TECHNICAL FIELD

The instant disclosure is in the field of biosciences, more particularly towards molecular and industrial biotechnology. The present disclosure relates to recombinant methanotrophic bacteria, a method of developing said recombinant methanotrophic bacteria, and methods and applications related to indigo biosynthesis by the recombinant methanotrophic bacteria.

BACKGROUND OF THE DISCLOSURE

Indigo and its derivative pigments are considered to be one of the oldest classes of dyes known to man and extensively used in the textile industry. Indigo has been prized since antiquity for its vibrancy and deep blue color. India was the leading producer of natural indigo until the chemical synthesis took over the market. Until 2011, about 50,000 tons of indigo has been synthesized per year, of which 95% is used to dye over 4 billion denim garments. Indigo is also used as a food colorant and in the cosmetic industry.

While there is a high and unprecedented demand for indigo, such a demand presents serious environmental concerns mainly because of two important reasons. First, industrial scale indigo synthesis (chemical/synthetic route) relies on aniline, a by-product derived from the petroleum product benzene. Further, the chemical synthesis involves use of hazardous chemicals like formaldehyde, hydrogen cyanide, and strong bases. Second, as indigo is insoluble in water, it is reduced to the water-soluble form by using hazardous reducing agents. Sodium dithionite is the widely used reducing agent in industries because of its low cost and short reduction time. Said reducing agent and its derivatives are major pollutants of effluents from textile industries and subsequently have hazardous influences on public health.

Indigo is thus majorly produced by chemical synthesis and said synthetic counterparts have replaced the natural production. However, due to the industrial importance of indigo and the drawbacks of chemical synthesis as discussed above, there exists a need to provide a scalable, cost-effective, biological and ecofriendly route for indigo synthesis. The present disclosure addresses said need. SUMMARY OF THE DISCLOSURE

The present disclosure relates to a recombinant methanotrophic bacterium capable of producing indigo from methane, comprising: a gene encoding enzyme for increasing concentration of indole; and a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by: a) conversion of tryptophan to indole, or b) reducing or preventing formation of tryptophan from indole, or both a) and b).

In some embodiments, the gene encoding enzyme for increasing concentration of indole is a gene encoding tryptophanase ( TnaA ), or a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB), or a combination thereof. In some embodiments, the conversion of tryptophan to indole is facilitated by TnaA, and the mutant TrpB reduces or prevents tryptophan formation from indole thereby enhancing accumulation of indole.

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding an oxidase or dehydrogenase. In some embodiments, the oxidase is an indole oxidase. In some embodiments, the indole oxidase is flavin-containing monooxygenase ( FMO ).

In some embodiments, the dehydrogenase is acyl-CoA dehydrogenase-like protein (facA ). or a combination thereof. In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding FMO or a gene encoding lac A . or a combination thereof.

In some embodiments, the recombinant methanotrophic bacterium comprises an overexpressed gene DAHP Synthase, AroF, AroB, AroD, AroE, AroK, AroA, AroC, and combinations thereof; a knocked-down gene selected from the group consisting of tryptophan operon regulator, tyrosine aminotransferase, aspartate aminotransferase and combinations thereof. The present method also describes a method for developing the recombinant methanotrophic bacterium as described above, comprising engineering a wild-type methanotrophic bacterium with one or more genes as defined above to obtain the recombinant methanotrophic bacterium.

The present disclosure further describes a process for producing indigo from methane, comprising culturing the recombinant methanotrophic bacterium as described above, in presence of a methane source.

In some embodiments, the culturing of the recombinant methanotrophic bacterium is carried out at a temperature ranging from about 30°C to 50°C, a pH ranging from about 3 to 8, and for a time-period ranging from 24 hours to 240 hours, and the culturing mode is selected from the group consisting of batch, fed batch, continuous process and combinations thereof.

The present disclosure further provides use of the recombinant methanotrophic bacterium as described above for production of indigo.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a flow-chart of the present disclosure indicating conversion of methane to indigo in recombinant methanotrophic bacteria via shikimic acid pathway.

Figure 2 illustrates the shuttle vector backbone for gene overexpression of tryptophanase or oxygenase or a combination thereof, for expression in methanotrophic bacteria.

Figure 3 illustrates a shuttle vector for overexpression of tryptophanase gene from Escherichia coli strain XL 1 -Blue from hps promoter in methanotrophic bacteria.

Figure 4 illustrates a shuttle vector for overexpression of tryptophanase gene from Escherichia coli strain S17-1 from hps promoter in methanotrophic bacteria.

Figure 5 illustrates a shuttle vector for overexpression of FMO and TnaA as an operon from ppqA promoter in methanotrophic bacteria. Figure 6 illustrates a shuttle vector for overexpression of TnaA from hps promoter and IacA from ppqA promoter in methanotrophic bacteria.

DESCRIPTION OF THE DISCLOSURE

As used herein, the term “genetic engineering”, “genetic manipulation”, “recombination”, “recombinant DNA technology” and the likes are used interchangeably and refers to the act of modifying the genetic makeup/DNA of an organism. This modification for example, by way of introduction of a foreign gene/DNA into the organism, or manipulation of existing gene/DNA of the organism, to arrive at a recombinant organism. Thus, the term “recombinant” or the likes and variant terminologies thereof is also within the purview of the above definition. In some embodiments, the present disclosure provides a recombinant methanotrophic bacterium.

As used herein, the term “native” gene(s) refers to the gene(s) known to be naturally present/existing in an organism. The term native gene(s) refers to the gene(s) known to be naturally present/existing in an organism, that may be further transformed to another organism to develop a recombinant organism. Thus, the term “native” gene(s) refers to any gene that occurs naturally in an organism, regardless of whether it is further modified or transformed into another organism to create such a recombinant organism. For example, the term native gene(s) refers to the gene(s) known to be naturally present/existing in a methanotrophic bacterium, that may be further employed for genetic engineering to develop a recombinant methanotrophic bacterium. Similarly, the term native gene(s) refers to the gene(s) known to be present/existing in the same methanotrophic bacterium which may be further modified for genetic engineering to modify the bacterium to a recombinant methanotrophic bacterium.

Additionally, when the gene employed for genetic engineering is a native gene, it means that the recombinant methanotrophic bacterium obtained by the genetic engineering has one or more additional copy of said native gene apart from the copy/copies already present or existing in the methanotrophic bacterium before genetic engineering.

As used herein, the term “heterologous” gene(s) refers to the gene that is not native, i.e. not present or existing naturally in an organism. Accordingly, the term heterologous gene(s) refers to the gene(s) that is not present or existing naturally in an organism, for example, a methanotrophic bacterium and a gene from another organism. Thus, the term heterologous gene(s) also refers to the gene(s) that is from a non-methanotrophic organism and is used for modifying a methanotrophic bacterium to obtain a genetically modified/recombinant methanotrophic bacterium. Further, the heterologous gene can be an unmodified heterologous gene, or a modified heterologous gene. In some embodiments, a modified heterologous gene comprises a codon-optimized heterologous gene, a mutated heterologous gene, or a combination thereof.

In some embodiments, the native gene, the heterologous gene or a combination thereof are expressed through genomic expression, extra chromosomal expression, episomal expression or any combinations thereof.

As used herein, the term “overexpression” of gene(s) refers to expression of one or more copies of a gene to produce one or more copies of corresponding protein. This overexpression can be of a single gene, or two or more genes. In some embodiments, a single gene is overexpressed. In some embodiments, the overexpression is of two or more genes expressed separately or expressed simultaneously. In some embodiments, expression of two or more genes simultaneously is also termed as ‘co-expression’ . Therefore, in some embodiments, the term overexpression also encompasses ‘co-expression’.

The overexpressed gene can be a native gene, a heterologous gene, or a combination thereof. In some embodiments, overexpression refers to the features including but not limiting to: expression of one or more copies of gene(s) native to methanotrophic bacterium; expression of one or more copies of gene(s) heterologous to methanotrophic bacterium; or expression of one or more copies of native gene(s) and heterologous gene(s) in methanotrophic bacterium.

In some embodiments, overexpression by expressing one or more copies of native gene(s) in a methanotrophic bacterium is achieved by transforming additional copies of said native gene(s) into the methanotrophic bacterium; enhancing the expression of the already existing (native) gene in the methanotrophic bacterium; or a combination thereof.

In some embodiments, overexpression by expressing of one or more copies of native gene(s) in methanotrophic bacterium is achieved by transforming one or more copies of said native gene(s) into a methanotrophic bacterium. In some embodiments, expression of a single additional copy of a native gene by transformation in methanotrophic bacterium refers to overexpression of said native gene in the methanotrophic bacterium. In some embodiments, expression of two or more additional copies of a native gene by transformation in methanotrophic bacterium refers to overexpression of said gene in the methanotrophic bacterium.

In some embodiments, overexpression refers to enhancing the expression of a native gene in a methanotrophic bacterium, wherein said enhancement is by producing one or more copies of the gene in addition to the copy/copies already produced in the methanotrophic bacterium. Such overexpression by enhancing the expression of the native gene is achieved by altering the strength of a promoter in methanotrophic bacterium, by deletion/overexpression/mutation of positive or negative regulators controlling the gene expression, by optimizing the gene sequence to enable better transcription, by optimizing the gene sequence to enable better translation, by optimizing the gene sequence to enhance protein expression and folding, by co-expression of chaperones to enable better expression and folding, by optimizing the gene sequence to enhance protein activity or any combinations thereof. In some embodiments, altering the strength of the promoter to enhance the expression of native gene comprises replacing the native promoter with promoter of higher strength, mutating the native promoter to optimize the promoter regulatory elements, overexpression/deletion/mutation of transcriptional or translational regulators driving protein expression, or any combinations thereof.

In some embodiments, overexpression by expression of one or more copies of heterologous gene(s) in methanotrophic bacterium is achieved by transforming the heterologous gene(s) in a methanotrophic bacterium. In some embodiments, expression of a single copy of a heterologous gene in methanotrophic bacterium refers to overexpression of said gene in the methanotrophic bacterium, especially when such a gene is not naturally present in the said bacterium. In some embodiments, expression of two or more copies of a heterologous gene in methanotrophic bacterium refers to overexpression of said gene in the methanotrophic bacterium.

In some embodiments, overexpression is achieved through expression of a transformed gene (either a native gene, a heterologous gene or both) in methanotrophic bacteria by genomic integration, extrachromosomal DNA expression, episomal expression, or any combination thereof.

In some embodiments, the level of overexpression of the gene can be varied based on number of factors regulating gene expression including but not limited to: whether the gene is present or expressed episomally, extra-chromosomally or on the genome; the nature of the promoter used to drive expression; codon optimization of the gene sequence; number of copies of the gene introduced; the sequence of the ribosomal binding site and so on.

As used herein, the term “co-expression” of gene(s) refers to simultaneous expression of two or more genes. The two or more genes are native genes or heterologous genes to an organism, or a combination of both native and heterologous genes. In some embodiments, the two or more genes are native genes to a methanotrophic bacterium. In some embodiments, the two or more genes are heterologous genes to a methanotrophic bacterium. In some embodiments, the two or more genes are a combination of native and heterologous genes to a methanotrophic bacterium.

In some embodiments, the co-expression genes are driven by same or different promoters. In some embodiments, the two or more co-expression genes can be expressed in a single vector or multiple vectors.

