Variant nitrile hydratases, microorganisms expressing same, and uses in amide synthesis - Patents.com

Abstract

The present invention relates to variant nitrile hydratases engineered to contain higher activity and / or stability, nucleic acids encoding said nitrile hydratases, and microorganisms engineered to express said novel nitrile hydratases. Additionally, the present invention relates to the use of said nitrile hydratases and microorganisms expressing said nitrile hydratases as biocatalysts, particularly in processes for producing amide compounds from nitrile compounds, preferably for use in converting acrylonitrile to acrylamide.

Description

[Technical field]

[0001] Related Applications This invention claims priority to U.S. Provisional Patent Application No. 63 / 322,203, filed March 21, 2022, the contents of which are incorporated by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (1149704.018013.xml, size: 20,675 bytes, and creation date: March 14, 2023) are incorporated herein by reference in their entirety.

[0003] The present invention relates to novel modified nitrile hydratases and / or operons engineered to contain higher activity and / or stability, nucleic acids encoding the modified nitrile hydratases and / or operons, and microorganisms engineered to contain and express the modified nitrile hydratases or modified nitrile hydratase-containing operons. Additionally, the present invention relates to the use of the modified nitrile hydratases and / or microorganisms engineered to express the modified nitrile hydratases as biocatalysts, particularly in processes for producing amide compounds from nitrile compounds, preferably for use in converting acrylonitrile to acrylamide. [Background technology]

[0004] Acrylamide is used as a monomer to form acrylamide polymers and copolymers. Aqueous solutions of acrylamide prepared by biotransformation can be used for these polymerization and copolymerization reactions. Since the discovery of nitrile hydratase, a microbial enzyme that hydrolyzes nitriles to amides, microorganisms with nitrile hydratase activity have been intensively used for the industrial production of amide compounds. Due to the milder reaction conditions compared to the chemical synthesis of amides, the use of nitrile hydratase-producing microorganisms as biocatalysts is increasingly increasing.

[0005] In fact, one of the best-known commercial examples of nitrile bioconversion by nitrile hydratase-producing microorganisms is the production of acrylamide (AMD) from acrylonitrile (AN). However, a difficult problem in using nitrile hydratase-producing microorganisms as biocatalysts is that both AN and AMD deactivate the biocatalyst. Therefore, it is problematic to provide a nitrile hydratase biocatalyst that remains stable for a long period of time. Also, nitrile hydratase biocatalysts that are modified to improve stability are often not as active as unmodified biocatalysts, i.e., they do not start converting AN to AMD as rapidly as unmodified biocatalysts.

[0006] Therefore, the technical problem underlying the present invention is to provide improved nitrile hydratases and nitrile hydratase producing microorganisms and use them as biocatalysts, i.e., biocatalysts when used to convert nitriles to amides, have both enhanced stability (resistance to AN and AMD deactivation) and desired activity (i.e., initiate the conversion of AN to AMD very rapidly, i.e., biocatalytic activity is the initial reaction rate).

[0007] This technical problem is solved by providing embodiments which are reflected in the claims, described in the description and shown in the following examples and figures. Summary of the Invention

[0008] The present invention relates to novel nitrile hydratases engineered to contain higher activity and / or stability, nucleic acids encoding said nitrile hydratases, and microorganisms engineered to express said novel nitrile hydratases. Additionally, the present invention relates to the use of said nitrile hydratases and microorganisms engineered to express said nitrile hydratases as biocatalysts, particularly in processes for producing amide compounds from nitrile compounds, preferably for use in converting acrylonitrile to acrylamide.

[0009] More specifically, the present invention relates to a novel nitrile hydratase from Pseudonocardia thermophila that has been engineered to contain mutations that provide enhanced stability and activity. In summary, the present inventors have created a DNA sequence encoding a novel nitrile hydratase enzyme that can be introduced into another microorganism, such as an industrial production microorganism, and used in the industrial production of acrylamide (AMD) from acrylonitrile (AN).

[0010] Even more specifically, the present invention provides a variant nitrile hydratase (enzyme) comprising an alpha subunit (nhhA) having an amino acid sequence with at least 98, 99, or 100% sequence identity to SEQ ID NO:2 and a beta subunit (nhhB) comprising an amino acid sequence with at least 98, 99, or 100% sequence identity to SEQ ID NO:1, with the proviso that the alpha subunit comprises one, two, or three of the following mutations: L6T, A19V, and F126Y, and the beta subunit comprises one or both of the following mutations: E108D and A200E, or comprises all three of the following mutations: E108R, A200E, and S212Y, and the variant nitrile hydratase has enhanced stability and / or activity compared to the nitrile hydratase enzyme produced by a wild-type strain (Pseudonocardia thermophila strain DSM43832).

