A method for adaptive evolution of a micro-organism

Abstract

The present disclosure provides a method of adaptive evolution of a wild type micro-organism involved in converting a precursor of L-glufosinate thereof to L-glufosinate, esters, or salts thereof. The present disclosure also provides an adapted micro-organism which has improved bioconversion capability as compared to the wild type micro-organism. Also provided herein is a process for preparation of L-glufosinate, esters, or salts thereof by bioconversion of a precursor of L-glufosinate thereof in presence of said adapted micro-organism.

Description

[0001] “A METHOD FOR ADAPTIVE EVOLUTION OF A MICRO-ORGANISM”

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a method of adaptive evolution of a wild type micro-organism. More particularly, the present invention relates to a method of precursor based adaptive evolution of said micro-organism for improving the synthesis of a herbicide, in particular L- glufosinate, esters, or salts thereof. The present invention also relates to an adapted microorganism obtained by said method and its use in synthesis of said herbicide. The present invention also relates to a process for preparation of said herbicide or a process for preparation of a composition comprising said herbicide produced from a precursor of said herbicide in presence of said adapted micro-organism.

[0004] BACKGROUND OF THE INVENTION:

[0005] Glufosinate is a non-selective herbicide belonging to the group of organophosphate and has been widely used around the world. It is generally used in the form of ammonium salt for total vegetation control and to control growth of weeds and grasses. It is considered to be one of the safest herbicides from a toxicological or environmental standpoint. Glufosinate is used as a racemic mixture of L- & D- glufosinate. However, it is well known that L-glufosinate i.e. (S)- 2-amino-4- (hydroxy(methyl)phosphonoyl)butanoic acid is much more potent than D- glufosinate. The L-isomer of glufosinate is a structural analogue of glutamate and, therefore, is a competitive inhibitor of the enzyme glutamine synthetase (GS) of bacteria and plants. The L-enantiomer of glufosinate acts by inhibition of glutamine synthetase thereby causing accumulation of toxic levels of ammonium ion and indirectly stopping photosynthesis.

[0006] 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is an essential precursor keto acid for the biosynthesis of herbicide L-glufosinate. Conversion of PPO to L-glufosinate is done using transamination reaction. The transamination reaction is an equilibrium reaction, which means that under certain conditions, some PPO will remain when the reaction is at equilibrium. The PPO so remaining represents a yield loss of L-glufosinate, and thus it is desirable to minimize the amount of PPO remaining in the reaction mixture, thereby increasing the yield of L-glufosinate.

[0007] Mainly there are three methods known in prior art for preparing optically pure L- glufosinate, namely by asymmetric chemical synthesis, by chiral separation and by biocatalytic method. Out of the three known methods, biocatalytic synthesis method is considered to be the perfect “green” technique. This method has many advantages such as mild reaction conditions, low toxicity, high stereoselectivity, and the production of eco-friendly waste; and is suitable for industrial scale production of optically pure L-glufosinate. The biocatalytic synthesis method involves use of isolated enzymes or whole cells (such as bacteria, fungi, microalgae and plants) as catalysts in organic reactions. However, wild- type of micro-organisms are capable of converting less than 50% of L-glufosinate precursor (PPO) to L-glufosinate. Thus, alternative approaches are of increasing interest. To improve the desired performance of microbial cells, different approaches exist, including approaches involving genetic modification. However, several objections have been raised regarding genetically modified cells and their uses, including in terms of safety and impact on other organisms, including humans.

[0008] Adaptive laboratory evolution (ALE) or adaptive evolution refers to the culture of cells or organisms under defined conditions leading to adaptive changes that accumulate in populations of cells or (microbial) organisms during selection under specified growth conditions. ALE is a useful experimental methodology for fundamental scientific research and industrial applications to create microbial cell factories. By using ALE, cells are adapted to the environment that researchers set based on their objectives through the serial transfer of cell populations in batch cultivations or continuous cultures and the fitness of the cells (i.e., cell growth) under such an environment increases. Then, omics analyses of the evolved mutants, including genome sequencing, transcriptome, proteome and metabolome analyses, are performed. (Refer: Takashi Hirasawa, and Tomoya Maeda. (2023). Adaptive Laboratory Evolution of Micro-organisms: Methodology and Application for Bioproduction. Microorganisms.; 11(1): 92).

[0009] Temporary adaptation is realized by modulating the expression of genes related to phenotypic alterations (Lopez-Maury et al., 2008), while adaptive evolution involves selection of mutations advantageous for survival in a particular new environment and represents permanent alteration in response to the changed environment.

[0010] For the bacterial adaptive evolution, there are several processes and mechanism reported e.g.: horizontal gene transfer, plasmids and phages as vectors for host evolution, coevolution of bacteria in the same community, gene duplication and amplification (GDA), insertion sequences (IS), specific gene mutations, bacterial parallel evolution etc. Horizontal gene transfer process involves gene transfer between different species or different organisms. Plasmids and phages as vectors for host evolution process is based on evolution- related genes transferred by plasmids or phages. In Gene duplication and amplification (GDA) process, bacteria is adapted to various environments by increasing the copy number of specific genes — can be flanked by insertion sequences (ISs). Insertion sequences process for host genome evolution works by mobilizing specific genes within or among different microorganisms yielding desired characteristics. In specific gene mutation process, adaptive evolution results from gain of function mutations in specific gene(s) that allow adaptation to a specific environment. In bacterial parallel evolution process, the same mutation arises in all independent selections for the desired phenotype. Coevolution of bacteria in the same community refers to the process, where bacteria that share the same environment can collectively engage in adaptive evolution in a way that mutually benefits the community as a whole.

[0011] Adaptive laboratory evolution (ALE) or adaptive evolution can be categorized into three types of long-term culture methods; serial transfer, colony transfer, and continuous culture experiments respectively.

[0012] Serial transfer methods are based on transferring an aliquot of the culture to a fresh medium at regular intervals for additional rounds of growth. Serial transfer-based ALE can also be applied to microbial co-cultures. The method is easy to automate and conduct high-throughput experiments. Disadvantage is that growth is inherently discontinuous, and control of the growth conditions is often limited and temporal.

[0013] Colony transfer method is often used when the serial transfer of liquid culture is not applicable for ALE. Representative examples of colony transfer include the analysis of mutation accumulation (MA) used for the analysis of mutation rate and identification of the molecular spectrum of spontaneous mutations. The disadvantage of the method includes usually low- throughput and limitation to automation.

[0014] Continuous culture- The advantage of chemostat culturing using ajar fermenter is that it can control constant growth rates, population densities, nutrient supply, and environmental conditions, such as pH and oxygen concentration. However, a disadvantage of the chemostat methodology is that it is difficult to maintain multiple replicates in parallel owing to the costs of operation.