In some embodiments, the co-expression genes can be expressed on the genome. In some embodiments, the co-expression genes are for extrachromosomal expression. In some embodiments, the co-expression genes are for episomal expression. In some embodiments, the co-expression genes are expressed through any combination of episomal expression, extrachromosomal expression and genomic expression.

In some embodiments, the level of co-expression of the gene can be varied based on number of factors regulating gene expression including but not limited to: whether the genes are present or expressed episomally, extra-chromosomally or on the genome; the nature of the promoter used to drive expression; codon optimization of the gene sequence; number of copies of the gene introduced; the sequence of the ribosomal binding site; modification of regulators driving gene expression, and so on. As used herein, the term “knock down” of gene(s) refers to reducing or down-regulating or completely terminating the expression of gene(s). In some embodiments, knock down of gene(s) can be achieved by complete removal or deletion of the gene, or partial removal or deletion of the gene. In some embodiments, knock down of gene(s) can also be achieved by introducing a transgene or point mutation to lower or terminate gene expression, by introducing a stop codon at alternate sites or other known approaches of gene knock-down. In some embodiments, knock down of gene(s) can be achieved by replacing the gene of interest with a homologue of lower activity or no activity.

As used herein, the term “enzyme” refers to a protein that helps to convert substrates to products in a biological reaction.

In some embodiments, an enzyme can be a “oxygenase” that are involved in the transfer of oxygen molecules to a substrate. During the course of the reaction, the substrate is oxidized to produce an intermediate product or final product or a combination of both. In some embodiments, the enzyme can be “dehydrogenase” that oxidizes a substrate by reducing an electron acceptor, usually NAD ⁺ /NADP ⁺ or a flavin coenzyme such as FAD or FMN.

As used herein, the term “indole” refers to an aromatic heterocyclic organic compound with a bicyclic structure, consisting of a six-membered benzene ring fused to a five- membered nitrogen-containing pyrrole ring with a molecular formula of CsFFN.

As used herein, the term “indoxyl” refers to an aromatic heterocyclic organic compound with a bicyclic structure, consisting of a six-membered benzene ring fused to a five- membered nitrogen-containing pyrrole ring having an hydroxyl group with a molecular formula of CsFFNO.

As used herein, the term “indigo” refers to an aromatic heterocyclic organic compound consisting of respectively of two six-membered benzene rings fused to a five-membered nitrogen-containing pyrrole ring a molecular formula of C16H10N2O2.

The present disclosure aims at providing a commercially scalable, cost-effective, biological and ecofriendly route for indigo synthesis. In particular, the present disclosure deviates from the traditional/conventional chemical synthesis of indigo and provides a sustainable and eco-friendly route for producing indigo in biological system.

Indigo is a derivative of the native aromatic amino acid biosynthesis pathway. In the present disclosure, recombinant methanotrophic bacteria are provided which have been engineered for synthesis of indigo from methane.

Methane is a sustainable source of carbon. Currently, solutions to convert methane into useful products are limited. Methanotrophic bacteria (methanotrophs) use significantly different metabolic pathways as they use only methane or C 1 substrates as the sole carbon and energy source. The present inventors were able to engineer methanotrophic bacteria to enable the developed recombinant methanotrophs to convert methane to indigo via. shikimic acid pathway.

Accordingly, the present disclosure provides engineered methanotrophs for conversion of methane to indigo. The present disclosure particularly provides recombinant methanotrophic bacteria capable of producing indigo from methane.

The present disclosure provides recombinant methanotrophic bacterium capable of producing indigo from methane, said bacterium comprising one or more genes capable of increasing concentration of indole followed by conversion of said indole to indoxyl during methane utilization via. shikimic acid pathway, wherein subsequent oxidation of indoxyl results in indigo.

In some embodiments, the present disclosure provides a recombinant methanotrophic bacterium capable of producing indigo from methane, comprising: a gene encoding enzyme for increasing concentration of indole; and a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by: a) conversion of tryptophan to indole, or b) reducing or preventing formation of tryptophan from indole, or both a) and b). In some embodiments, the present disclosure provides a recombinant methanotrophic bacterium capable of producing indigo from methane, comprising: a gene encoding enzyme for increasing concentration or levels of indole; and a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased, among others, by conversion of tryptophan to indole, or by preventing or reducing the rate of formation of tryptophan from indole, or both.

In some embodiments, the concentration of indole is increased in the recombinant methanotrophic bacterium by at least about five-fold compared to a corresponding wild- type methanotrophic bacterium.

In some embodiments, the concentration of indole is increased in the recombinant methanotrophic bacterium by about five-fold to fifty fold compared to a corresponding wild-type methanotrophic bacterium.

In some embodiments, the concentration of indole is increased in the recombinant methanotrophic bacterium by about five-fold, eight-fold, ten-fold, fifteen fold, twenty fold, thirty fold, forty fold or fifty fold compared to a corresponding wild-type methanotrophic bacterium.

In some embodiments of the present disclosure, the gene encoding enzyme for increasing concentration of indole, and the gene encoding enzyme for converting the indole to indoxyl, are heterologous genes i.e. genes not native to methanotrophic bacterium.

In some embodiments, the increase in concentration of indole is caused by overexpression, codon optimization, mutation or any combination thereof, of the gene encoding enzyme for converting the tryptophan to indole.

In some embodiments, the conversion of the indole to indoxyl is caused by overexpression, codon optimization, mutation or any combination thereof, of the gene encoding enzyme for converting the indole to indoxyl. In some embodiments, the gene encoding enzyme for increasing concentration of indole is a gene encoding tryptophanase ( TnaA ).

In some embodiments, the gene encoding enzyme for increasing concentration of indole is a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB).

In some embodiments, the gene encoding enzyme for increasing concentration of indole is a gene encoding tryptophanase (TnaA) and a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB).

In some embodiments, increasing concentration of indole comprises conversion of tryptophan to indole. In some embodiments, said conversion of tryptophan to indole is facilitated or catalyzed by TnaA.

In some embodiments, increasing concentration of indole comprises reducing or preventing formation of tryptophan from indole. In some embodiments, preventing formation of tryptophan refers to preventing the conversion of indole to tryptophan. In some embodiments, reducing formation of tryptophan from indole refers to reducing the rate of conversion of indole to tryptophan. In some embodiments reducing formation of tryptophan from indole refers to reducing the rate of conversion of indole to tryptophan completely. In some embodiments, reducing formation of tryptophan from indole refers to reducing the rate of conversion of indole to tryptophan by about at least two fold when compared to the conversion of indole to tryptophan in presence of an enzyme catalyzing conversion of indole to tryptophan. In some embodiments, the enzyme which catalyzes conversion of indole to tryptophan is a wild-type or native TrpB enzyme. Accordingly, in some embodiments, reducing formation of tryptophan from indole refers to reducing the rate of conversion of indole to tryptophan by about at least two fold when compared to the conversion of indole to tryptophan in presence of a wild-type or native TrpB enzyme.

In some embodiments, reducing the formation of tryptophan from indole refers to reducing the rate of conversion of indole to tryptophan by about at least three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, ten-fold or completely, when compared to the conversion of indole to tryptophan in presence of a wild-type or native TrpB enzyme. In some embodiments, preventing the formation of tryptophan from indole refers to preventing the conversion of indole to tryptophan completely, when compared to the conversion of indole to tryptophan occurring in presence of a wild-type or native TrpB enzyme.

In some embodiments, the formation of tryptophan from indole is reduced or prevented by mutating native TrpB.

In some embodiments, the mutant TrpB refers to a TrpB gene which is mutated within the methanotrophic bacterium by known mutation techniques to obtain a mutant TrpB gene, or a mutant TrpB gene developed and transformed into the methanotrophic bacterium, or a combination thereof.

In some embodiments, the B domain (TrpB) of tryptophan synthase catalyzes the condensation of serine and indole to form tryptophan. Mutation in TrpB prevents tryptophan formation (irrespective of presence of active alpha subunit) and leads to accumulation of indole.

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding an oxidase or dehydrogenase.

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding an indole oxidase.

In some embodiments, the indole oxidase is a flavin-containing monooxygenase ( FMO ).

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding a dehydrogenase.

In some embodiments, the dehydrogenase is a acyl-CoA dehydrogenase-like protein (IacA). In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is selected from a gene encoding flavin-containing monooxygenase ( FMO ), a gene encoding acyl-CoA dehydrogenase-like protein (IacA). or a combination thereof.

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding flavin-containing monooxygenase (FMO).

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding acyl-CoA dehydrogenase-like protein ( IacA ).

In some embodiments, the gene encoding enzyme for converting the indole to indoxyl is a gene encoding flavin-containing monooxygenase (FMO) and a gene encoding acyl-CoA dehydrogenase -like protein (IacA).

In some embodiments, the genes encoding TnaA, mutant TrpB, FMO and IacA are heterologous genes. In some embodiments, said heterologous genes TnaA, mutant TrpB, FMO and IacA are unmodified, codon-optimized, mutated, or any combination of heterologous genes thereof. In some embodiments, mutated or codon-optimized genes are employed to achieve the desired expression of said genes TnaA, mutant TrpB, FMO and IacA . thereby leading to indigo synthesis in the recombinant methanotrophic bacterium. In embodiments of the present disclosure, wild-type/naturally occurring methanotrophic bacteria do not have indigo biosynthesis pathway genes including TnaA, mutant TrpB, FMO and IacA and therefore does not produce any indigo.

In some embodiments, the heterologous genes encoding TnaA, mutant TrpB, FMO and IacA are sourced from bacteria, plant, yeast, or any combination thereof.

In some embodiments, the TnaA gene is sourced from E. coli. In some embodiments, the TnaA gene is sourced from E. coli strain K-12. In some embodiments, the TnaA gene is sourced from E. coli strain K-12 of genotype XL 1 -Blue. In some embodiments, the TnaA gene is sourced from E. coli strain K-12 of genotype SI 7-1.

In some embodiments, the TnaA gene is sourced from organisms belonging to Eubacteria, Archaebacteria, fungi, plants and combinations thereof. In some embodiments, the TnaA gene is sourced from E. coli species, Kleibsella oxytoca, Shigella species and/or other organisms comprising TnaA gene.

In some embodiments, the TrpB gene is sourced from a methanotrophic bacterium, wherein said TrpB gene is either mutated in the methanotrophic bacterium by known mutation techniques to obtain a mutant TrpB gene, or a mutant TrpB gene is developed and transformed into a methanotrophic bacterium. Accordingly, said mutant TrpB gene is a heterologous gene for the methanotrophic bacterium. In embodiments discussed above, the mutant TrpB reduces or prevents the reversible formation of tryptophan from indole.

In some embodiments, the TrpB gene is sourced from Methylococcus capsulatus and the recombinant methanotrophic bacterium of the present disclosure comprises a mutant TrpB gene.

In some embodiments, the TrpB gene is sourced from organisms belonging to Eubacteria, Archaebacteria, fungi, plants, and combinations thereof.

In some embodiments, the TrpB gene is sourced from Arabidopsis thaliana, Bacillus subtilis, E.coli K12, Saccharomyces cerevisiae and/or other organisms comprising TrpB gene.

In some embodiments, the FMO gene is sourced from Methylophaga sp or Corynebacterium sp.