[0011] In an exemplary embodiment, the invention provides a variant nitrile hydratase enzyme, wherein the alpha subunit comprises the following mutations: L6T, A19V, and F126Y, and the beta subunit comprises the following mutations: E108D and A200E, or E108R, A200E, and S212Y.

[0012] In an exemplary embodiment, the invention provides a variant nitrile hydratase enzyme, in which alpha and beta enzyme subunits are expressed together with an nhhG activator protein, optionally an nhhG activator protein comprising an amino acid sequence that is at least 98, 99, or 100% sequence identical to SEQ ID NO:3.

[0013] In some exemplary embodiments, the invention provides variant nitrile hydratases according to any of the foregoing, including soluble enzymes.

[0014] In some exemplary embodiments, the variant nitrile hydratases according to any of the foregoing may be immobilized on a solid support.

[0015] In some exemplary embodiments, the invention provides a variant nitrile hydratase according to any of the foregoing that is encapsulated in a vesicle, sol-gel matrix, or other material that provides improved thermal stability, e.g., compared to the enzyme in solution.

[0016] In other exemplary embodiments, the invention provides a nucleic acid comprising a sequence encoding a nitrile hydratase comprising an alpha subunit and a beta subunit, and optionally an activator protein, according to any of the foregoing, wherein the nucleic acid encoding one or more of the alpha subunit, the beta subunit, and optionally the activator protein, is codon-optimized for increased expression in a desired microorganism, optionally a yeast, fungus, or bacterium.

[0017] In other exemplary embodiments, the present invention provides a method for the production of a desired microorganism, optionally comprising the steps of: Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Bacterium ... Such nucleic acids are provided that are codon-optimized for expression in a bacterium selected from Revibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus. In exemplary embodiments of the invention, the microorganism may be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, or optionally from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobiumoligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp.RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putid, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula、Nocardia rhodochrous、Pseudonocardia thermophila、Trichoderma、Myrothecium verrucaria、Aureobasidium pullulans、Candida famata、Candida guilliermondii、Candida tropicalis、Cryptococcus flavus、Cryptococcus spUFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp. 163, and in certain exemplary embodiments may include bacteria of the species Corynebacterium glutamicum, and further optionally C. glutamicum ATCC13032, or a derivative thereof, strain MB001(DE3).

[0018] In another exemplary embodiment, the invention provides a nucleic acid as described above, i) the beta subunit is encoded by the nucleic acid of SEQ ID NO: 4; ii) the alpha subunit is encoded by the nucleic acid of SEQ ID NO:5; iii) the activator protein is encoded by the nucleic acid of SEQ ID NO: 6; or iv) Any combination of (i) to (iii).

[0019] In other exemplary embodiments, the invention provides a nitrile hydratase operon comprising a nucleic acid encoding a variant nitrile hydratase alpha (nhhA) and beta (nhhB) subunit, and optionally an activator protein (nhhG) according to any of the foregoing, optionally the nucleic acid being any one of the foregoing, and optionally the operon being derived from a yeast, fungus, or bacterium expressing a nitrile hydratase, optionally a Pseudonocardia bacterium, and further optionally a Pseudonocardia thermophila bacterium.

[0020] In another exemplary embodiment, the present invention provides a nitrile hydratase operon comprising a nucleic acid encoding the alpha (nhhA) and beta (nhhB) subunits of a variant nitrile hydratase, and an activator protein (nhhG) according to any of the foregoing, optionally including SEQ ID NO:7.

[0021] In other exemplary embodiments, the invention provides one or more extrachromosomal sequences, optionally plasmids, comprising at least one nucleic acid or operon according to any of the above.

[0022] In another exemplary embodiment, the present invention provides a microorganism, optionally a yeast, fungus, or bacteria, further optionally an industrial microorganism, which optionally does not endogenously express nitrile hydratase or endogenously expresses nitrile hydratase, the microorganism being engineered to contain a nucleic acid encoding a variant nitrile hydratase comprising alpha (nhhA) and beta (nhhB) subunits of the variant nitrile hydratase according to any of the above, and optionally an activator protein (nhhG), or an operon, optionally comprising said nucleic acid, as described above, and optionally one or more of said nucleic acids are included in one or more extrachromosomal sequences (plasmids) or integrated (in one or more copies) into the chromosomal DNA of the microorganism.

[0023] In some exemplary embodiments, the microorganism is an industrial microorganism that endogenously expresses a nitrile hydratase, and the nucleic acid or operon replaces an endogenous nitrile hydratase gene or an operon that includes an endogenous nitrile hydratase gene. The microorganism encoding the nitrile hydratase includes microbial species such as Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Art. hrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasi These include Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus.