[0015] Surprisingly, the inventors of the present invention have found that using “coevolution of bacteria in same community” as the ALE process and “serial transfer” as the ALE method for adaptive evolution of micro-organsim, it resulted in an unexpected enhancement in the % conversion of a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof in a bioconversion process in comparison to the % conversion resulted using wild type microorganism.

[0016] Therefore, the inventors of the present invention have developed a precursor-based method of adaptive evolution of a wild type micro-organism for improving the yield of L-glufosinate, esters, or salts thereof, thereby gaining all the advantages of biocatalytic synthesis and still avoiding the aforementioned hindrances of other processes used for preparing L-glufosinate.

[0017] OBJECTIVES OF THE INVENTION:

[0018] A primary objective of the present invention is to provide a method of adaptive evolution of a wild type micro-organism involved in converting a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof. Another objective of the present invention is to provide a method of adaptive evolution of a wild type micro-organism of class Actinomycetes involved in converting a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof.

[0019] Another objective of the present invention is to provide a method of precursor of L-glufosinate based adaptive evolution of said micro-organism for bioconversion of said precursor to L- glufosinate, esters or salts thereof.

[0020] An objective of the present invention is to provide an adapted micro-organism prepared using said method. It is an objective of the present invention to provide an adapted micro-organism which has improved bioconversion capability as compared to the wild type micro-organism.

[0021] Yet another objective of the present invention is to provide a bioconversion process using said adapted micro-organism for preparing L-glufosinate, esters, or salts thereof, having high yield and purity. Another objective is to achieve maximum bioconversion of said precursor to L- glufosinate, esters, or salts thereof using the adapted micro-organism. An objective of the present invention is to provide a green process for obtaining L-glufosinate, esters, or salts thereof.

[0022] Another objective of the present invention is to provide a simple, inexpensive and efficient bioconversion process for obtaining L-glufosinate, esters, or salts thereof.

[0023] Yet another objective of the present invention is to provide an environment-friendly process for obtaining L-glufosinate, esters, or salts thereof.

[0024] An objective of the present invention is to provide a process for preparation of L-glufosinate, esters, or salts thereof by bioconversion of a precursor of L-glufosinate in presence of said adapted micro-organism. It is an objective of the present invention to improve the yield of L- glufosinate, esters or salts thereof in the bioconversion process.

[0025] Yet another objective of the present invention is to provide a process of preparation of L- glufosinate, esters, or salts thereof, wherein the resulting bioconversion using the adapted micro-organism is enhanced in comparison to the bioconversion resulting due to the wild type micro-organism.

[0026] An objective of the present invention is to provide an agrochemical composition comprising L-glufosinate, esters, or salts thereof, wherein L-glufosinate is prepared from precursor of L- glufosinate through bioconversion using said adapted micro-organism.

[0027] SUMMARY OF THE INVENTION:

[0028] An aspect of the present invention provides a method of adaptive evolution of a wild type micro-organism involved in converting a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof. In an embodiment, said micro-organism belongs to class Actinomycetes. In an embodiment, said method is a precursor based adaptive evolution of said micro-organism. In another embodiment, said precursor is a L-glufosinate precursor. In a further embodiment, said precursor is (2- oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid).

[0029] In an aspect, there is provided an adapted micro-organism obtainable by the method of adaptive evolution. In an embodiment, said method is a precursor based adaptive evolution of wild type micro-organism. In a further embodiment, said adapted micro-organism is capable of bioconversion of said precursor to L-glufosinate, esters or salts thereof. In an embodiment, said adapted micro-organism is capable of converting >= 50% of the precursor of L-glufosinate to L-glufosinate, salts or esters thereof. In an embodiment, said adapted micro-organism is capable of converting >= 90% of the precursor of L-glufosinate to L-glufosinate, salts or esters thereof.

[0030] An aspect of the present invention provides an adapted micro-organism selected from Amycolatopsis rifamycinica (MTCC No. 15) or Streptomyces griseus (MTCC No. 4734), wherein said adapted micro-organism is involved in bioconversion of a precursor of L- glufosinate to L-glufosinate, esters, or salts thereof, and wherein said adapted micro-organism is capable of converting at least 50% of the precursor of L-glufosinate to L-glufosinate, esters, or salts thereof and / or assimilating at least 30% of the precursor of L-glufosinate.

[0031] An aspect of the present invention provides a process of bioconversion of precursor of L- glufosinate to L-glufosinate, esters or salts thereof using said adapted micro-organism. In an embodiment, said process is capable of converting >=50 % (2-oxo-4-

[0032] [(hydroxy)(methyl)phosphinoyl]butyric acid) to L-glufosinate, esters or salts thereof.

[0033] In a further embodiment, said bioconversion process when done using the adapted microorganism is improvised in comparison to the bioconversion process done using wild type micro-organism.

[0034] An aspect of the present invention provides L-glufosinate, esters or salts thereof obtainable by process of bioconversion of L-glufosinate, esters, or salts thereof precursor to L-glufosinate using said adapted micro-organism. In an embodiment, the adapted micro-organism of the present invention is obtainable by the method of adapted evolution embodied in the present invention.

[0035] An aspect of the present invention also provides a composition comprising L-glufosinate, esters or salts thereof, wherein L-glufosinate, esters or salts thereof are obtained by a bioconversion process comprising converting a precursor of L-glufosinate to L-glufosinate, esters or salts thereof using the adapted micro-organism of the present invention. In an embodiment, the adapted micro-organism of the present invention is obtainable by the method of adapted evolution embodied in the present invention. In an embodiment, said composition further comprises at least one agrochemically acceptable excipient.

[0036] DETAILED DESCRIPTION OF THE INVENTION:

[0037] Those skilled in art will be aware that invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and methods referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more said steps or features.

[0038] For convenience, before providing further description of the present invention, certain terms employed in the specification, examples are described here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. The terms used herein are defined as follows.

[0039] As used in the specification and the claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only.

[0040] The term “about” shall be interpreted to mean “approximately” or “reasonably close to” and any statistically insignificant variations therefrom. “About” or “approximately” as used herein is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ±10% or ±5% of the stated value. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0041] As used herein, the terms “comprising”, “including”, “having”, “containing”, “involving”, and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0042] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. In an embodiment, the aspects and embodiments described herein shall also be interpreted to replace the clause “comprising” with either “consisting of’ or with “consisting essentially of’ or with “consisting substantially of’.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0044] The term “Glufosinate” refers to an isomeric mixture of L-glufosinate and D-glufosinate.