In some embodiments, the FMO gene is sourced from Methylophaga sp. selected from a group com prising Methylophaga aminisulfidivorans MP, Methylophaga sp strain SKI and a combination thereof. In some embodiments, the FMO gene is sourced from Corynebacterium sp. selected from a group comprising Corynebacterium glutamicum, Corynebacterium jeikeium, Corynebacterium striatum, Corynebacterium bovis, and combinations thereof.

In some embodiments, the FMO gene is sourced from Methylophaga sp. and codon- optimized for expression in a methanotrophic bacterium. In some embodiments, the FMO gene is sourced from Methylophaga sp. and codon-optimized for expression in Methylococcus capsulatus, Methylomicrobium buryatense, or a combination thereof.

In some embodiments, the FMO gene is sourced from Corynebacterium glutamicum and codon-optimized for expression in a methanotrophic bacterium. In some embodiments, the FMO gene is sourced from Corynebacterium glutamicum and codon-optimized for expression in Methylococcus capsulatus, Methylomicrobium buryatense, or a combination thereof.

In some embodiments, the IacA gene is sourced from Acinetobacter baumannii.

In some embodiments, the IacA gene is sourced from Acinetobacter baumannii and codon- optimized for expression in Methylococcus capsulatus , Methylomicrobium buryatense, or a combination thereof.

In some embodiments, the TnaA comprises a nucleic acid sequence set forth as SEQ ID NO. 1 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 1; and a corresponding amino acid sequence set forth as SEQ ID NO. 2 or an amino acid sequence having at least 80% identity to SEQ ID NO. 2. In some embodiments, the TnaA gene comprising a nucleic acid sequence set forth as SEQ ID NO. 1 is a gene from Escherichia coli XL 1 -Blue strain. In some embodiments, the TnaA gene comprising a nucleic acid sequence set forth as SEQ ID NO. 1 is a gene from Escherichia coli S17-1 strain.

In some embodiments, the mutant TrpB comprises a nucleic acid sequence set forth as SEQ ID NO. 3 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 3; and a corresponding amino acid sequence set forth as SEQ ID NO. 4 or an amino acid sequence having at least 80% identity to SEQ ID NO. 4. In some embodiments, the mutant TrpB comprises a nucleic acid sequence set forth as SEQ ID NO. 3 wherein a wild-type/native TrpB gene is sourced from Methylococcus capsulatus and is mutated to develop SEQ ID NO. 3.

In some embodiments, the FMO comprises a nucleic acid sequence set forth as SEQ ID NO. 5 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 5; and a corresponding amino acid sequence set forth as SEQ ID NO. 6 or an amino acid sequence having at least 80% identity to SEQ ID NO. 6. In some embodiments, the FMO gene comprises a nucleic acid sequence set forth as SEQ ID NO. 5 which is a codon-optimized gene for expression in methanotrophic bacteria. In some embodiments, the FMO gene is sourced from Methylophaga aminisulfidivorans and codon-optimized to develop SEQ ID NO. 5 for expression in methanotrophic bacteria.

In some embodiments, the FMO comprises a nucleic acid sequence set forth as SEQ ID NO. 36 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 36; and a corresponding amino acid sequence set forth as SEQ ID NO. 37 or an amino acid sequence having at least 80% identity to SEQ ID NO. 37. In some embodiments, the FMO gene comprises a nucleic acid sequence set forth as SEQ ID NO. 36 which is a codon-optimized gene for expression in methanotrophic bacteria. In some embodiments, the FMO gene is sourced from Corynebacterium glutamicum and codon-optimized to develop SEQ ID NO. 36 for expression in methanotrophic bacteria.

In some embodiments, the IacA comprises a nucleic acid sequence set forth as SEQ ID NO. 7 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 7; and a corresponding amino acid sequence set forth as SEQ ID NO. 8 or an amino acid sequence having at least 80% identity to SEQ ID NO. 8. In some embodiments, the IacA gene comprises a nucleic acid sequence set forth as SEQ ID NO. 7 which is a codon-optimized gene for expression in methanotrophic bacteria. In some embodiments, the IacA gene is sourced from Acinetobacter baumannii and codon-optimized to develop SEQ ID NO. 7 for expression in methanotrophic bacteria.

In some embodiments, the nucleic acid sequence or corresponding amino acid sequence that possesses at least 80% identity with respect to the nucleic acid or amino acid sequences described above ( TnaA , mutant TrpB, FMO and IacA) encompasses nucleic acid or amino acid sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, including all values falling within the range of 80% to 99.99%.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises an overexpressed gene, said overexpressed gene selected from a group comprising 3-Deoxy-D-arabinoheptulosonate 7-phosphate synthase ( DAHP synthase), Phospho-2-dehydro-3-deoxyheptonate aldolase (A ml· ^' ). 3-Dehydroquinate synthase ( AroB ), 3-dehydroquinate dehydratase ( AroD ), Shikimate dehydrogenase (A ml·.). Shikimate kinase ( AroK ), 3-Phosphoshikimatel-carboxyvinyltransferase (AroA). Chorismate synthase (AroC) and combinations thereof.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises an overexpressed gene of shikimic acid or shikimate pathway, said overexpressed gene of shikimate pathway selected from a group comprising 3-Deoxy-D- arabinoheptulosonate 7-phosphate synthase ( DAHP synthase), Phospho-2 -dehydro-3 - deoxyheptonate aldolase ( AroF ), 3-Dehydroquinate synthase (AroB), 3-dehydroquinate dehydratase (AroD), Shikimate dehydrogenase (AroE), Shikimate kinase (AroK), 3- Phosphoshikimatel-carboxyvinyltransferase (Am A). Chorismate synthase (AroC). and combinations thereof.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene DAHP synthase. In some embodiments, the DAHP synthase is a native gene of methanotrophic bacterium, or a heterologous gene from a non- methanotrophic source. In some embodiments, the DAHP synthase is a native gene of Methylococcus capsulatus.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroF. In some embodiments, the AroF is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroF is a heterologous gene sourced from E. coli. In some embodiments, the AroF is a heterologous gene sourced from E. coli, wherein said AroF is mutated to develop a mutant AroF to alter regulation of the shikimate pathway.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroB. In some embodiments, the AroB is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroB is a native gene of Methylococcus capsulatus.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroD. In some embodiments, the AroD is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroD is a native gene of Methylococcus capsulatus.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroE. In some embodiments, the AroE is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroE is a native gene of Methylococcus capsulatus.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroK. In some embodiments, the AroK is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroK is a native gene of Methylococcus capsulatus.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroA. In some embodiments, the AroA is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroA is a native gene of Methylococcus capsulatus.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises the overexpressed gene AroC. In some embodiments, the AroC is a native gene of methanotrophic bacterium, or a heterologous gene from a non-methanotrophic source. In some embodiments, the AroC is a native gene of Methylococcus capsulatus.

In some embodiments, the genes DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA and AroD are derived from methanotrophic bacteria. In some embodiments, the genes DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA and AroD are derived from methanotrophic bacterium selected from a group comprising Methylococcus capsulatus, Methylomicrohium huryatense, Methylomicrohium alcaliphilum, Methylomicrohium kenyanse, Methylomicrohium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrohium japanense and Me thy lococcaceae bacterium. In some embodiments, the overexpression of the gene in the recombinant methanotrophic bacterium of the present disclosure is achieved by altering promoter strength of native gene(s) of methanotrophic bacterium. In some embodiments, the overexpression of the gene in the recombinant methanotrophic bacterium of the present disclosure is achieved by altering promoter strength of native gene of methanotrophic bacterium selected from the group consisting of DAHP Synthase, AroF, AroB, AroD, AroE, AroK, AroA, AroC and combinations thereof.

In some embodiments, the overexpression of the gene in the recombinant methanotrophic bacterium of the present disclosure is achieved by transforming a gene selected from the group consisting of DAHP Synthase, AroF, AroB, AroD, AroE, AroK, AroA, AroC and combinations thereof, and wherein the transformed gene is a native gene to methanotrophic bacterium, or is a heterologous gene. In some embodiments, overexpression is achieved in the recombinant methanotrophic bacterium by transforming a native gene selected from the group consisting of DAHP Synthase, AroB, AroD, AroE, AroK, AroA, AroC and combinations thereof, into a methanotrophic bacterium. In some embodiments, overexpression is achieved in the recombinant methanotrophic bacterium by transforming a heterologous gene such as AroF, into a methanotrophic bacterium. In some embodiments, the native gene, the heterologous gene or both are either unmodified, codon-optimized, mutated or any combination thereof. In some embodiments, mutated or codon-optimized genes are employed to achieve the desired expression of said genes, thereby leading to indigo synthesis in the recombinant methanotrophic bacterium.

In some embodiments, the recombinant methanotrophic bacterium of the present disclosure comprises a knocked-down gene. In some embodiments, said knocked-down gene is selected from the group consisting of genes downregulating the transcription of tryptophan operon such as Tryptophan operon regulator, tyrosine aminotransferase, aspartate aminotransferase, or any combinations thereof.

In some embodiments, aspartate transaminase and aspartate aminotransferase are used interchangeably and refer to enzymes that catalyze the reversible transfer of an amino group between aspartate and glutamate. These enzymes play a key role in amino acid metabolism. In some embodiments, the gene encoding DAHP synthase comprises a nucleic acid sequence set forth as SEQ ID NO. 9 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 9; and a corresponding amino acid sequence set forth as SEQ ID NO. 10 or an amino acid sequence having at least 80% identity to SEQ ID NO. 10.

In some embodiments, the gene encoding AroF comprises a nucleic acid sequence set forth as SEQ ID NO. 11 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 11; and a corresponding amino acid sequence set forth as SEQ ID NO. 12 or an amino acid sequence having at least 80% identity to SEQ ID NO. 12.

In some embodiments, the gene encoding AroF comprises a nucleic acid sequence set forth as SEQ ID NO. 38 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 38; and a corresponding amino acid sequence set forth as SEQ ID NO. 39 or an amino acid sequence having at least 80% identity to SEQ ID NO. 39.

In some embodiments, the gene encoding AroF comprises a nucleic acid sequence set forth as SEQ ID NO. 40 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 40; and a corresponding amino acid sequence set forth as SEQ ID NO. 41 or an amino acid sequence having at least 80% identity to SEQ ID NO. 41.

In some embodiments, the gene encoding AroF comprises a nucleic acid sequence set forth as SEQ ID NO. 42 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 42; and a corresponding amino acid sequence set forth as SEQ ID NO. 43 or an amino acid sequence having at least 80% identity to SEQ ID NO. 43.

In some embodiments, the gene encoding AroF comprises a nucleic acid sequence set forth as SEQ ID NO. 44 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 44; and a corresponding amino acid sequence set forth as SEQ ID NO. 45 or an amino acid sequence having at least 80% identity to SEQ ID NO. 45.

In some embodiments, the gene encoding AroB comprises a nucleic acid sequence set forth as SEQ ID NO. 13 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 13; and a corresponding amino acid sequence set forth as SEQ ID NO. 14 or an amino acid sequence having at least 80% identity to SEQ ID NO. 14.

In some embodiments, the gene encoding AroD comprises a nucleic acid sequence set forth as SEQ ID NO. 15 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 15; and a corresponding amino acid sequence set forth as SEQ ID NO. 16 or an amino acid sequence having at least 80% identity to SEQ ID NO. 16.

In some embodiments, the gene encoding AroE comprises a nucleic acid sequence set forth as SEQ ID NO. 17 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 17; and a corresponding amino acid sequence set forth as SEQ ID NO. 18 or an amino acid sequence having at least 80% identity to SEQ ID NO. 18.

