based on amino acid auxotrophism

[0079] • Gene expression control of genetically enhanced yeast Pichia for protein production, based on amino acid auxotrophism as kill-switch control, Lysine auxotrophism as example of amino acid auxotrophism.

[0080] • Lysin biosynthesis pathway is central pathway in bacteria and yeast, from where few other amino acids also are generated. Mutations in Diaminopimelate epimerase (coded by dapF / lysF), causes DAP accumulation and requires Lysine supplementation for proper growth, as described in figure 3.

[0081] • Starting from the culture inoculation until the culture harvest after 96 hours of culturing, the required Amino acid-Lysine was added in the culture medium, as the amino acid biosynthesis pathway was disabled.

[0082] • After 96 hours of growth and protein expression, lysine addition in media was stopped. Due to the disabled Lysine biosynthesis pathway, cell growth and multiplication was arrested.

[0083] Result:

[0084] Pichia pastoris strain was manipulated with respect to deletion of dapF gene by homologous gene integration method. For culture maintenance ad further protein expression, Lysine was contimupisly maintained in culture medium

[0085] The grown culture was shifted to medium without external lysine, wherein the growth was arrested as observed by the unchanged culture density

[0086] SEQUENCE LISTING:

[0087] SEQUENCE LISTING INFORMATION:

[0088] DTD VERSION: VI 3

[0089] FILE NAME: SEQUENCE LISTING.XML

[0090] SOFTWARE NAME: WIPO SEQUENCE

[0091] SOFTWARE VERSION: 2.3.0

[0092] PRODUCTION DATE: 2025-09-22

[0093] GENERAL INFORMATION:

[0094] CURRENT APPLICATION / IP OFFICE: IN

[0095] CURRENT APPLICATION / APPLICATION NUMBER: 202441022164

[0096] CURRENT APPLICATION / FILING DATE: 2024-09-22

[0097] CURRENT APPLICATION / APPLICANT FILE REFERENCE: 202441022164

[0098] EARLIEST PRIORITY APPLICATION / IP OFFICE: IN

[0099] EARLIEST PRIORITY APPLICATION / APPLICATION NUMBER:

[0100] 202441022164

[0101] EARLIEST PRIORITY APPLICATION / FILING DATE: 2024-09-22

[0102] APPLICANT NAME: FERTIS INDIA PVT. LTD.

[0103] APPLICANT NAME / LANGUAGE: EN

[0104] INVENTION TITLE: NOVEL IN VIVO EXPRESSION SYSTEM FOR GENE

[0105] EXPRESSION CONTROL AND APPLICATIONS THEREOF ( EN )

[0106] SEQUENCE TOTAL QUANTITY: 3

[0107] SEQUENCES:

[0108] SEQUENCE NUMBER (ID): 1

[0109] LENGTH: 121

[0110] MOLECULE TYPE: DNA FEATURES LOCATION / QUALIFIERS:

[0111] - SOURCE, 1..121

[0112] > MOL TYPE, OTHER DNA

[0113] > ORGANISM, GUANIDINE SPECIFIC 5UTR REGION

[0114] RESIDUES:

[0115] AAAATAGAAT AAATACTCCA CCGGGAGTTA AATCGTATGA

[0116] ACGATTGTTT GCATTTCAGT 60

[0117] AGGTCTGAGA AGAAATGTAG ATAGTCGTTC TTTTTTTAGC

[0118] TGAAGGAGAG TGAAACCCAT 120

[0119] G 121

[0120] SEQUENCE NUMBER (ID): 2

[0121] LENGTH: 278

[0122] MOLECULE TYPE: DNA

[0123] FEATURES LOCATION / QUALIFIERS:

[0124] - SOURCE, 1..278

[0125] > MOL TYPE, OTHER DNA

[0126] > ORGANISM, TUF PROMOTER (PROMOTER OF TRANSLATION ELONGATION FACTOR TU) WITH GUANIDINE SPECIFIC 5UTR

[0127] RESIDUES:

[0128] TGCTCGATCT CGGCGAGCGC CGCGGCGGCG CCCCGGAGGC

[0129] GCAGCGGCTC TACGAGCAGG 60

[0130] TCGGCGAGGC CGCGTCCTGA CCCCTCATTC GGCGCGATCC

[0131] GCCCTGCCGG CAAACCTGCC 120

[0132] GCTTGATCAT CGTCGCACAA GCCTCTAATC GGTCCGCAAA

[0133] ATAGAATAAA TACTCCACCG 180

[0134] GGAGTTAAAT CGTATGAACG ATTGTTTGCA TTTCAGTAGG

[0135] TCTGAGAAGA AATGTAGATA 240

[0136] GTCGTTCTTT TTTTAGCTGA AGGAGAGTGA AACCCATG

[0137] 278 SEQUENCE NUMBER (ID): 3

[0138] LENGTH: 456

[0139] MOLECULE TYPE: DNA

[0140] FEATURES LOCATION / QUALIFIERS:

[0141] - SOURCE, 1..456

[0142] > MOL TYPE, OTHER DNA

[0143] > ORGANISM, T7 LYSOZYME

[0144] RESIDUES:

[0145] ATGGCTCGTG TACAGTTTAA ACAACGTGAA TCTACTGACG

[0146] CAATCTTTGT TCACTGCTCG 60

[0147] GCTACCAAGC CAAGTCAGAA TGTTGGTGTC CGTGAGATTC

[0148] GCCAGTGGCA CAAAGAGCAG 120

[0149] GGTTGGCTCG ATGTGGGATA CCACTTTATC ATCAAGCGAG

[0150] ACGGTACTGT GGAGGCAGGA 180

[0151] CGAGATGAGA TGGCTGTAGG CTCTCACGCT AAGGGTTACA

[0152] ACCACAACTC TATCGGCGTC 240

[0153] TGCCTTGTTG GTGGTATCGA CGATAAAGGT AAGTTCGACG

[0154] CTAACTTTAC GCCAGCCCAA 300

[0155] ATGCAATCCC TTCGCTCACT GCTTGTCACA CTGCTGGCTA

[0156] AGTACGAAGG CGCTGGTCTT 360

[0157] CGCGCCCATC ATGAGGTGGC GCCGAAGGCT TGCCCTTCGT

[0158] TCGACCTTAA GCGTTGGTGG 420

[0159] GAGAAGAACG AACTGGTCAC TTCTGACCGT GGATAA

[0160] 456

[0161] Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.

Claims

We claim,1. A unique in vivo expression system for gene expression control and biocontainment of genetically engineered microbes based on naturally occurring gaseous and / or non-gaseous molecules present in in vivo environments in plants, aquatic species, mammalian species, wherein the expression results in activation or suppression of cell multiplication, metabolism, product synthesis / formation, breakdown of external substrate, cell lysis, and the like.

2. The expression system as claimed in claim 1, wherein the expression system can be inducible and / or constitutive system and involves the expression elements such as and not limited to promoter, operator, Riboswitch elements and Translation regulators like UTR (UnTranslated regions), RBS (Ribosome binding site), enhancers of transcription / translation, and the like.

3. The expression system as claimed in claim 1 and 2, wherein the expression elements are regulated based on sensing the presence and / or absence of the naturally occurring gaseous and / or non-gaseous compounds.

4. The expression system as claimed in claim 3, wherein the gaseous compounds that are sensed by the expression system includes but not limited to CO, CO2, CH4, NO, N2O, ethylene, acetylene, H2S, NH3, TMA- Trimethyl amine, and the like.

5. The expression system as claimed in claim 3, wherein the non-gaseous compounds that are sensed by the expression system includes but not limited to sugars, sugar isomers, organic acids, amino acids, fatty acids, peptides, proteins, phenolic acids, hormones, urea, guanidine, methanol, formic acid, formaldehyde, acetaldehyde, and the like.

6. The expression system as claimed in claim 1, wherein the gene expression control in genetically engineered microbes comprises alteration of cellular process such as and not limited to:• Activation of gene / pathway expression for product synthesis / formation• Inactivation / Repression of gene / pathway expression for product synthesis / formation• Activation of gene / pathway expression for external substrate breakdown• Inactivation / Repression of gene / pathway expression for external substrate breakdown• Enhancing cell multiplication• Arresting cell multiplication• Activating cell lysis (self-destruction)• Altering / delaying cell metabolism7. The expression system as claimed in claim 1, wherein the gene expression control enables biocontainment of the microbe in a specific environment and prevents the microbe survival in non-specific environment.