[0045] “L-glufosinate” is also known as L-phosphinothricin or (S)-2-amino-4- (hydroxy(methyl)phosphoryl)butanoic acid. The term can generically refer to any form of L- glufosinate such as solvates, hydrates, anhydrous form, polymorph forms, pseudo polymorph forms, amorphous form or mixture thereof, and derivatives such as esters; and salts. The term may also refer to racemic glufosinate, wherein the content of L-glufosinate is 70% or greater, preferably 80% or greater, and more preferably 90% or greater. Typically, the ratio of L- glufosinate: D-glufosinate can be in the range from about 90: 10 to about 100:0.

[0046] The term "Precursor" or "Substrate" refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. According to the present invention, said precursor is a precursor of L-glufosinate. Particularly, said precursor is 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO). All the referred terms are used interchangeably throughout the description. The term “% Enantiomeric excess” or “% ee” means the enantiomeric purity of a sample, that is, the percentage of one enantiomer that exceeds the other enantiomer in the sample. For example, the enantiomeric excess of L-glufosinate is the percentage of L-glufosinate that exceeds D-glufosinate in the glufosinate.

[0047] As used herein, “Biocatalyst” refers to a whole cell, particularly, a micro-organism, comprising a natural catalyst or one or more enzymes to perform chemical transformations on organic compounds; or isolated enzymes, partially purified enzymes, cell-free extracts or crude cell extract liquid / powder / immobilized or fixed form, permeabilized cells, whole cells, whole fermentation broths, lyophilized cells, from such micro-organisms.

[0048] The term “Micro-organism” as used herein refers to microscopic biological entities that are typically unicellular and capable of independent life or replication. Terms like “microbe” or “microbial cell” or “microbial strain” or “strain” or “micro-organism” can be used interchangeably throughout the document. Microorganisms include, but are not limited to, bacteria, viruses, yeasts, fungi, protozoa, algae, hybridomas, fused plant cells, animal cells or tissues, stem cells, and tumor cells. These entities may occur naturally or may be genetically modified or engineered through recombinant DNA technology, cell fusion, or other biotechnological methods.

[0049] The term “Wild type micro-organism” as used herein refers to a micro-organism that occurs in nature, i.e. a micro-organism that has not been genetically modified.

[0050] The term “Adaptive evolution” as used herein refers to a method during which a population of living cells is exposed to a selective diet promoting the acquisition of a phenotype of interest through the accumulation of advantageous physiological modifications. Physiological changes can occur as a result of genetic modification (point mutation, loss or acquisition of genetic material) or epigenetic modification and can result from stress or any other factor that can have a lasting impact on the behavior of living cells in culture. The purpose of the said method is to promote the emergence of living cells having acquired a phenotype of interest, without prior knowledge of the genetic and physiological modifications leading to said phenotype, then to collect the living cells having acquired a phenotype of interest, said phenotype giving them a competitive advantage over other cells, such as in particular to survive stress, to grow under given conditions, possibly to grow more rapidly under given conditions, to make better use of the culture medium, or any another characteristic satisfying industrial criteria.

[0051] The term “Precursor-based” refers to a method of adaptive evolution carried out using a precursor of the desired end product. Said method comprises exposing the biocatalytic microorganism to a precursor-induced growth medium to increase the capability of the microorganism of assimilation and bioconversion of said precursor to its end product.

[0052] The term “Adapted micro-organism” refers to micro-organism which is being produced using the method of adaptive evolution.

[0053] The term 'Actinomycetes'' as used herein refers to the class of bacteria which belongs to phylum: Actinomycetota, order: Pseudonocardiales or Kitasatosporales and family: Pseudonocardiaceae or Treptomycetaceae Particularly, as per the present invention, said class refers to bacteria of genus Amycolatopsis or Streptomyces.

[0054] The term “Culture medium” also called growth medium refers to the chemical environment for the cultivation and proliferation of microbes.

[0055] The term "Serial propagation" or "Propagation" refers to a method of iterative cultivation wherein a microorganism, such as a bacterium or virus, is grown in a defined environment for a predetermined period, followed by the transfer of a portion of the grown culture into a fresh medium or host environment. This process is repeated across multiple cycles, thereby enabling the microorganism to adapt, mutate, or evolve under controlled conditions. In an embodiment, said serial propagation comprises inoculating the culture medium with the wild-type Actinomycetes, wherein the culture medium comprises increasing concentrations of precursor of L-glufosinate for adaptive evolution of said Actinomycetes. The increase in concentration of said precursor is carried out in subsequent culturing cycles of serial propagation.

[0056] The term “% Assimilation” as used herein refers to the percentage of precursor consumed by biocatalyst in relation to the total precursor added at the start of reaction. In an embodiment, % assimilation of precursor refers to % PPO assimilation or % 2- oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid assimilation. The term “bioconversion” refers to a biologically mediated process wherein a precursor (substrate) is enzymatically or metabolically transformed into a structurally distinct end product through the activity of living organisms, such as microorganisms, plant cells, or isolated enzymes. It is measured as % bioconversion which refers to the quantitative measure of the efficiency with which a precursor or substrate is converted into a desired end product. In an embodiment, % L-Glufosinate (L-GF) conversion refers to % of 2- oxo-4-(hydroxy (methyl) phosphinoyl) converted into L-Glufosinate (L-GF).

[0057] The term “Yield” as used herein refers to the amount of product obtained per unit weight of raw material or precursor and may be expressed as gram product per gram precursor (g / g).

[0058] The term “Productivity” or “Volumetric productivity” or “Production rate” as used herein refers to the amount of product formed per volume of medium per unit of time. It may be expressed in gram per litre per hour (g / L / h).

[0059] An embodiment of the present invention provides a method of adaptive evolution of a wild type micro-organism.

[0060] In an embodiment, present invention provides a method of adaptive evolution of a wild type micro-organism involved in converting a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof.

[0061] In another embodiment, the present invention provides a precursor-based method for adaptive evolution of said micro-organism.

[0062] In a preferred embodiment of the present invention, said method comprises propagating the wild type micro-organism with different concentrations of the precursor of L-glufosinate in the culture medium for adaptative evolution of said micro-organism.

[0063] In another preferred embodiment, said method comprises (a) culturing said micro-organism in a suitable culture medium; and (b) serially propagating the cultured micro-organism prepared in step (a) in a suitable culture medium; wherein, the serial propagation comprises inoculating the culture medium using different concentrations of precursor of L-glufosinate for adaptive evolution of the micro-organism. In an embodiment, serial propagation of a micro-organism or serially propagating a microorganism refers to a process characterised by i) inoculating a first culture medium with a selected micro-organism under sterile conditions and incubating the inoculated culture medium under conditions suitable for growth of the micro-organism for a predetermined period; ii) initiating a first propagation cycle by transferring a portion of the grown culture to a fresh culture medium and incubating the inoculated culture medium under conditions suitable for growth of the culture; iii) repeating the transfer and incubation steps for one or more additional cycles to achieve serial propagation.