[0024] In some exemplary embodiments, the microorganism used to express the variant nitrile hydratase or nitrile hydratase operon is a microbe selected from Rhodococcus, Pseudomonas, Escherichia, and Geobacillus. Typically, biocatalysts include Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serrati a, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluy The microorganisms used to express the variant nitrile hydratase or nitrile hydratase operon are selected from the group consisting of: veromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus, or any part of said microorganisms having nitrile hydratase activity, or variant nitrile hydratase or nitrile hydratase operon, and are selected from the group consisting of: Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium,The bacterium is selected from Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus.

[0025] In more specific exemplary embodiments of the invention, the microorganism may be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or from the following species: Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp.RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putid, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacteriumimperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp.163.

[0026] In a further specific exemplary embodiment, the biocatalyst is a bacterium of the species Corynebacterium glutamicum, optionally the C. glutamicum strain ATCC13032 or its derivative MB001(DE3), deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071.

[0027] In some exemplary embodiments, the present invention provides a method for producing an amide compound from a nitrile compound, the method comprising contacting the nitrile compound with a nitrile hydratase according to any of the above or a bacterium expressing the nitrile hydratase. The bacterium may optionally be in a dried form. The enzyme may be in a soluble form or immobilized on a solid support or the like. Further optionally, the nitrile hydratase may be encapsulated in a material such as a gel-sol matrix that enhances thermal stability during amide synthesis.

[0028] In some exemplary embodiments, the present invention provides a method of producing an amide compound from a nitrile compound, the method comprising contacting the nitrile compound with a nitrile hydratase or a bacterium expressing said nitrile hydratase according to any of the above, wherein the amide compound is selected from the group consisting of acrylamide, methacrylamide, acetamide, and nicotinamide, preferably acrylamide, and the nitrile compound is selected from the group consisting of acrylonitrile, methacrylonitrile, acetonitrile, and 3-cyanopyridine, preferably acrylonitrile.

[0029] In some exemplary embodiments, these amide synthesis methods employ soluble nitrile hydratase, encapsulated nitrile hydratase, immobilized nitrile hydratase, or employ whole or lysed microbial cell biocatalysts.

[0030] In some exemplary embodiments, these amide synthesis methods use an intact microbial biocatalyst, which can optionally be fresh (i.e., directly from fermentation), stored (e.g., stored frozen (wet frozen)), or dried, e.g., lyophilized.

[0031] Exemplary methods for producing amide compounds from nitrile compounds using biocatalysts according to the present invention are further described and exemplified below. [Brief description of the drawings]

[0032] The invention will be explained in more detail with reference to the accompanying drawings, which are described in detail below.

[0033] [Figure 1] Contains an alignment of wild-type (strain DSM43832) and variant nitrile hydratase beta subunit (nhhB) polypeptide sequences. [Diagram 2] Contains an alignment of wild-type (strain DSM43832) and variant nitrile hydratase alpha subunit (nhhA) polypeptide sequences. [Diagram 3] Included is an alignment of wild-type (strain DSM43832) and variant nhhBAG operon nucleic acid sequences. The variant nhhBAG operon contains the NdeI / XhoI restriction sites used to clone the strain DSM43832 nhhBAG operon and lacks the amidase gene present in the endogenous operon upstream of nhhB. In the wild-type operon sequence, the TGA stop codon of nhhB overlaps with the ATG start codon of nhhA, and similarly, the TGA stop codon of nhhA overlaps with the GTG start codon of nhhG. In contrast, in the variant operon, this overlap has been resolved by the introduction of an intergenic ribosome binding site (seen as a gap in the alignment). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Prior to describing the invention, the following definitions are provided. Unless otherwise specified, all terms should be interpreted as they would be by one of ordinary skill in the art.

[0035] definition As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. 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, unless clearly indicated otherwise.

[0036] As used herein, the term "biocatalyst" refers to any biocatalyst having nitrile hydratase activity. The biocatalyst capable of converting acrylonitrile to acrylamide can be a microorganism encoding an enzyme having nitrile hydratase activity or any part of the microorganism having nitrile hydratase activity. The biocatalyst can be selected from the microorganism, lysed cells of the microorganism, cell lysate of the microorganism, or any combination thereof. In a very specific embodiment, the biocatalyst is a variant nitrile hydratase disclosed in the examples, or a bacterial strain expressing it (Corynebacterium glutamicum).

[0037] As used herein, the term "biomass" generally refers to harvested cells, typically microbial cells, and most typically bacterial cells, obtained after fermentation, typically after excess broth has been removed, optionally by filtration or centrifugation, resulting in a biomass composition having a dry content typically in the range of about 10-35%, more typically about 25-30%.