8. The expression system as claimed in claim 1, wherein said gene expression controls comprises invitro handling of engineered microbes for enhancing product formation, enhancing microbial multiplication, in bioreactors, shake flasks, Fermenters, and the like.

9. The expression system as claimed in claim 1, wherein said gene expression controls are useful for in situ / in vivo applications including but not limited to soil environment, plant foliar and rhizosphere environment, animal gut, aquaculture, water treatment plant, etc., for enhancing cell multiplication, timely activation of product formation, activation of cell metabolism, timely repression of cell multiplication, kill-switch for cell lysis, and the like.

10. The expression system as claimed in claim 1, wherein said microbes include but limited to Enterobacter, E. coll. Serratia, Azorhizobium, cyanobacteria, Gluconacetobacter, Acetobacter, Beijerinckia, Duganella, Delftia, Sinorhizobium, Bradyrhizobiun, Halomonas, Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Methylomonas, Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, Acetabacterium, Streptomyces, Rhodococcus, Frankia sp, Microbacterium, Curtobacterium, Bradyrhizobium japonicum, Ralstonia eutropha, Epichloe typhina,Rhodococcus, cyanobacteria, methylobacteria, methanogens, methylotrophs, Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azotobacter, Azorhizobium, Beijerinckia, Duganella, Bradyrhizobiun, Sinorhizobium, Methylomonas, Methylosarcina, Methylococcus, Nitrospira, Nitromonas, Nictrobacter, Bacillus, Paenibacillus, Streptomyces, Rhodobacter sphaeroides, Cupriavidus necator, Rhodobacter spp, Azospirillum lipoferum, Rhodopseudomonas palustris, Flavobacterium, Xanthobacter spp; algae such as chlorella, Chlamydomonas, and the like., fungi, yeasts such as Pichia, saccharomyces, Rhodosporidium, myceliopthora, Schizosaccharomyces, Priformospora indica. Aspergillus, Pichia stipitis, and the like.

11. The expression system as claimed in claim 1 and 6, wherein the gene expression control by cell lysis comprises gene manipulations in microbial genome for intracellular expression of killer compounds such as and not limited to lytic enzymes (such as lysozyme, lyticase, zymolyase, chitinase, protease, nucleases), dsRNA, RNA inhibitors, anti-sense RNA, and the like.

12. The expression system as claimed in claims 1 and 2, wherein the gene expression control can be under control of constitutive promoter such as and not limited to promoters of enzymes involved in Glycolysis, TCA cycle, etc., and / or inducible promoter such as and not limited to lac, tac, trc, lactose / arabinose / raffmose / galactose based promoter, and the like.

13. The expression system as claimed in claims 1, 2 and 12, wherein the gene expression under inducible promoter requires induction compounds such as and not limited to sugars, sugar analogues, sugar isomers, proteins, peptides, nitrogen compounds like urea, guanidine, aminoacids, and the like.

14. The expression system, as claimed in claim 1, wherein the expression is for activation of the external substrates or compounds such as and not limited to sugars (mono / di / tri / oligo / polysaccharides), sugar isomers, proteins,peptides, fattyacids, lipids, phenolic compounds, TMA, polymers, and the like.

15. The expression system as claimed in claim 1, wherein the gene expression control comprises generating auxotrophism which requires the presence of essential key compounds for cell survival such as but not limited to aminoacids, nucleotides and / or their precursors or derivatives, etc.

16. The expression system as claimed in claim 1, wherein the gene expression control comprises incorporation of CRISPR-Cas based compounds, such as but not limited to, variants of Cas9, Casl2, Casl3, based gene control elements for arresting microbe metabolism or growth or gene expression.

17. The expression system as claimed in claim 1, wherein gene expression control process comprises combination of one or more processes such as and not limited to presence and absence of inducer compounds, and / or Auxotrophism and / or in situ compounds and / or environmental factors like abiotic / biotic / pH / temperature stress, and the like.

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