[0064] In another embodiment, the serial propagation comprises inoculating the culture medium with the wild type micro-organism wherein the culture medium comprises increasing concentrations of precursor of L-glufosinate for adaptive evolution of the wild type micro-organism. Said increase in concentrations of said precursor is carried out in subsequent culturing in a given cycle of serial propagation.

[0065] Therefore, in an embodiment, the present invention provides a method of adaptive evolution of a wild-type micro-organism involved in converting a precursor of L-glufosinate to L- glufosinate, esters, or salts thereof, said method comprising i) inoculating a first culture medium with said wild type micro-organism and incubating the inoculated culture medium under conditions suitable for growth of the microorganism; ii) initiating a first propagation cycle by transferring a portion of the grown culture from step i) to a fresh culture medium comprising the precursor of L-glufosinate and incubating the inoculated culture medium under conditions suitable for adaptation of the micro-organism to the precursor; iii) repeating the transfer and incubation step ii for one or more additional cycles to obtain adapted micro-organism; wherein the culture medium of every additional cycle comprises the precursor in an amount incremental to the previous cycle. In a preferred embodiment, said precursor is (2- oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid).

[0066] In an embodiment, said method is a precursor-based method for adaptive evolution of said micro-organism and wherein said precursor is (2- oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid).

[0067] In an embodiment, amount of a precursor of L-glufosinate used in the culture medium for adaptive evolution of the wild type micro-organism ranges from about 0.1% w / w to about 15% w / w. In an embodiment, amount of the precursor ranges from about 0.25% w / w to about 12% w / w. In an embodiment, amount of the precursor ranges from about 0.5% w / w to about 9% w / w. In an embodiment, amount of the precursor ranges from about 0.75% w / w to about 6% w / w. In an embodiment, amount of the precursor ranges from about 1% w / w to about 3% w / w. In an embodiment, amount of the precursor ranges from about 1.25% w / w to about 2.25% w / w. In an embodiment, amount of the precursor ranges from about 1.5% w / w to about 2% w / w.

[0068] In a preferred embodiment, amount of the precursor ranges from about 0.1% w / w to about 5% w / w.

[0069] In a preferred embodiment, amount of the precursor ranges from about 0.1% w / w to about 2.5% w / w.

[0070] In an embodiment, the amount of the precursor in the first propagation cycle ranges from about 0.1% w / w to about 15% w / w, preferably about 0.1% w / w to about 10% w / w, more preferably about 0.1% w / w to about 5% w / w, even more preferably 0.1% w / w to about 2.5% w / w.

[0071] In an embodiment, the amount of the precursor in the one or more additional cycles is increased as compared to the previous cycle. Therefore, the amount of the precursor in the one or more additional cycles is incremental to the amount of the precursor in previous cycle. For example, the amount of the precursor in second propagation cycle is more than the amount of the precursor in the first propagation cycle, the amount of the precursor in third propagation cycle is more than the amount of the precursor in the second propagation cycle, and so on.

[0072] In an embodiment, the amount of the precursor in the one or more additional cycles ranges from about 0.1% w / w to about 15% w / w, preferably about 0.1% w / w to about 10% w / w, more preferably about 0.1% w / w to about 5% w / w, even more preferably about 0.1% w / w to about 2.5% w / w.

[0073] In a preferred embodiment of the present invention, said propagation cycles are performed three times. In each successive propagation cycle, the amount of said precursor used is increased by a factor of approximately 2.5 relative to the amount used in the immediately preceding propagation cycle.

[0074] In a preferred embodiment, the amount of said precursor in the first propagation cycle of serial propagation is 0.1%w / w, in the second propagation cycle of serial propagation is 0.25%w / w and in the third propagation cycle of serial propagation is 0.5%w / w.

[0075] In an embodiment, the wild type micro-organism used for adaptive evolution comprises the micro-organism of class Actinomycetes.

[0076] In an embodiment, the micro-organism of class Actinomycetes is selected from the group consisting of order Pseudonocardiales, order Kitasatosporales, and combinations thereof

[0077] In an embodiment, the micro-organism of class Actinomycetes is selected from the group consisting of family Pseudonocardiaceae, family Streptomycetaceae, and combinations thereof

[0078] In a preferred embodiment, class micro-organism is selected from the genus Amycolatopsis or Streptomyces.

[0079] In a yet another preferred embodiment, said wild type micro-organism is of Amycolatopsis species.

[0080] In a yet another preferred embodiment, said wild type micro-organism is of Streptomyces species.

[0081] In an embodiment, said method comprising the micro-organism of class Actinomycetes wherein said Actinomycetes class micro-organism is further selected from the genus or species comprising Amycolatopsis or Streptomyces. In a preferred embodiment, said micro-organism of Amycolatopsis species is Amycolatopsis rifamycinica.

[0082] In another preferred embodiment, said micro-organism of Streptomyces species is Streptomyces griseus.

[0083] In a preferred embodiment, said micro-organism of Amycolatopsis species is Amycolatopsis rifamycinica (M CC Slo. 15).

[0084] In another preferred embodiment, said micro-organism of Streptomyces species is Streptomyces griseus (MTCC No. 4734).

[0085] An embodiment of the present invention provides a culture medium which can be used for culturing and adaptive evolution of the micro-organism of class Actinomycetes, wherein said medium can be a complex medium, i.e. a medium that contains a mixture of components that are not defined and / or of which the quantities are not defined. Alternatively, the culture medium can be a defined medium, i.e. a medium of which all compounds and their quantities are known. Preferably, the culture medium is a defined medium.

[0086] In an embodiment, said micro-organism is grown and serially propagated in a culture medium including but not limited to International Streptomyces Project medium no. l (ISP medium no.l), International Streptomyces Project medium no.2 (ISP medium no.2), International Streptomyces Project medium no.3 (ISP medium no.3), International Streptomyces Project medium no.4 (ISP medium no.4), International Streptomyces Project medium no.5 (ISP medium no.5), International Streptomyces Project medium no.6 (ISP medium no.6), International Streptomyces Project medium no.7 (ISP medium no.7), tryptone soy broth (TSB), caso medium, glucose yeast extract peptone (GYEP), modified tryptone soya broth medium (MTBS) etc.

[0087] In an embodiment, the culture medium used for culture, propagation, and adaptive evolution of the wild type microorganism can be same culture medium or different culture medium. Preferably, the present invention provides the same culture medium for both cultivation, culture, propagation, growth, and adaptive evolution of said micro-organism. In an embodiment, the culture medium used for culture and adaptive evolution of Actinomycetes can be same culture medium or different culture medium. Preferably, the present invention provides the same culture medium for both cultivation and adaptive evolution of said micro-organism.