In some embodiments, the gene encoding AroK comprises a nucleic acid sequence set forth as SEQ ID NO. 19 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 19; and a corresponding amino acid sequence set forth as SEQ ID NO. 20 or an amino acid sequence having at least 80% identity to SEQ ID NO. 20.

In some embodiments, the gene encoding AroA comprises a nucleic acid sequence set forth as SEQ ID NO. 21 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 21; and a corresponding amino acid sequence set forth as SEQ ID NO. 22 or an amino acid sequence having at least 80% identity to SEQ ID NO. 22.

In some embodiments, the gene encoding AroC comprises a nucleic acid sequence set forth as SEQ ID NO. 23 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 23; and a corresponding amino acid sequence set forth as SEQ ID NO. 24 or an amino acid sequence having at least 80% identity to SEQ ID NO. 24.

In some embodiments, the gene encoding a tryptophan operon regulator comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO. 30, and a corresponding amino acid sequence having at least 80% identity to SEQ ID NO. 31. In some embodiments, the gene encoding tyrosine aminotransferase comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO. 59; and a corresponding amino acid sequence having at least 80% identity to SEQ ID NO. 60.

In some embodiments, the gene encoding aspartate transaminase comprises a nucleic acid sequence set forth as SEQ ID NO. 34 or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 34; and a corresponding amino acid sequence set forth as SEQ ID NO. 35 or an amino acid sequence having at least 80% identity to SEQ ID NO. 35.

In some embodiments, the nucleic acid sequence or corresponding amino acid sequence that possesses at least 80% identity with respect to the nucleic acid or amino acid sequences described above ( DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD, tryptophan operon regulator and aspartate transaminase) encompasses nucleic acid or amino acid sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, including all values falling within the range of 80% to 99.99%.

In some embodiments, the recombinant methanotrophic bacteria comprising TnaA gene improves indole level by converting tryptophan to indole, and thereby reduces tryptophan levels in the shikimic acid pathway.

In some embodiments, the recombinant methanotrophic bacteria comprising mutated TrpB gene improves flux towards indigo formation in the shikimic acid pathway by reducing or preventing the formation of tryptophan from indole.

In some embodiments, the recombinant methanotrophic bacteria comprising gene selected from FMO, IacA, or a combination thereof, improves flux towards indigo formation in the shikimic acid pathway by catalyzing the conversion of indole to indoxyl.

In some embodiments, the overexpressed gene(s) in the recombinant methanotrophic bacteria increases the endogenous pool of shikimic acid pathway metabolites, thereby enhancing indigo formation in the recombinant methanotrophic bacteria. In some embodiments, the recombinant methanotrophic bacteria of the present disclosure comprising overexpressed gene(s) selected from DAHP Synthase, AroF, AroE, AroK, AroB, AroC, A roA . AroD, or any combination of the genes thereof, improve flux towards chorismate formation in the shikimic acid pathway.

In some embodiments, the knocked-down gene(s) in the recombinant methanotrophic bacteria improves the flux towards indigo biosynthesis by reducing the formation of tyrosine and phenyl alanine in the shikimic acid pathway. In some embodiments, the knocked-down gene(s) in the recombinant methanotrophic bacteria improves the flux towards indole formation from chorismate.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium is selected from the group consisting of Methylococcus capsulatus, Methylomicrobium buryatense, Methylomicrobium alcaliphilum, Methylomicrobium kenyanse, Methylomicrobium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrobium japanense and Methylococcaceae bacterium.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium is selected from a group comprising Methylococcus capsulatus, Methylomicrobium buryatense, Methylosinus trichosporium, Methylomicrobium alcaliphilum and Methylomicrobium kenyanse.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium is Methylococcus capsulatus.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium is Methylococcus capsulatus Bath strain.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium is Methylomicrobium buryatense . In some embodiments of the present disclosure, the recombinant methanotrophic bacterium is Methylomicrobium buryatense strain 5G.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding tryptophanase ( TnaA ) and a gene encoding flavin-containing monooxygenase ( FMO ).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding tryptophanase (TnaA) and a gene encoding acyl-CoA dehydrogenase -like protein (IacA).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding tryptophanase (TnaA), a gene encoding flavin-containing monooxygenase (FMO), and a gene encoding acyl-CoA dehydrogenase-like protein (IacA).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB) and a gene encoding flavin-containing monooxygenase (FMO).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB) and a gene encoding acyl-CoA dehydrogenase-like protein (IacA).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB), a gene encoding flavin-containing monooxygenase (FMO), and a gene encoding acyl-CoA dehydrogenase -like protein (IacA).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding tryptophanase (TnaA), a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB) and a gene encoding acyl-CoA dehydrogenase-like protein (IacA). In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises a gene encoding tryptophanase ( TnaA ), a gene encoding mutant beta subunit of tryptophan synthase (mutant TrpB) and a gene encoding flavin-containing monooxygenase ( FMO ).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises genes encoding TnaA, mutant TrpB, IacA and FMO.

In all the above embodiments describing different gene combinations, the recombinant methanotrophic bacterium is selected from the group consisting of Methylococcus capsulatus, Methylomicrobium buryatense, Methylomicrobium alcaliphilum, Methylomicrobium kenyanse, Methylomicrobium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrobium japanense and Methylococcaceae bacterium.

In all the above embodiments describing different gene combinations, the recombinant methanotrophic bacterium is Methylococcus capsulatus.

In all the above embodiments describing different gene combinations, the recombinant methanotrophic bacterium is Methylomicrobium buryatense .

In some embodiments, the present disclosure provides recombinant methanotrophic bacteria comprising:

(i) a gene encoding enzyme for increasing concentration of indole, and a gene encoding enzyme for converting the indole to indoxyl; and

(ii) optionally, an overexpressed gene, a knocked-down gene, or a combination of the overexpressed gene and the knocked-down gene.

In some embodiments, the present disclosure provides recombinant methanotrophic bacteria comprising: (i) a gene encoding enzyme for increasing concentration of indole, and a gene encoding enzyme for converting the indole to indoxyl; and

(ii) an overexpressed gene.

In some embodiments, the present disclosure provides recombinant methanotrophic bacteria comprising:

(i) a gene encoding enzyme for increasing concentration of indole, and a gene encoding enzyme for converting the indole to indoxyl; and

(ii) a knocked-down gene.

In some embodiments, the present disclosure provides recombinant methanotrophic bacteria comprising:

(i) a gene encoding enzyme for increasing concentration of indole, and a gene encoding enzyme for converting the indole to indoxyl; and

(ii) a combination of overexpressed gene and knocked-down gene.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- a gene selected from TnaA, mutant TrpB , and a combination thereof;

- a gene selected from FMO, IacA, and a combination thereof; and

- a gene selected from an overexpressed gene, a knocked-down gene, or a combination thereof, wherein the overexpressed gene is selected from the group consisting of DAHP Synthase, AroF, AroB, AroD, AroE, AroK, AroA, AroC and combinations thereof, and the knocked-down gene is selected from the group consisting of tryptophan operon regulator genes, tyrosine aminotransferase, aspartate aminotransferase, and combinations thereof.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- a gene selected from TnaA, mutant TrpB, and a combination thereof;

- a gene selected from FMO, IacA, and a combination thereof; and

- an overexpressed gene selected from the group consisting of DAHP Synthase, AroF, AroB, AroD, AroE, AroK, AroA, AroC and combinations thereof. In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- a gene selected from TnaA, mutant TrpB , and a combination thereof;

- a gene selected from FMO, IacA, and a combination thereof; and

- a knocked-down gene selected from the group consisting of tryptophan operon regulator genes, tyrosine aminotransferase, aspartate aminotransferase and combinations thereof.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene; FMO gene; and

- overexpressed gene of shikimate pathway.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises: TnaA gene;

- FMO gene; and

- one or more overexpressed genes selected from a group consisting of aroA, aroC, aroK, aroE, aroD, DAHP synthase, aroB and aroF.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene;

- FMO gene; and

- overexpressed genes aroA, aroC, aroK, aroE, aroD, DAHP synthase, aroB and aroF

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene;

- FMO gene; and

- one or more overexpressed genes selected from a group consisting of aroA, aroC, aroK, aroE, DAHP synthase, aroB and aroF. In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene or mutant TrpB gene in combination with FMO gene or lac gene; and

- knocked-down gene comprising tryptophan operon regulator gene(s).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene;

- FMO gene; and

- knock-down of tryptophan operon regulator gene(s).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene;

- FMO gene;

- knock-down of tryptophan operon regulator gene(s); and

- overexpressed gene(s) of shikimate pathway.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TrpB gene;

- FMO gene; and

- overexpressed gene(s) of shikimate pathway.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TrpB gene;

- FMO gene; and

- knock-down of tryptophan operon regulator gene(s).

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- TnaA gene;

- IacA gene; and overexpressed gene(s) of shikimate pathway.

In some embodiments of the present disclosure, the recombinant methanotrophic bacterium comprises:

- mutant TrpB gene;

- IacA gene; and

- overexpressed gene(s) of shikimate pathway.

In all the above embodiments describing different gene combinations comprising: a gene encoding enzyme for increasing concentration of indole, a gene encoding enzyme for converting the indole to indoxyl, and optionally an overexpressed gene and/or knocked- down gene, the recombinant methanotrophic bacterium is selected from the group consisting of Methylococcus capsulatus, Methylomicrobium buryatense, Methylomicrobium alcaliphilum, Methylomicrobium kenyanse, Methylomicrobium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrobium japanense and Methylococcaceae bacterium.

In all the above embodiments describing different gene combinations comprising: a gene encoding enzyme for increasing concentration of indole, a gene encoding enzyme for converting the indole to indoxyl, and optionally an overexpressed gene and/or knocked- down gene, the recombinant methanotrophic bacterium is Methylococcus capsulatus.

In all the above embodiments describing different gene combinations comprising: a gene encoding enzyme for increasing concentration of indole, a gene encoding enzyme for converting the indole to indoxyl, and optionally an overexpressed gene and/or knocked- down gene, the recombinant methanotrophic bacterium is Methylomicrobium buryatense.

In various embodiments of the present disclosure, the recombinant methanotrophic bacterium can comprise any of the genes encoding enzyme for increasing concentration of indole and genes encoding enzyme for converting the indole to indoxyl, in combination with any of the overexpressed gene and/or knocked-down gene described herein.

In some embodiments of the present disclosure, any homologue of the genes described herein can be employed for engineering the recombinant methanotrophic bacterium.

Thus, the present disclosure provides recombinant methanotrophic bacterium as described above which convert methane to indigo. In particular, the present disclosure describes expression or overexpression of heterologous genes, overexpression of native genes, knock down of genes, and combinations thereof, involved in shikimic acid pathway and intermediate metabolites as an approach to synthesize indigo in methanotrophic bacterium.