[0088] In a preferred embodiment, the suitable culture medium or the first culture medium is a modified tryptone soya broth medium (MTBS) medium.

[0089] In a preferred embodiment, the suitable culture medium or the fresh culture medium for serial propagation is a modified tryptone soya broth medium (MTBS) medium.

[0090] In yet another preferred embodiment, the culture medium is a tryptone soya broth medium modified with addition of glucose within said medium.

[0091] In another preferred embodiment, Actinomycete culture medium comprises the following components in parts by weight / volume: 15-25 parts of glucose (g / L), 3-4 parts of tryptone (g / L), 1.2- 1.7 parts of soy peptone (g / L) and 3-7 parts of sodium chloride (g / L).

[0092] In an embodiment, the precursor based adaptive evolution of said micro-organism according to the present invention is carried out at a temperature ranging from about 28°C to about 40°C, preferably at 37°C.

[0093] In an embodiment, the precursor based adaptive evolution of said micro-organism according to the present invention is carried out at a pH ranging from about 6 to 9, preferably at pH 7.2.

[0094] In an embodiment, the precursor based adaptive evolution of said micro-organism according to the present invention is carried out with an aeration ranging from about 0.4 vvm to about 1.6 vvm, preferably at 0.6 vvm.

[0095] In an embodiment, the present invention provides an adapted micro-organism obtainable by the method of adaptive evolution embodied in the present invention In an embodiment, said method is a precursor based adaptive evolution of wild type microorganism. In a further embodiment, said adapted micro-organism is capable of bioconversion of precursor of L-glufosinate, salts or esters thereof to L-glufosinate, esters or salts thereof.

[0096] In an embodiment, said adapted micro-organism is capable of converting atleast 50% of precursor of L-glufosinate, salts or esters thereof to L-glufosinate, salts or esters thereof. In an embodiment, said adapted micro-organism is capable of converting atleast 60% of said precursor to L-glufosinate, salts or esters thereof. In an embodiment, said adapted microorganism is capable of converting atleast 70% of said precursor to L-glufosinate, salts or esters thereof. In an embodiment, said adapted micro-organism is capable of converting atleast 80% of said precursor to L-glufosinate, salts or esters thereof. In an embodiment, said adapted micro-organism is capable of converting atleast 90% of said precursor to L-glufosinate, salts or esters thereof. In an embodiment, said adapted micro-organism is capable of converting atleast 95% of said precursor to L-glufosinate, salts or esters thereof.

[0097] In an embodiment, the % assimilation of precursor is at least 30%. In an embodiment, the % assimilation of precursor is at least 50%. In an embodiment, the % assimilation of precursor is at least 60%. In an embodiment, the % assimilation of precursor is at least 70%. In an embodiment, the % assimilation of precursor is at least 75%. In an embodiment, the % assimilation of precursor is at least 80%. In an embodiment, the % assimilation of precursor is at least 85%. In an embodiment, the % assimilation of precursor is at least 90%. In an embodiment, the % assimilation of precursor is at least 92%. In an embodiment, the % assimilation of precursor is at least 95%.

[0098] In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 30%. In an embodiment, the % assimilation of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid is at least 50%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 60%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 70%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 75%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 80%. In an embodiment, the % assimilation of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid is at least 85%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 90%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 92%. In an embodiment, the % assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 95%.

[0099] In a preferred embodiment, the present invention provides an adapted micro-organism of class Actinomycetes. wherein said micro-organism is prepared using 2-oxo-4- (hydroxymethylphosphinyl)butyric acid based adaptive evolution of a wild type Actinomycetes.

[0100] In another preferred embodiment, the present invention provides an adapted micro-organism selected from Amycolatopsis or Streptomyces species, wherein said micro-organism is prepared using 2-oxo-4-(hydroxymethylphosphinyl)butyric acid based adaptive evolution of a wild type a micro-organism of said species.

[0101] In an embodiment, the present invention provides an adapted micro-organism selected from Amycolatopsis rifamycinica (MTCC No. 15) or Streptomyces griseus (MTCC No. 4734), wherein said adapted micro-organism is involved in bioconversion of a precursor of L- glufosinate to L-glufosinate, esters, or salts thereof, and wherein said adapted micro-organism is capable of converting at least 50% of the precursor of L-glufosinate to L-glufosinate, esters, or salts thereof and / or assimilating at least 30% of the precursor of L-glufosinate.

[0102] In an embodiment, the present invention provides a process of bioconversion of precursor of L-glufosinate to L-glufosinate, esters or salts thereof using said adapted micro-organism.

[0103] In a preferred embodiment, the present invention provides a process of bioconversion of 2-oxo- 4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters or salts thereof using said adapted micro-organism, wherein said adapted micro-organism belongs to class Actinomycetes.

[0104] In a preferred embodiment, said adapted micro-organism belongs to Amycolatopsis or Streptomyces species. In an embodiment, the present invention provides a process for bioconversion of a precursor of L-glufosinate, esters or salts thereof, to L-glufosinate, esters or salts thereof in the presence of an adapted micro-organism obtained using the process of adaptive evolution of the wild type microorganism as embodied in the present invention. In an embodiment, said process for bioconversion comprises inoculating a bioconversion medium comprising the precursor of L- glufosinate, esters or salts thereof with the adapted micro-organism of the present invention to obtain L-glufosinate, esters or salts thereof. In an embodiment, the amount of L-glufosinate, esters or salts thereof obtained in the bioconversion process using the adapted micro-organism is enhanced in comparison to the amount of L-glufosinate, esters or salts thereof obtained in the bioconversion process using the wild type micro-organism. In an embodiment, the amount of L-glufosinate, esters or salts thereof obtained from a bioconversion process using the adapted micro-organism is atleast 10% or atleast 20 % or atleast 50% more than the amount of L-glufosinate, esters or salts thereof obtained from a bioconversion process using the wild type micro-organism.

[0105] In an embodiment, the present invention provides a process for bioconversion of precursor of L-glufosinate, esters or salts thereof, to L-glufosinate, esters or salts thereof, using microorganism obtained from the method of adaptive evolution of wild- type of micro-organism as embodied in the present invention, wherein said process comprising:

[0106] (i) culturing wild type micro-organism in a suitable culture medium;

[0107] (ii) inoculating and serially propagating the cultured micro-organism cells obtained in step (i) into suitable culture medium in the presence of different concentration of precursor of L-glufosinate to adapt the micro-organism;

[0108] (iii) cultivating adapted microbial cells obtained in step (ii) into culture medium; and

[0109] (iv) carrying out bioconversion of said precursor to L-glufosinate, esters or salts thereof, using adapted cells obtained in step (iii) into bioconversion medium.