Shikimate pathway derived metabolites act as precursors for a wide variety of natural products that not only play crucial role in growth and physiological response, but also have high economical value. Intermediates from this pathway are channeled towards different metabolic branches for formation of diverse end-products including aromatic amino acids and their derivatives. Based on aromatic amino acid metabolism and methane metabolism, the present inventors have carefully employed target genes (heterologous genes and native genes) for overexpression and optionally knock down to further enhance the levels of the metabolites. Wild-type methanotrophic bacteria do not synthesize indigo. Particularly, the wild-type methanotrophic bacteria do not have a mechanism to utilize indole for indigo biosynthesis. For manipulation of indigo production in methanotrophic bacteria, the present invention employs/targets genes for increasing indole concentration in the cells and to convert the indole to indoxyl, which further leads to indigo biosynthesis in recombinant methanotrophs. Genes encoding the enzymes TnaA, mutant TrpB, IacA and FMO in different combinations were particularly employed. Optionally, genes of shikimate pathway regulating flux to chorismate are also targeted for overexpression thereby leading to increased concentrations of chorismate. Said genes include but are not limited to DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, and AroD. Other optional target genes are genes of shikimate pathway regulating flux to aromatic amino acid biosynthesis (tryptophan, tyrosine and phenyl alanine). In particular, the genes related to the tryptophan operon regulators, tyrosine aminotransferase, aspartate transaminase are knocked-down to improve the flux towards indigo biosynthesis in recombinant methanotrophs. Accordingly, in some embodiments of the present disclosure, the conversion of methane to indigo in the recombinant methanotrophic bacteria occurs via the following shikimic acid pathway/mechanism: methane is converted to chorismate followed by conversion of chorismate to tryptophan (a key aromatic amino acid). The levels/concentrations of indole is increased by converting tryptophan to indole, or alternatively by reducing or preventing formation of tryptophan from indole, or both. The conversion of tryptophan to indole is catalyzed by employing enzymes such as TnaA. The mutant TrpB prevents the formation of tryptophan from indole and hence overexpression of mutant TrpB results in indole accumulation. The indole thus synthesized/accumulated is converted to indoxyl in the presence of oxidase or dehydrogenase such as FMO, IacA or a combination thereof. Indoxyl undergoes oxidation in air to form indigo, the blue colored dye.

In an embodiment of the present disclosure, the conversion of methane to indigo in the recombinant methanotrophic bacteria via. shikimic acid pathway is illustrated in Figure 1.

In embodiments of the present disclosure, the source of methane used by the recombinant methanotrophic bacteria for the production of target metabolites is selected from a group comprising biogas, natural gas, landfill gas, organic waste, pure methane, any source comprising methane and combinations thereof.

The present disclosure thus describes engineering of methane, central carbon metabolism and shikimic acid pathway to improve availability of branch point metabolites and aromatic amino acids for production of indigo in methanotrophic bacteria. This enables the production of indigo by employing the recombinant methanotrophic bacteria described herein using a cost-effective substrate (methane) through a sustainable and ecofriendly process. More particularly, said biosynthesis of indigo is accomplished by genetic transformation of methanotrophic bacteria for expression of specific genes described herein that catalyze the synthesis and accumulation of target compounds from methane.

While the aforementioned aspects have been described for developing a recombinant methanotrophic bacterium for production of indigo compound, each of the aforementioned aspects are identically applicable for a recombinant methanotrophic bacterium developed for production of indoxyl compound. Thus, in some embodiments, the present disclosure also provides a recombinant methanotrophic bacterium capable of producing indoxyl from methane, comprising:

- a gene encoding enzyme for increasing concentration of indole; and

- a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by conversion of tryptophan to indole, or by reducing or preventing formation of tryptophan from indole, or both.

Further, while indoxyl is known to readily undergo spontaneous dimerisation in presence of molecular oxygen to produce indigo, a skilled person will understand and know the techniques to isolate said indoxyl for industrial use/applications (prior to its conversion to indigo), and each of such techniques/methodologies to isolate indoxyl are within the purview of the present disclosure and claims.

The present disclosure further provides a method for developing the recombinant methanotrophic bacteria as described above, said method comprising: a) transforming or mutating a gene encoding enzyme for increasing concentration of indole, in a wild-type methanotrophic bacteria; and b) transforming a gene encoding enzyme for converting the indole to indoxyl, in a wild- type methanotrophic bacteria.

In some embodiments, the method for developing the recombinant methanotrophic bacteria comprises: a) transforming a gene encoding an enzyme for conversion of tryptophan to indole, transforming a mutated gene encoding an enzyme for reducing or preventing formation of tryptophan from indole, mutating a gene encoding enzyme for conversion of indole to tryptophan, or any combinations thereof; and b) transforming a gene encoding enzyme for converting the indole to indoxyl.

In some embodiments, the method for developing the recombinant methanotrophic bacteria comprises: a) transforming a TnaA gene for conversion of tryptophan to indole, transforming a mutant TrpB gene for reducing or preventing formation of tryptophan from indole, or any combinations thereof; and b) transforming a gene encoding oxidase or dehydrogenase for converting the indole to indoxyl.

In some embodiments of the method for developing the recombinant methanotrophic bacteria, gene encoding oxidase or dehydrogenase is FMO, IacA or a combination thereof.

In some embodiments, the method for developing the recombinant methanotrophic bacteria comprises: a) transforming a TnaA gene, a mutant TrpB gene, or a combination thereof; and b) transforming FMO gene, IacA gene, or a combination thereof.

In some embodiments, the method for developing the recombinant methanotrophic bacteria comprises: a) designing vector(s) for expression of TnaA gene, a mutant TrpB gene, or a combination thereof, and FMO gene, IacA gene, or a combination thereof; and b) transforming the vector into a wild-type methanotrophic bacteria, to obtain the recombinant methanotrophic bacteria.

In some embodiments, the vector is a single vector expressing TnaA gene.

In some embodiments, the vector is a single vector expressing mutant TrpB gene.

In some embodiments, the vector is a single vector expressing FMO gene.

In some embodiments, the vector is a single vector expressing IacA gene.

In some embodiments, the vector is a co-expression vector expressing a combination of TnaA gene and mutant TrpB.

In some embodiments, the vector is a co-expression vector expressing a combination of FMO gene and IacA gene.

In some embodiments, the vector is a co-expression vector expressing a combination of TnaA gene and a gene selected from FMO gene and IacA gene. In some embodiments, the vector is a co-expression vector expressing a combination of TnaA gene, FMO gene and IacA gene.

In some embodiments, the vector is a co-expression vector expressing a combination of mutant TrpB and a gene selected from FMO gene and IacA gene.

In some embodiments, the vector is a co-expression vector expressing a combination of mutant TrpB, FMO gene and IacA gene.

In some embodiments, the method for developing the recombinant methanotrophic bacteria further comprises: a) overexpressing genes selected from a group comprising DAHP Synthase, AroF, AroE, AroK, AroB, AroC, Aro and AroD, ; b) knocking-down of genes selected from a group comprising tryptophan operon regulator, tyrosine aminotransferase, aspartate transaminase and combinations thereof; or c) overexpressing genes and knocking-down of genes as defined in steps (a) and (b), to obtain the recombinant methanotrophic bacteria.

In some embodiments, the method for developing the recombinant methanotrophic bacteria further comprises: a) designing vector(s) for expression of one or more overexpression genes selected from a group comprising DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD and; and transforming the vector into methanotrophic bacteria; and/or b) knocking-down of genes selected from tryptophan operon regulator, tyrosine aminotransferase, aspartate transaminase and combinations thereof to obtain the recombinant methanotrophic bacteria.

In some embodiments, the vector is a single vector expressing each overexpression gene separately, or a co-expression vector expressing two or more overexpression genes, wherein the overexpression genes are selected from a group comprising DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA and AroD. In some embodiments, the method for developing the recombinant methanotrophic bacteria comprises: a) transforming a TnaA gene, transforming a mutant TrpB gene, and/or mutating a native TrpB gene in a methanotrophic bacterium; b) transforming an FMO gene and/or a lac A gene; and c) optionally, overexpressing genes selected from a group comprising DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD and combinations thereof, and/or knocking-down of genes selected from tryptophan operon regulator, tyrosine aminotransferase, aspartate transaminase and combinations thereof, in a wild-type methanotrophic bacterium, to obtain the recombinant methanotrophic bacterium.

In some embodiments, overexpressing the genes selected from a group comprising DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA and AroD, is carried out by transforming said genes into methanotrophic bacteria, altering promoter strength of native genes, or a combination of both.

In some embodiments, expression, overexpression and/or knock-down of the genes described herein in the methods of the present disclosure can be performed in any sequence/order to develop the recombinant methanotrophic bacteria of the present disclosure. Thus, in other words, the sequence/order of engineering the genes to develop the recombinant methanotrophic bacteria does not adversely impact the end result i.e. obtaining of the recombinant methanotrophic bacteria of the present disclosure.

In some embodiments of the method for developing recombinant methanotrophic bacteria described herein, the developed recombinant methanotrophic bacterium is selected from a group comprising Methylococcus capsulatus, Methylomicrohium huryatense, Methylomicrohium alcaliphilum, Methylomicrohium kenyanse, Methylomicrohium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrohium japanense and Methylococcaceae bacterium. In some embodiments of the method for developing recombinant methanotrophic bacteria described herein, the developed recombinant methanotrophic bacterium is selected from a group comprising Methylococcus capsulatus, Methylomicrobium buryatense, Methylomicrobium alcaliphilum, Methylosinus trichosporium and Methylomicrobium kenyanse.

In some embodiments of the method for developing recombinant methanotrophic bacteria described herein, the developed recombinant methanotrophic bacterium is Methylococcus capsulatus.

In some embodiments of the method for developing recombinant methanotrophic bacteria described herein, the developed recombinant methanotrophic bacterium is Methylomicrobium buryatense .

In some embodiments of the present disclosure, the method for developing recombinant methanotrophic bacteria comprises the following general steps:

Designing single or multiple vectors comprising gene of interest selected from TnaA, mutant TrpB, FMO, IacA, DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD, genes of tryptophan operon regulators, tyrosine aminotransferase and aspartate aminotransferase

Transforming a host cell the vector(s)

Selection of host cell transfo (eg. E. coli transformants)

Transforming gene of interest in me 1 bacterium from the host cell transformants (eg. E. coli transformants) using conjugation technique (eg. solid mating technique or liquid mating technique)

Selection of methanotrophic bacteria t 1 (recombinant methanotrophic bacteria) In some embodiments of the present disclosure, the method for developing recombinant methanotrophic bacteria comprises the following general steps:

Designing single or multiple vectors comprising gene of interest selected from TnaA, mutant TrpB, FMO, IacA, DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD, genes of tryptophan operon regulators, tyrosine aminotransferase and aspartate aminotransferase

Transforming methanotrophic bacterium with the vector(s) by transformation techniques

Selection of methanotrophic bacteria t 1 (recombinant methanotrophic bacteria)

In some embodiments of the present disclosure, exemplary methods/protocols for developing recombinant methanotrophic bacteria is described in the examples below.

While the aforementioned aspects have been described for a method to develop a recombinant methanotrophic bacterium for production of indigo compound, each of the aforementioned method aspects are identically applicable for a recombinant methanotrophic bacterium developed for production of indoxyl compound. Thus, in some embodiments, the present disclosure also provides a method of developing a recombinant methanotrophic bacterium capable of producing indoxyl from methane, comprising:

- a gene encoding enzyme for increasing concentration of indole; and

- a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by conversion of tryptophan to indole, or by reducing or preventing formation of tryptophan from indole, or both.

Further, while indoxyl is known to readily undergo spontaneous dimerisation in presence of molecular oxygen to produce indigo, a skilled person will understand and know the techniques to isolate said indoxyl for industrial use/applications (prior to its conversion to indigo), and each of such techniques/methodologies to isolate indoxyl are within the purview of the present disclosure and claims. The present disclosure further describes recombinant plasmid or vector for expression of the genes described herein.

In some embodiments, the present disclosure provides vector(s) for expression of genes selected from a group comprising TnaA gene, mutant TrpB gene, FMO gene, IacA gene and combinations thereof.

In some embodiments, the present disclosure provides vector(s) for expression of genes selected from a group comprising DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA and AroD.

In some embodiments, the present disclosure provides vector(s) for knocking-down genes selected from a group comprising tryptophan operon regulators, tyrosine aminotransferase, and aspartate transaminase and combinations thereof.

In some embodiments, an expression cassette is provided comprising a promoter, operator, regulator, the gene of interest, terminator, ribosomal binding site and antibiotic resistance genes. In some embodiments, the gene of interest is selected from a group comprising TnaA gene, mutant TrpB gene, FMO gene, IacA gene, DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD, genes of tryptophan operon regulators, tyrosine aminotransferase, aspartate aminotransferase and combinations thereof.

In some embodiments, the vector described herein comprises elements including:

(i) the expression cassette comprising promoter, regulator, operator, gene of interest (genes selected from a group comprising TnaA gene, mutant TrpB gene, FMO gene, IacA gene, DAHP Synthase, AroF, AroE, AroK, AroB, AroC, AroA, AroD and combinations thereof), terminator, ribosomal binding site and antibiotic resistance genes,

(ii) origins of replication compatible with the assembly/transient host organism used for cloning and the final expression host,

(iii) antibiotic resistance markers for selection of vector,

(iv) transfer genes required for conjugation, and combinations thereof. In some embodiments, the promoter for regulating expression of gene of interest is selected from a group comprising formaldehyde activating enzyme- 1 (Fae-1), formaldehyde activating enzyme-2 (Fae-2), coenzyme PQQ synthesis protein (ppqA), methanol dehydrogenase (pmxaf), 3-hexulose-6-phosphate synthase (hps), particulate methane monooxygenase (s70), soluble methano monooxygenase (s54) and combinations thereof. Said promoter drives the expression of the gene of interest described above either alone or as an operon for gene overexpression.

In some embodiments, the promoters described herein can be isolated or derived from any source. In some embodiments, the promoters described herein are either isolated from methantrophic bacteria selected from Methylococcus capsulatus, Methylomicrobium buryatense, Methylomicrobium alcaliphilum, Methylomicrobium kenyanse, Methylomicrobium album, Methylocapsa acidiphila, Methylocellasilvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrobium japanense and Methylococcaceae bacterium, or from other bacteria such as Escherichia coli. In some embodiments, the promoters can be native promoters or mutated promoters.

In some embodiments, the promoters are selected from a group comprising SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48 and SEQ ID NO. 49.

In some embodiments, the vector is selected from a group comprising of broad host range shuttle vectors that have origin of replication, selection markers, conjugative transfer genes for expression and propagation in methanotrophs and E. coli. In one embodiment of the present disclosure, the vectors include IncP/IncQ Origin of replication based vectors, pBBR origin based vectors or similar vectors for achieving the purpose set forth in the present invention (developing recombinant methanotrophic bacteria).

In some embodiments of the present disclosure, the target genes (gene of interest) and promoters are PCR amplified and cloned in vectors. Subsequently, operons containing any combination of target genes described herein are generated for increasing the metabolite pool and target metabolite biosynthesis using methane as substrate. In another embodiment of the present disclosure, the knock-down of genes selected from tryptophan operon regulator genes, asparatate transaminase or a combination thereof is carried out by techniques selected from homologous recombination, promoter replacement, point mutagenesis, RNA interference (RNAi), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and any other known technique of gene knock-down.

The present disclosure further relates to a method of producing indigo from methane.

The present disclosure particularly relates to a method of producing indigo from methane, comprising culturing the recombinant methanotrophic bacteria described herein in presence of a methane source. More particularly, the present disclosure describes fermentation/conversion of methane to indigo by employing the recombinant methanotrophic bacteria described herein.

In some embodiments, the methane is a sole carbon and energy source for the recombinant methanotrophic bacterium of the present disclosure. In other words, the recombinant methanotrophic bacterium of the present disclosure only uses methane as a carbon and energy source. In some embodiments, the recombinant methanotrophic bacterium only uses methane as a carbon and energy source, and does not use any other carbon and energy source for growth/metabolism.

In some embodiments of the present disclosure, the methane source employed for the production of indigo is selected from the group comprising pure methane, biogas, natural gas, landfill gas, organic waste, any source comprising methane and combinations thereof.

In some embodiments, the method of producing indigo comprises:

(i) receiving methane source as an input/substrate;

(ii) culturing the recombinant methanotrophic bacteria described herein in presence of methane, thereby converting methane to indigo; and

(iii) optionally, isolating or purifying the indigo from the culture.

In some embodiments, the method of producing indigo comprises: (i) receiving input selected from a group comprising biogas, natural gas, landfill gas, organic waste, pure methane, any methane containing source, and combinations thereof;

(ii) culturing the recombinant methanotrophic bacteria described herein in presence of said input, thereby converting methane present in the input to indigo; and

(iii) optionally, isolating or purifying the indigo from the culture.

In some embodiments of the method of producing indigo according to the present disclosure, the recombinant methanotrophic bacterium employed is selected from the group consisting of Methylococcus capsulatus, Methylomicrobium buryatense, Methylomicrobium alcaliphilum, Methylomicrobium kenyanse, Methylomicrobium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrobium japanense and Methylococcaceae bacterium.

In some embodiments, the method of producing indigo comprises:

(i) receiving input selected from a group comprising biogas, natural gas, landfill gas, organic waste, pure methane, any methane containing source, and combinations thereof;

(ii) culturing recombinant Methylococcus capsulatus described herein in presence of said input, thereby converting methane present in the input to indigo; and

(iii) optionally, isolating or purifying the indigo from the culture.

In some embodiments, the method of producing indigo comprises:

(i) receiving input selected from a group comprising biogas, natural gas, landfill gas, organic waste, pure methane, any methane containing source, and combinations thereof;

(ii) culturing recombinant Methylomicrobium buryatense described herein in presence of said input, thereby converting methane present in the input to indigo; and

(iii) optionally, isolating or purifying the indigo from the culture. In some embodiments, the method of producing indigo as described above is carried out at a temperature ranging from about 30°C to 50°C.

In some embodiments, the method of producing indigo as described above is carried out at a pH ranging from about 3.0 to about 8.0.

In some embodiments, the method of producing indigo as described above is carried out for a time-period ranging from 24 hours to 240 hours.

In some embodiments, the method of producing indigo as described above is carried out for a time-period ranging from 24 hours to 120 hours.

In some embodiments, the method of producing indigo is carried out by a culturing mode selected from a group comprising batch culturing, fed-batch culturing, continuous culturing, and combinations thereof.

In some embodiments, the culturing of the recombinant methanotrophic bacterium is carried out at a temperature ranging from about 30°C to 50°C, a pH ranging from about 3 to 8, for a time-period ranging from 24 hours to 240 hours, and by a culturing mode selected from a group comprising batch culturing, fed-batch culturing, continuous culturing, and combinations thereof.

In some embodiments, the growth of recombinant methanotrophic bacterial cells of the present disclosure under suitable culturing conditions facilitate formation of indigo from methane resulting in the microbial synthesis of isolatable quantities of indigo. In some embodiments, the present disclosure also describes optimization of the process of fermentation of recombinant methanotrophic bacterium in the presence of gaseous substrate (methane) to enhance the biomass and product (indigo) yield.

It will be understood by a person of ordinary skill in the art that any recombinant methanotrophic bacterium comprising the genes (target gene or gene of interest) described above can be suitably subjected to culturing at a temperature, pH values, time period, media concentrations, culturing mode and other process/culturing parameters, for indigo production. While the aforementioned aspects have been described for methods of producing indigo from methane, each of the aforementioned aspects of said method are identically applicable for a method for producing indoxyl by culturing the recombinant methanotrophic bacterium described herein in presence of a methane source. Thus, in some embodiments, the present disclosure also provides a method for producing indoxyl from methane, comprising culturing the recombinant methanotrophic bacterium, said bacterium comprising:

- a gene encoding enzyme for increasing concentration of indole; and

- a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by conversion of tryptophan to indole or by preventing formation of tryptophan from indole, or both.

Further, while indoxyl is known to readily undergo spontaneous dimerisation in presence of molecular oxygen to produce indigo, a skilled person will understand and know the techniques to isolate said indoxyl for industrial use/applications (prior to its conversion to indigo), and each of such techniques/methodologies to culture the recombinant methanotrophic bacterium and isolate indoxyl are within the purview of the present disclosure and claims.

The present disclosure further relates to use of a recombinant methanotrophic bacterium for production of indigo from methane, said bacterium comprising: a gene encoding enzyme for increasing concentration of indole; and a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by conversion of tryptophan to indole, or by reducing or preventing formation of tryptophan from indole, or both.

In embodiments relating to the use of recombinant methanotrophic bacterium for production of indigo from methane, the features of said recombinant methanotrophic bacterium is according to the embodiments described above and is incorporated herein in its entirety.

While the aforementioned aspects have been described for use of the recombinant methanotrophic bacterium for conversion of methane to indigo, each of the aforementioned aspects of use are identically applicable for use of said recombinant methanotrophic bacterium for conversion of methane to indoxyl. Thus, in some embodiments, the present disclosure also provides use of a recombinant methanotrophic bacterium for production of indoxyl from methane, said bacterium comprising: a gene encoding enzyme for increasing concentration of indole; and a gene encoding enzyme for converting the indole to indoxyl, wherein the concentration of indole is increased by conversion of tryptophan to indole, or by reducing or preventing formation of tryptophan from indole, or both.

Further, while indoxyl is known to readily undergo spontaneous dimerisation in presence of molecular oxygen to produce indigo, a skilled person will understand and know the techniques to isolate said indoxyl for industrial use/applications (prior to its conversion to indigo), and each of such techniques/methodologies to use the recombinant methanotrophic bacterium and isolate indoxyl are within the purview of the present disclosure and claims.

The present disclosure also provides a method of enhancing the production of indigo in a methanotrophic bacterium comprising: developing a recombinant methanotrophic bacterium as described herein; and culturing the recombinant methanotrophic bacterium in presence of a methane source.

In embodiments relating to the method of enhancing production of indigo in a methanotrophic bacterium, the features of developing a recombinant methanotrophic bacterium and culturing the recombinant methanotrophic bacterium are according to the embodiments described above and is incorporated herein in its entirety.

In some embodiments of the recombinant methanotrophic bacterium and methods of the present disclosure, the recombinant methanotrophic bacterium described herein produces at least about 0.01 g/L of indigo when compared to the corresponding wild-type methanotrophic bacterium which does not produce any indigo. In some embodiments, the recombinant methanotrophic bacterium of the present disclosure produces at least about 0.1 g/L of indigo when compared to the corresponding wild-type methanotrophic bacterium which does not produce any indigo. In some embodiments the recombinant methanotrophic bacterium of the present disclosure produces at least about 0.5 g/L of indigo when compared to the corresponding wild-type methanotrophic bacterium which does not produce any indigo. In some embodiments, the recombinant methanotrophic bacterium of the present disclosure produces about 0.01 g/L to about 50 g/L of indigo.