[0110] In an embodiment, the present invention provides a process of bioconversion of a precursor of L-glufosinate to L-glufosinate, esters or salts thereof comprising:

[0111] (i) culturing a wild type micro-organism capable of converting a precursor of L- glufosinate to L-glufosinate, esters or salts thereof in a suitable culture medium;

[0112] (ii) inoculating and serially propagating the wild type micro-organism cultured in step (i) into a suitable culture medium wherein, the serial propagation comprises inoculating the wild type micro-organism in a suitable culture medium comprising different concentrations of said precursor of L-glufosinate for adaptive evolution of the wildtype micro-organism ;

[0113] (iii) obtaining the adapted micro-organism from step ii) and propagating the said adapted micro-organism in a fresh culture medium; and

[0114] (iv) inoculating a bioconversion medium comprising the precursor of L-glufosinate, esters or salts thereof with the adapted micro-organism to obtain L-glufosinate, esters or salts thereof, wherein the amount of L-glufosinate, esters or salts thereof obtained in the bioconversion process using the adapted micro-organism is enhanced in comparison to the amount of L-glufosinate, esters or salts thereof obtained in the bioconversion process using the wild type micro-organism.

[0115] In an embodiment, bioconversion medium used in step (iv) comprising addition of enzyme cofactor and amine donor.

[0116] In an embodiment, said enzyme cofactor is PLP (Pyridoxal 5 phosphate).

[0117] In an embodiment, said amine donor is L-Glutamic acid.

[0118] In an embodiment, amount of enzyme cofactor used in the bioconversion medium ranges from about 0.0005 % w / w to 0.05 % w / w. In an embodiment, the amount of enzyme cofactor ranges from about 0.0010 % w / w to 0.01 % w / w. In a preferred embodiment, the amount of enzyme cofactor ranges from about 0.0012 % w / w to 0.025 % w / w.

[0119] In an embodiment, amount of amine donor used in the bioconversion medium ranges from about 1 % w / w to about 70 % w / w. In an embodiment, the amount of amine donor ranges from about 2 % w / w to about 65 % w / w. In a preferred embodiment, the amount of amine donor ranges from about 5 % w / w to about 50 % w / w.

[0120] In an embodiment, said process of bioconversion is carried out at temperature range of about 28°C to about 55 °C, preferably at 40°C. In an embodiment, said process of bioconversion is carried out at a pH ranging from about 6 to 9, preferably at pH 8.

[0121] The inventors of present invention found that the adapted micro-organism used remain catalytically competent for several days and can be reused or recycled multiple times to catalyse the bioconversion of precursor of L-glufosinate to L-glufosinate, esters, or salts thereof.

[0122] In an embodiment, adapted micro-organism used for bioconversion process as described herein can be reused or recycled for about 5 cycles of 5 days each.

[0123] In an embodiment, said bioconversion process is a biocatalytic process.

[0124] In an embodiment, the adapted micro-organism obtainable by adaptive evolution method of present invention may produce multiple enzymes, preferably one or more enzymes which acts as biocatalyst for the bioconversion process. In another embodiment, the adapted microorganism produces at least one enzyme capable of catalysing the conversion of a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof.

[0125] In another embodiment, one or more enzymes produced by adapted micro-organism is / are selected from aminotransferase and / or amino-acid dehydrogenase and / or aldo-keto reductases, or combinations thereof.

[0126] According to an embodiment, the biocatalyst is selected from, but not limited to, isolated enzymes; partially purified enzymes; cell-free extract or crude cell extract in liquid, powder, or immobilized / fixed form; permeabilized cells, whole cells, whole fermentation broths, lyophilized cells, or combinations thereof.

[0127] In a preferred embodiment, the biocatalyst comprises whole cells, whole fermentation broths, permeabilized cells, or lyophilized cells. In a more preferred embodiment, the biocatalyst comprises whole cells or whole fermentation broths.

[0128] In an embodiment, the amount of adapted micro-organism in terms of wet cell weight is in a range from about 20 g / L to about 300 g / L. In an embodiment, the amount of adapted micro- organism in terms of wet cell weight is in a range from about 50 g / L to about 250 g / L. In an embodiment, the amount of adapted micro-organism in terms of wet cell weight is in a range from about 80 g / L to about 200 g / L. In an embodiment, the amount of adapted micro-organism in terms of wet cell weight is in a range from about 110 g / L to about 150 g / L.

[0129] In a preferred embodiment, the amount of adapted micro-organism in terms of wet cell weight is 200 g / L.

[0130] In an embodiment, the enantiomeric excess of the L-glufosinate, esters, or salts thereof obtained, having L-form may be, for example, 10% ee or more, 20% ee or more, 30% ee or more, 40% ee or more, 50% ee or more, 60% ee or more, 70% ee or more, 80% ee or more, 90% ee or more, 91% ee or more, 92% ee or more, 93% ee or more, 94% ee or more, 95% ee or more, 96% ee or more, 97% ee or more, 98% ee or more, or 99% ee or more or 99.2% ee or more or 99.4% ee or more or 99.6% ee or more.

[0131] In an embodiment, the present invention provides a bioconversion process of converting precursor of L-glufosinate to L-glufosinate, esters, or salts thereof wherein the resulted bioconversion using the adapted micro-organism is enhanced in comparison to the bioconversion resulted due to wild type micro-organism.

[0132] An embodiment of the present invention provides a bioconversion process of converting more than 50% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 55% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 60% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 65% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 70% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 75% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 80% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 85% of precursor to L-glufosinate, esters, or salts thereof. In an embodiment, the process is capable of converting more than 90% of precursor to L-glufosinate, esters, or salts thereof.

[0133] An embodiment of the present invention provides a bioconversion process of converting more than 50% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 55% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 60% of 2-oxo- 4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 65% of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 70% of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 75% of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 80% of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the bioconversion process is capable of converting more than 85% of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof. In an embodiment, the process is capable of converting more than 90% of 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof.

[0134] An embodiment of the present invention provides enhanced productivity of L-glufosinate, esters, or salts thereof synthesis using its precursor adapted micro-organism in comparison to the productivity resulted due to L-glufosinate, esters, or salts thereof synthesis using wild type micro-organism.

[0135] In an embodiment, productivity of L-glufosinate, esters, or salts thereof synthesis ranges from about 2g / L / day to about 50g / L / day. In an embodiment, the productivity of L-glufosinate, esters, or salts thereof synthesis ranges from about 4g / L / day to about 40g / L / day. In an embodiment, the productivity of L-glufosinate, esters, or salts thereof synthesis ranges from about 5g / L / day to about 30g / L / day. In an embodiment, the productivity of L-glufosinate, esters, or salts thereof synthesis ranges from about 6g / L / day to about 20g / L / day. In a preferred embodiment, the productivity of L-glufosinate, esters, or salts thereof synthesis ranges from about 8g / L / day to about 12g / L / day.