Thus, as shown in the above disclosure and embodiments, the approach of indigo biosynthesis described herein enables a greener and more sustainable solution for manufacturing indigo. The present disclosure simultaneously provides a solution to address the environmental concerns related to discharge of toxic byproducts that are produced during chemical synthesis of indigo, and at the same time utilizes methane (a powerful greenhouse gas with a global warming potential) to produce a valuable product such as indigo.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description and embodiments provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES

Biological Materials: The wild-type methanotrophic bacteria Methylococcus capsulatus was procured from Professor Colin Murrell at University of East Anglia, UK. Methylomicrobium buryatense used for transformation experiments was a gift from Dr. Yuri Trotsenko, at Pushchino Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Russia. E. coli XUl-Blue and S 17 strains for sourcing TnaA gene were a gift from Prof. Colin Murrell, University of East Anglia, UK. Wild-type TrpB gene was sourced from Methylococcus capsulatus which was obtained as mentioned above. Genes for FMO and lac A were codon optimized and chemically synthesized. EXAMPLE 1

Cloning of Indigo pathway genes into a shuttle vector

Indigo biosynthesis requires tryptophan as a precursor molecule. Tryptophan can be converted to indole by tryptophanase enzyme ( TnaA ) . Alternatively, a mutant beta subunit of tryptophan synthase enzyme (mutant Trp B) can aid in indole accumulation. Indole thus formed can be converted to indoxyl in presence of a suitable oxygenase enzyme (eg. FMO) or a dehydrogenase (eg. IacA). The indoxyl molecule undergoes spontaneous dimerisation in presence of molecular oxygen producing indigo. Different sets of genes were generated to engineer recombinant methanotrophic strains for indigo production as listed in Table 1.

Table 1: Vectors for Indigo Biosynthesis

As shown in Table 1, to engineer recombinant strains for indigo production, Flavin- monooxygenase (FMO) gene from Methylophaga sp. was codon optimised (SEQ ID NO. 5) for expression in Methylococcus capsulatus Bath. All genes were synthesized and sequences were confirmed by nucleotide sequencing. FMO was PCR amplified with primers containing BamHI restriction sites on both ends. PCR amplified gene was cloned downstream to pyrroloquinoline quinone biosynthesis protein A (ppqA) gene promoter in a shuttle vector using sequence and ligation independent cloning (SLIC) method. The shuttle vector consisted of a kanamycin resistance gene cassette (SEQ ID NO. 50) as a selection marker. The shuttle vector backbone used for cloning the gene of interest and regulatory sequences is provided under SEQ ID NO. 27. Said vector backbone was suitably employed in all the present examples. Colonies were screened by performing PCR. The resultant vector vecA was verified using DNA sequencing. Next, tryptophanase gene (TnaA) (SEQ ID NO. 1) from E. coli was amplified using genomic DNA from strains XL 1 -Blue and S- 17. The TnaA genes were cloned downstream to FMO gene using SLIC method. The vectors thus generated were called vecAl and vecA2 containing FMO gene along with TnaA genes from XLl-Blue (VecAl) and S-17 strain (VecA2), respectively.

The gene coding for beta subunit of tryptophan synthase (TrpB) enzyme was PCR amplified from genomic DNA of Methylococcus capsulatus. Said wild-type TrpB gene (SEQ ID NO. 25) was cloned in a commercially procured pET21a vector

(https://www.snapgene.com/resoiirces/plasmid fi{es/?set=pet_and_duet_vectors_(novagen)&p¾asmid=pET-2 1a(%2.B) at BamHI site using the SLIC method. The clone was confirmed by sequencing the vector. The vector generated was labelled vecC. Site-directed mutagenesis was performed on TrpB gene in vecC to introduce R389P and K392M double mutation in the coding region of the gene. The mutation was verified by sequencing. The generated mutant TrpB gene (SEQ ID NO. 3) was PCR amplified for cloning in vecA downstream to FMO gene using the SLIC method of cloning. The resulting vector vecA3 was verified by PCR and DNA sequencing. Accordingly, the indigo biosynthesis pathway was cloned under a constitutive ppqA promoter as an operon.

Another set of vectors using TnaA genes (SEQ ID NO. 1) from E. coli were generated. The TnaA genes were PCR amplified using genomic DNA from strains XLl-Blue and S-17 using primers containing BamHI restriction sites on both ends. The PCR amplified TnaA genes were cloned downstream to constitutive 3-hexulose-6-phosphate synthase (hps) gene promoter using the SLIC method of cloning. The resultant vectors, vecB and vecC containing TnaA gene from XLl-Blue and S-17 respectively were screened using PCR and verified by DNA sequencing.

Next, acyl-CoA dehydrogenase-like protein (IacA) from Acinetobacter baumannii was codon optimised (SEQ ID NO. 7) for Methylococcus capsulatus. IacA was PCR amplified with primers containing BamHI restriction sites on both ends. ppqA promoter containing shuttle vector was restriction digested using BamHI enzyme for cloning IacA gene. Clones were screened by PCR and confirmed by sequencing (vecD). Subsequently, ppqA promoter-IacA gene-T7 terminator cassette (SEQ ID NO. 46-SEQ ID NO. 7-SEQ ID NO. 32) was mobilised from vecD into vecB and vecC. The primers designed to amplify this promoter-gene -terminator cassette added Is pi restriction sites on both ends of the PCR product. Vectors vecB and vecC were digested using Fspl enzyme. PCR amplified ppqA promoter-iacA gene-T7 terminator cassette was cloned using the SLIC method. The resultant vectors vecBl and vecCl were verified by PCR and DNA sequencing.

EXAMPLE 2

Indole/Indigo production using recombinant Methylococcus capsulatus

Wild-type methanotrophs, such as Methylococcus capsulatus do not have the genes required for the conversion of indole to indigo. Hence, the native/wild-type methanotrophic bacteria strains do not produce any indigo.

For indigo production in recombinant methanotrophs, the relevant genes described in Example 1 were transformed into M. capsulatus. Said transformation into M. capsulatus was carried out by first transforming the desired plasmid to a host cell ( E . coli), followed by transforming the genes in M. capsulatus from the E. coli transformants using conjugation technique (solid mating technique). The detailed procedure of transformation to obtain recombinant M capsulatus is as follows:

E. coli XL 1 -Blue competent cells were separately transformed with vectors vecAl, vecA2, vecA3, vecBl and vecCl containing indigo biosynthetic pathway genes (Example 1). The transformants were selected by plating on Luria Bertani media plates supplemented with about 50 pg/ml kanamycin. One colony from each plate was inoculated in about 5 ml Luria Bertani media containing about 50 pg/ml kanamycin to generate a primary culture and incubated overnight in a shaker incubator at about 37°C and about 180 rpm. Next day, about 20 ml of sterile Luria Bertani media supplemented with tryptophan, glycerol and about 50 pg/ml kanamycin was inoculated with about 1% of the respective primary cultures. Flasks were incubated again in a shaker incubator for about 48 hours at about 37°C and 180 rpm. Gradual change in colour of the media from yellow to blue of indigo was observed during the incubation. After about 48 hours of incubation, cells were pelleted by centrifugation at about 4,700 rpm for about 15 minutes. Cell pellets and media supernatants were analysed for indole and indigo production using HPLC analysis. In clones transformed with vecAl, vecA2 and vecA3, HPLC analysis revealed indole accumulation in the supernatant at levels up to 100 mg/L and blue colored indigo accumulation up to 80 mg/L in the pellets. Most of the indole was present in the supernatant. Further, clones transformed with vecB 1 and vecCl containing IacA gene, showed an indigo titre of 94 mg/L. Said results confirm the success of the present strategy of employing gene encoding enzyme for increasing concentration of indole (TnaA or mutant TrpB) and gene encoding enzyme for catalysing indole to indoxyl conversion ( FMO or lac A) and thereby indigo production.

Bioreactor run was performed for E. coli XL 1 -Blue competent cells transformed with vecBl comprising TnaA and IacA. The transformants were selected by plating on Luria Bertani media plates supplemented with about 50 pg/ml kanamycin. One colony from the transformation plate was inoculated in about 100 ml of sterile Luria Bertani media containing about 50 pg/ml kanamycin to generate primary culture for a bioreactor run. The flask was incubated overnight at about 37°C and about 180 rpm.

The recombinant strains were run in about 5000 ml non-jacketed bioreactor containing 4000 ml sterile Luria Bertani media supplemented with tryptophan, glycerol and about 50 pg/ml kanamycin. Entire primary culture of about 100 ml was inoculated in the reactor and grown for about 48 hours at about 37°C with dissolved oxygen maintained at about 20%. Indigo production was visible by the change in colour of the reactor media from yellow to blue of indigo. After about 48 hours of reactor run, cells were harvested by centrifugation at about 6000 rpm for about 15 minutes at about 4°C. Cell pellets and media supernatant were analysed for indigo titre determination. HPLC analysis revealed an indigo titre of 115 mg/L.

The above indole and indigo production results substantiate that the genes (i.e. the gene encoding enzyme for increasing concentration of indole such as TnaA and mutant TrpB ; and the gene encoding enzyme for converting the indole to indoxyl such as FMO and IacA) actively function to result in indole and indigo synthesis.

Based on the successful confirmation of the activity of target genes (TnaA, mutant TrpB, FMO and IacA) described above, wild-type Methylococcus capsulatus was now transformed with different indigo pathway clones generated in E. coli (i.e. E. coli transformants) using a solid mating protocol (conjugation technique). In this technique, transformation was initiated by plating a loopful ofM capsulatus cells on nitrate minimal salt (NMS) media plate supplemented with about 0.02% protease peptone. Plates were incubated at about 37°C for about 24 to 48 hours in an anaerobic jar fed with methane. E. coli S17 competent cells transformed with vectors of interest were incubated overnight at about 37°C. Next day, a loopful of A. coli transformants (comprising vectors vecAl, vecA2, vecA3, vecBl, vecCl, vecB and vecC) were uniformly spread onM capsulatus containing NMS-protease peptone media plates. Plates were incubated at about 37°C for about 48 hours in an anaerobic jar fed with methane. After about 48 hours of incubation, a loopful of culture from NMS-protease peptone plate was spread on NMS plates supplemented with kanamycin for selection of transformants. Plates were incubated at about 45 °C in an anaerobic jar fed with methane. Colonies appearing after 5 to 6 days of transformation were screened by PCR and further verified using DNA sequencing. Confirmed transformant colonies were patched on NMS-kanamycin plates and incubated at about 45°C in an anaerobic jar containing methane for about 3 to 4 days or till the complete visible growth of the patch.

For growth studies, primary culture was generated by inoculating a loopfiil of culture from each transformant in about 20 ml sterile NMS media supplemented with 30 pg/ml of kanamycin. Flasks were sealed using sterile suba seals and about 20% v/v methane was fed in the flasks. Flasks were incubated at about 45°C for about 12 to 16 hours at about 160 rpm. Next day, in 500 ml flasks containing about 100 ml sterile NMS media supplemented with about 30 pg/ml of kanamycin was inoculated with the primary culture. Flasks were sealed and methane feeding was performed. Cultures were incubated at about 45°C for as mentioned about 24 to 72 hours at about 160 rpm. Post incubation, cells were pelleted by centrifugation at about 4,700 rpm for about 15 minutes. Cell pellets and media supernatants were analysed to confirm the presence of indole and indigo from recombinant Methylococcus capsulatus cells, and were subjected to further determination of the titres of said compounds.