[0136] In an embodiment, the present invention provides L-glufosinate, esters or salts thereof obtainable by process of bioconversion of precursor of L-glufosinate to L-glufosinate, esters or salts thereof using the adapted micro-organism of the present invention. In an embodiment, the adapted micro-organism of the present invention is obtainable by the method of adapted evolution embodied in the present invention.

[0137] In an embodiment, the present invention provides L-glufosinate, esters or salts thereof obtainable by process of bioconversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters or salts thereof using the adapted micro-organism of class Actinomycetes.

[0138] In a preferred embodiment, said adapted micro-organism belongs to Amycolatopsis or Streptomyces genus.

[0139] An embodiment of the present invention provides a composition comprising L-glufosinate, esters or salts thereof, wherein L-glufosinate, esters or salts thereof are obtained by a bioconversion process comprising converting a precursor of L-glufosinate, salts or esters thereof to L-glufosinate, esters or salts thereof using the adapted micro-organism of the present invention.

[0140] In a preferred embodiment, the present invention provides a composition comprising L- glufosinate, esters or salts thereof, wherein L-glufosinate, esters or salts thereof are obtained by a bioconversion process comprising converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters or salts thereof using the adapted micro-organism of class Actinomycetes.

[0141] In another embodiment, the composition further comprises at least one agrochemically acceptable excipient.

[0142] According to an embodiment of present invention, agrochemically acceptable excipients includes but not limited to diluents, adjuvants, co-solvents, surfactants, colorants, dispersants, emulsifiers, thickeners, antifreeze agents, biocides, anti-foam agents, stabilizers, wetting agents, chelating agents, buffering agents or a mixture thereof. However, those skilled in the art will appreciate that it is possible to utilize additional agrochemically acceptable excipients without departing from the scope of the present invention.

[0143] In an embodiment, the present invention provides use of L-glufosinate, esters, or salts thereof; or an agrochemical composition comprising L-glufosinate, esters, or salts thereof, prepared according to the bioconversion process of the present invention for controlling unwanted plants or weeds.

[0144] Advantages of invention:

[0145] 1. The present invention provides a “green” technique to obtain L-glufosinate, esters, or salts thereof.

[0146] 2. The present invention provides a simple and efficient method to obtain L-glufosinate, esters, or salts thereof.

[0147] 3. The bio-catalytic process of the present invention efficiently converts 2-oxo-4- (hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof, at a high precursor concentration.

[0148] 4. The biocatalyst used in present invention can be reused or recycled multiple times without affecting the efficiency of the process.

[0149] 5. Adapted micro-organism of the present invention demonstrates enhanced conversation rate of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters, or salts thereof, as compared to wild type micro-organism.

[0150] 6. Adapted micro-organism of the present invention is tolerant to precursor and therefore allows for better assimilation.

[0151] 7. The present invention provides enantioselective synthesis of L-glufosinate using 2-oxo-4- (hydroxymethylphosphinyl)butyric acid achieved over bio-catalysis system.

[0152] 8. The present invention provides an adapted non-GMO micro-organism.

[0153] EXAMPLES:

[0154] The present invention is more specifically explained by below examples. However, it should be understood that the scope of the present invention is not limited by the examples in any manner. It will be appreciated by any person skilled in this art that the present invention includes below examples and further can be modified and altered within the technical scope of the present invention.

[0155] Example 1 : Adaptation of wild type Actinomycetes strain(s) with 0,5% PPO solution at laboratory scale

[0156] (i) Preparation of 5% PPO solution:

[0157] 14 g of (2-oxo-4-(hydroxymethylphosphinyl)butyric acid) PPO (72%) dissolved in 30 g of chilled distilled water and pH was adjusted to 8. The volume of the solution was adjusted to 100 mL using chilled distilled water, after which it was autoclaved. From this sterilized solution, concentrations of 0.1%, 0.25%, and 0.5% were prepared by adding necessary volumes directly into the fermenter used for adaptation of the strains.

[0158] (ii) Revival of Actinomycetes strain(s):

[0159] Wild type Amycolatopsis rifamycinica (MTCC No. 15) and Streptomyces griseus (MTCC No. 4734) procured from MTCC Chandigarh, India. These strains were revived from lyophilized stocks using MTSB medium and were used for adaptation and subsequent bioconversion reaction.

[0160] (iii) Adaptation of Actinomycetes strain(s) with PPO:

[0161] For adaptation of the culture, 10% primary inoculum of wild type Actinomycetes culture / biocatalyst was inoculated in sterile 100 ml MTSB (Modified Tryptic Soy Broth) with 0.1% sterile PPO. The culture was incubated in an orbital shaker at 30 °C for 48 h at 150 rpm. Further, 10% inoculum of 0.1% PPO adapted culture was inoculated in sterile MTSB containing 0.25% PPO and was incubated at 30 °C for 48 h in an orbital shaker at 150 rpm. Further, 10% of 0.25% adapted biocatalyst was inoculated in sterile MTSB containing 0.5% PPO and was incubated under the same conditions. The final 0.5% PPO adapted culture was harvested by centrifugation at 7000 rpm for 10 minutes and the obtained cell pellet was used to set up PPO to L-GF reaction. The final 0.5% PPO adapted culture was preserved in glycerol stocks at -80°C for further use.

[0162] (iv) Comparative analysis of biocatalysis: wild type vs. adapted Actinomycetes strains

[0163] In a baffled reaction vessel, was added 16.2g (1.08M) of L-Glutamic acid (in concentration of 162g / L), 0.2mM of pyridoxal 5’ phosphate and 5g of the above-described Actinomycetes / bio- catalyst (in concentration of 200g / L). To this reaction system, was added 14g (72% purity) (0.55mM) of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) (in concentration of 140 g / L). The reaction system was then diluted to 100 ml volume with water. The pH of the reaction system was adjusted to 8. The reaction was maintained at 40 °C with stirring speed at 300 rpm. Comparative analysis of wild type Actinomycetes strains and adapted Actinomycetes strains as % PPO assimilation and % L-GF conversion is shown in Table 1 below.

[0164] Table 1 : Comparative analysis: wild type vs. adapted Actinomycetes strain's reaction with 5% biocatalyst loading

[0165] From Table 1, it is evident that the adapted strains of Amycolatopsis rifamycinica and Streptomyces griseus outperforms both in terms of % PPO assimilation and % L-GF conversion when compared to the respective wild type Amycolatopsis rifamycinica and Streptomyces griseus strains.

[0166] Example 2: Biomass production of adapted Hctz / w ce strain(s) at fermenter scale

[0167] Semi-continuous batch production of adapted Actinomycetes strains of example 1, i.e. Amycolatopsis rifamycinica (MTCC No. 15) and Streptomyces griseus (MTCC No. 4734) was carried out at fermenter scale (4 L) using the process mentioned below. a.100 ml of 48 hours grown 0.5% PPO adapted Actinomycetes strain(s) / biocatalyst was inoculated into 1.3 L of sterile MTSB (0.5% PPO) and grown for 24 h at 30 C, 300 rpm and 2.5 L of air flow per minute. b. Fresh media with 0.5% PPO was then added to bring the volume up to 4 L and it was allowed to grow for another 24 h. c. Once the culture is grown, 3 L of fermentation broth was harvested from the fermenter, and Actinomycetes cells were separated using centrifuge at 7000 rpm for 10 mins. These harvested cells were used as biocatalyst for Example 3 reactions. d. Fresh 3L of sterile MTSB with 0.5% PPO was added back to the fermenter for further growth of 24h. In similar manner, semi -continuous fermentation batch was maintained for consecutive 8-10 cycles by repeating (c) and (d).

[0168] Example 3 : Bioconversion of PPO to L-GF using adapted Actinomycetes strains grown at fermenter scale

[0169] Adapted Actinomycetes cells of Example 2 which were grown in semi -continuous batch mode in the fermenter were centrifuged at 7000 rpm for 15 minutes and obtained biocatalyst was used for further reaction PPO to L-GF reaction was set up as described in example 1, step (iv). Comparative studies for wild-type Actinomycetes strains under similar conditions have been tabulated in Table 2.

[0170] Table 2: Comparative analysis of wild type vs adapted strain's reaction grown at fermenter scale

[0171] From Table 2, it is evident that the adapted strain of Amycolatopsis rifamycinica has shown significant % PPO assimilation and % L-GF conversion as compared to that of adapted Streptomyces griseus strain. Thus, 0.5 % PPO adapted strain of Amycolatopsis rifamycinica was further selected for cell recycle study.

[0172] Example 4: Bioconversion of PPO to L-GF with recycled adapted strain of Amycolatopsis rifamycinica

[0173] The stability and efficiency of whole cell biocatalyst utilized in biotransformation reaction were investigated through the implementation of cell recycling studies. A series of three consecutive recycles was carried out using the same cells and maintaining same reaction parameters as specified in Example 3. After completion of the first reaction cycle for 120 h, cells were harvested by performing centrifugation at 7000 rpm for 15 mins. The obtained cell pellet was then transferred to fresh reaction medium along with 15% fresh cells and allowed to react for next 120 h. The same protocol was used for the duration of three recycle runs. Sample was withdrawn for HPLC analysis to examine the substrate and product's assimilation and conversion pattern every 24h and shown in Table 3.

[0174] Table 3: Bioconversion of PPO to L-GF using adapted Actinomycetes strain (Amycolatopsi rifamycinica) grown at fermenter scale

[0175] From Table 3, it is evident that the recycled Amycolatopsis rifamycinica strain shows good % PPO assimilation and % L-GF conversion in repeated cycles.

Claims

We Claim:

1. A method of adaptive evolution of a wild type micro-organism involved in converting a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof, said method comprising: a. culturing said wild type micro-organism in a suitable culture medium; b. serially propagating the wild type micro-organism cultured in step (a) in a suitable culture medium; wherein, the serial propagation comprises inoculating the wild type micro-organism in a suitable culture medium comprising different concentrations of said precursor of L-glufosinate for adaptive evolution of the wild type micro-organism.

2. As claimed in claim 1, wherein the precursor of L-glufosinate is (2- oxo-4-(hydroxy (methyl) phosphinoyl) butyric acid).

3. As claimed in claim 1 , wherein the micro-organism belongs to the class Actinomycetes.

4. As claimed in claim 3, wherein the micro-organism belonging to the class Actinomycetes is selected from Amycolatopsis or Streptomyces.

5. As claimed in claim 4, wherein said micro-organism is Amycolatopsis rifamycinica or Streptomyces griseus.

6. As claimed in claim 1, wherein the different concentrations of said precursor of L- glufosinate used in the culture medium for adaptive evolution of the wild type microorganism comprises amounts ranging from about 0.1% w / w to about 5% w / w.

7. As claimed in claim 1, wherein said method of adaptive evolution produces an adapted micro-organism capable of converting at least 50% of the precursor of L-glufosinate to L-glufosinate, esters, or salts thereof.

8. As claimed in claim 1, wherein said method of adaptive evolution produces an adapted micro-organism capable of assimilating at least 30% of the precursor of L-glufosinate.

9. A method of adaptive evolution of a wild type micro-organism involved in converting a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof, said method comprising:(i) inoculating a first culture medium with said wild type micro-organism and incubating the inoculated culture medium under conditions suitable for growth of the micro-organism;ii) initiating a first propagation cycle by transferring a portion of the grown culture from step i) to a fresh culture medium comprising the precursor of L-glufosinate and incubating the inoculated culture medium under conditions suitable for adaptation of the micro-organism to the precursor; iii) repeating the transfer and incubation of step ii) for one or more additional cycles to obtain adapted micro-organism; wherein the culture medium of every additional cycle comprises the precursor in an amount incremental to the previous cycle.

10. An adapted micro-organism obtainable by the method of adaptive evolution as claimed in any of the preceding claims.

11. An adapted micro-organism selected from Amycolatopsis rifamycinica (MTCC No. 15) or Streptomyces griseus (MTCC No. 4734), wherein said adapted micro-organism is involved in bioconversion of a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof, and wherein said adapted micro-organism is capable of converting at least 50% of the precursor of L-glufosinate to L-glufosinate, esters, or salts thereof and / or assimilating at least 30% of the precursor of L-glufosinate.

12. A process of bioconversion of a precursor of L-glufosinate to L-glufosinate, esters, or salts thereof, in the presence of the adapted micro-organism as claimed in claim 10 or claim 11.

13. As claimed in claim 12, wherein said process is carried out by inoculating a bioconversion medium comprising said precursor of L-glufosinate with the adapted micro-organism, and wherein the bioconversion medium further comprises an enzyme cofactor and an amine donor.

14. L-glufosinate, esters or salts thereof obtained by a bioconversion process as claimed in claim 12 or claim 13.

15. As claimed in claim 14, wherein productivity of L-glufosinate, esters or salts thereof obtained ranges from about 2g / L / day to about 50g / L / day.

16. A composition comprising L-glufosinate, esters or salts thereof, as claimed in claim 14.

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