EXAMPLE 3

Bioreactor run (batch fermentation) for indole/indigo production in recombinant M. capsulatus expressing indigo biosynthesis pathway genes

Methylococcus capsulatus cells were transformed with vector vecB 1 harboring TnaA and IacA genes. The same procedure as outlined under Example 2 was followed to obtain transformed M. capsulatus cells. Transformants were patched on a fresh NMS plate containing kanamycin for selection of transformants. Primary culture was prepared by inoculation of about 100 ml sterile NMS media supplemented with 30 pg/ml kanamycin with a loopful of recombinant M. capsulatus containing vecB 1. Flask was sealed using suba seal and about 20% v/v methane was fed in the flask as a sole carbon source for growth. Flask was incubated at about 45°C for about 12 to 16 hours at about 160 rpm. Next day, cells from primary culture was used as inoculum for about 200 ml NMS media. Flasks were sealed and methane feeding and incubation was performed as mentioned above. The secondary culture generated was used as an inoculum for batch fermentation in bioreactor.

In a 5L reactor, about 3000 ml sterile NMS media supplemented with 30 pg/ml kanamycin was inoculated with about 1000 ml of secondary culture. The reactor temperature was maintained at about 45°C throughout the reactor run. About 0.1 to 0.2 1pm (litres per minute) of methane gas was fed into the reactor. The dissolved oxygen level was maintained between 15 to 20%. pH of the media was maintained by addition of acid and base solutions. Anti-foam was added as and when required. The reactor was run in batch mode for about 72 hours. At the end of the run, cells were harvested by centrifugation at about 6000 rpm for about 15 minutes at about 4°C. Cell pellets and media supernatants were analysed to confirm the presence of indole and indigo from recombinant Methylococcus capsulatus cells, and were subjected to further determination of the titres of said compounds.

EXAMPLE 4

Indole/Indigo production using recombinant Methylomicrobium buryatense

Methylomicrobium buryatense cells were transformed with vectors containing indigo biosynthesis pathway genes (vectors - vecAl, vecA2, vecA3, vecBl, vecCl, vecB and vecC). The transformations were performed using the solid mating technique as described below. Wild type M. buryatense strain was spread in DSMZ media and incubated in anaerobic jar fed with methane. The anaerobic jar was incubated at about 30°C for about 24 to 48 hours. A loopful ofM buryatense was spread on to mating plate (DSMZ media with 15% nutrient agar) and further incubated at about 30°C for about 24 hours. After the incubation period, a loopful of E. coli containing appropriate vector was spread onto mating plate containing M. buryatense and incubated at about 30°C for about 48 hours. For selecting recombinant M buryatense, a loopful of cells from mating plate was spread on to DSMZ media with about 30 mg/1 kanamycin. Recombinant clones were confirmed by PCR and restriction digestion.

For growth studies, primary culture was generated by inoculating a loopful of culture from each transformant in about 20 ml sterile DSMZ media supplemented with about 30 pg/ml of kanamycin. Flasks were sealed using sterile suba seals and about 20% v/v methane was fed in the flasks. Flasks were incubated at about 45 °C for about 12 to 16 hours at about 160 rpm. Next day, 500 ml flasks containing 100 ml sterile DSMZ media supplemented with about 30 pg/ml of kanamycin was inoculated with the primary culture. Flasks were sealed and methane feeding was performed as mentioned before. Cultures were incubated at about 45°C for about 48 to 50 hours at about 160 rpm. Post incubation, cells were pelleted by centrifugation at about 4,700 rpm for about 15 minutes. Cell pellets and media supernatants were analysed to confirm the presence of indole and indigo from recombinant Methylococcus buryatense cells, and were subjected to further determination of the titres of said compounds.

EXAMPLE 5

Overexpression of Shikimic Acid pathway genes and effect on Indigo production

Shikimic acid pathway provides flux for aromatic amino acid biosynthesis pathway. Hence, shikimic acid biosynthesis pathway enzymes were overexpressed to improve the intracellular levels of chorismate and aromatic amino acids like tryptophan. Genes aroA, aroC, aroK, aroE, aro Q, DAHP synthase and aroB were PCR amplified from genomic DNA of Methylococcus capsulatus. The aroF gene from Escherichia coli strains XL 1 -Blue and S-17 was PCR amplified using respective genomic DNAs. Each of the PCR amplified genes aroA (SEQ ID NO. 21), aroC (SEQ ID NO. 23), aroX (SEQ ID NO. 19), aroE (SEQ ID NO. 17), DAHP synthase (SEQ ID NO. 9), aroB (SEQ ID NO. 13) and aroF (SEQ ID NO. 11) was cloned downstream to constitutive formaldehyde activating enzyme 2 (fae2) gene promoter at BamHI site using the SLIC method of cloning (as explained in Example 1). Clones were verified by PCR and DNA sequencing.

Next, each ‘fae2 promoter-GeneX-T7 terminator’ cassette comprising one or more genes (Gene X) selected from aroD, aroA, aroC, aroK, aroE, DAHP synthase, aroB and aroF was PCR amplified and cloned into vecAl (harboring TnaA and FMO genes) at the Narl site using the SLIC method. Clones were confirmed by performing PCR and DNA sequencing.

The above developed vector comprising TnaA, FMO, aroA, aroC, aroK, aroE, aroD, DAHP synthase, aroB and aroF genes was transformed in E. coli similar to the protocol described in Example 2. The E. coli transformants were used to conjugate the vector into M. capsulatus. The recombinant M. capsulatus strains with the overexpressed genes were analyzed for increase in indigo and indole production. Cell pellets and media supernatants were analysed using HPLC analysis to confirm the presence of indole and indigo from recombinant Methylococcus capsulatus cells, and were subjected to further determination of the titres of said compounds.

EXAMPLE 6

Knock-down of trvR and effect on Indigo production

Tryptophan is a precursor for indigo formation. TrpR protein (Trp operon repressor) encoded in the genome of E. coli regulates the intracellular levels of tryptophan and few other amino acids. It was hypothesized that the knock-down or deletion of the trpR gene can increase the intracellular free tryptophan levels. Higher tryptophan levels can lead to higher levels of indole and indigo. Tryptophan can be converted to indole by TnaA which can feed into indigo biosynthesis pathway and consequently increase the indigo titre.

Deletion of trpR gene (SEQ ID NO. 61) was carried out by replacing it with ampicillin resistance gene cassette from pUC57. Ampicillin resistance gene cassette was amplified using primers with 50 bp homology to upstream and downstream sequences of trpR coding region. The PCR amplified cassette was gel purified. E. coli XLl-Blue competent cells were transformed with the purified ampicillin resistance gene cassette. After 3 hours of recovery in Luria Bertani media, cells were plated on LB plates supplemented with about 100 pg/ml ampicillin. Plates were incubated overnight at about 37°C. Colonies were screened by PCR and verified by genomic DNA sequencing to select E. coli deltrpR cells (i.e. cells with trpR deleted). The E. coli deltrpR cells were used for transforming plasmids containing indigo biosynthesis pathway genes.

E. coli deltrpR competent cells were transformed with vectors vecAl, vecA2, vecA3, vecBl and vecCl in individual experiments. Transformation of vectors in E. coli was performed as described in Example 2. Transformants were selected on LB agar plates containing about 50 pg/ml kanamycin. Recombinant E. coli cells containing the genomic copy of trpR and transformed with above mentioned vectors were used as controls to compare the changes in indigo yields. One colony from each plate was inoculated in about 5 ml Luria Bertani media containing about 50 pg/ml kanamycin to generate a primary culture and incubated overnight at about 37°C and about 180 rpm. Next day, about 20 ml of sterile Luria Bertani media supplemented with about 2% glycerol and about 50 pg/ml kanamycin was inoculated with about 1% of the respective primary cultures. Flasks were incubated for about 48 hours at about 37°C and about 180 rpm. Gradual change in colour of the media from yellow to blue of indigo was observed during the incubation. After about 48 hours of incubation, cells were pelleted by centrifugation at about 4,700 rpm for 15 minutes. Cell pellets and media supernatants were analysed using HPLC analysis to confirm the presence of indole and indigo, and were subjected to further determination of the titres of said compounds. Accordingly, a similar recombinant methanotrophic bacterium is obtained with deletion of tryptophan operon regulator gene(s) and transformation with individual vectors vecAl, vecA2, vecA3, vecBl and vecCl.

EXAMPLE 7

Analysis of transcript levels of Indigo pathway genes

Recombinant Me thylococcus capsulatus cells containing individual vectors vecAl, vecA2, vecA3, vecBl and vecCl with indigo pathway genes were grown in about 20 ml NMS media supplemented with 30 pg/ml of kanamycin and about 20% v/v methane for about 48 hours at about 45 °C and about 160 rpm. Cells were harvested by centrifugation at about 4,700 rpm for about 15 minutes. Total RNA was extracted using TRI reagent as per manufacturer’s instructions. DNase treatment was performed to remove DNA contamination from RNA samples. About 2 pg of DNase treated RNA was used for cDNA synthesis as per manufacturer’s instructions (Takara - PrimeScript 1st strand cDNA Synthesis kit). cDNA was used for the next round of semi-quantitative PCR amplification for full length TnaA, TrpB, FMO and IacA genes. PCR samples were gel electrophoresed and visualized in gel documentation unit. Bands were observed at expected sizes for full length TnaA, TrpB, FMO and IacA genes in recombinant clones indicating a functional pathway for indigo biosynthesis in M. capsulatus. Said results further indicate the capability of the recombinant M capsulatus to produce indigo.

Methylococcus capsulatus and Methylomicrobium buryatense are model methanotrophic bacteria which share similar biochemical pathways to other methanotrophic bacteria. Hence, a successful metabolic engineering of these model methanotrophic bacteria to produce indole and indigo can be used for other methanotrophic bacteria. Accordingly, the successful development of rc co m b i n an t Me thylococci is capsulatus and Methylomicrobium buryatense comprising indigo biosynthesis pathway genes and the functionality/activity of said indigo biosynthesis genes as shown in the above examples indicate that other methanotrophs such as Methylomicrobium alcaliphilum, Methylomicrobium kenyanse, Methylomicrobium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylosinus sporium, Methylocella palustris, Methylocystis parvus, Methylovulum miyakonense, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomicrobium japanense and Methylococcaceae bacterium comprising shikimate metabolic pathway for biosynthesis of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) can be similarly genetically engineered for indigo biosynthesis.

As regards all the embodiments/examples characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. As an example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations: A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; and C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims or plurality of embodiments, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to anyone of claims

1 to 3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4,

2 and 1, of claims 4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed. The above considerations apply mutatis mutandis to all claims and embodiments of the present specification. To give a few examples, the combination of claims 3, 5 and 1 is clearly and unambiguously envisaged in view of the claim structure/claimed subject- matter. The same applies for the combinations of claims 12, 10, 3 and 5, and, to give a few further examples which are not limiting, the combination of claims 14, 13, 9 and 8 and the combination of claims 13, 9 and 8.

The foregoing description of the specific embodiments and examples reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “including” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Additionally, throughout the specification, “a group comprising” of a particular stated element, integer or step, or group of elements, integers or steps also envisages “a group consisting” of said stated element, integer or step, or group of elements, integers or steps without inclusion of any other element, integer or step, or group of elements, integers or steps.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.