[0038] As used herein, the term "microorganism" as used herein encompasses a "nitrile hydratase-producing microorganism", which endogenously expresses and / or is engineered to express a variant nitrile hydratase according to the present invention. Such a microorganism in the context of the present invention is preferably a bacterium, a fungus, or a yeast. In the present invention, a "nitrile hydratase-producing microorganism" is used or is for use as a biocatalyst for converting a nitrile compound to the corresponding amide compound.

[0039] As used herein, the term "nitrile compound" refers to a compound that is converted to an amide compound by the action of the nitrile hydratase according to the present invention or a microorganism expressing the nitrile hydratase according to the present invention. The nitrile compound is any organic compound having a -C=N functional group, such as methacrylonitrile, acetonitrile, or 3-cyanopyridine, preferably acrylonitrile.

[0040] As used herein, the term "amide compound" refers to a compound produced by nitrile hydratase from a nitrile compound. An amide compound is a compound that has a functional group R n C(O) x It has the formula NR'2, where R and R' refer to H or an organic group, or an organic amide, and n = 1, x = 1. Examples of such amide compounds include methacrylamide, acetamide, or nicotinamide, and preferably includes acrylamide.

[0041] As used herein, the term "nitrile hydratase-producing microorganism" may be any microorganism capable of producing the variant nitrile hydratase of the present invention. In the context of the present invention, "nitrile hydratase-producing microorganism" includes non-naturally encoded nitrile hydratase, which has been genetically engineered (e.g., via transformation, transduction, transfection, conjugation, or other suitable methods for introducing or inserting polynucleotides into cells known in the art) to contain a gene or polynucleotide encoding a nitrile hydratase (see Sambrook and Russell 2001, Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), thus allowing the microorganism to produce and stably maintain the nitrile hydratase enzyme. For this purpose, it may further be necessary to insert additional polynucleotides that may be necessary to enable the transcription and translation of the nitrile hydratase gene or mRNA, respectively. Such additional polynucleotides may include, inter alia, promoter sequences, or origins of replication, or other plasmid control sequences. In this context, such genetically engineered microorganisms that do not naturally contain a gene encoding a nitrile hydratase but are engineered to contain a polynucleotide encoding a nitrile hydratase can be prokaryotic or eukaryotic. Examples of such prokaryotic microorganisms include, for example, Escherichia coli and Corynebacterium species. Examples of such eukaryotic microorganisms include, for example, yeasts (e.g., Saccharomyces cerevisiae or Pichia pastoris).

[0042] A "nitrile hydratase-producing microorganism" that encodes a nitrile hydratase (naturally or non-naturally) can, in some embodiments, produce and stably maintain a nitrile hydratase. However, according to the present invention, such a microorganism can also produce only nitrile hydratase during microbial culture (or fermentation). Such microorganisms include, among others, Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthritis, and the like. In exemplary embodiments of the present invention, the microorganism may be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia, and Geobacillus. In addition, the "nitrile hydratase-producing microorganism" includes, among others, the following species: Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacteriumradiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp.RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putid, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candidafamata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas These include testosterone, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp.163.

[0043] In certain exemplary embodiments, the "nitrile hydratase producing microorganism" is a bacterium of the species Corynebacterium glutamicum, preferably a Corynebacterium strain, C. glutamicum strain ATCC13032 or its derivative MB001(DE3) deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071.

[0044] In the context of the present invention, "nitrile hydratase" refers to a microbial enzyme that catalyzes the hydration of nitriles to the corresponding amides (IUBMB Enzyme Nomenclature EC 4.2.1.84). As used herein, the terms "nitrile hydratase" and "nitrile hydratase" also encompass modified or enhanced enzymes that, for example, can convert nitrile compounds (e.g., acrylonitrile) to amide compounds (e.g., acrylamide) faster, can be produced at a higher yield / time ratio, or are more stable, so long as they are able to catalyze the conversion (i.e., hydration) of nitrile compounds (e.g., acrylonitrile) to amide compounds (e.g., acrylamide). The enzyme generally includes an alpha subunit (nhhA) and a beta subunit (nhhB), which are optionally expressed together with an activator protein (nhhG). Exemplary embodiments In one embodiment, it refers to a variant nitrile hydratase (enzyme) comprising an alpha subunit (nhhA) having an amino acid sequence with at least 98, 99, or 100% sequence identity with SEQ ID NO:2 and a beta subunit (nhhB) comprising amino acids with at least 98, 99, or 100% sequence identity with SEQ ID NO:1, with the proviso that the alpha subunit comprises one, two, or three of the following mutations: L6T, A19V, and F126Y, and the beta subunit comprises one or both of the following mutations: E108D and A200E, or comprises all three of the following mutations: E108R, A200E, and S212Y, and the variant nitrile hydratase has enhanced stability and / or activity compared to the nitrile hydratase enzyme produced by a wild-type strain (Pseudonocardia thermophila strain DSM43832).

[0045] In the context of the present invention, "nitrile hydratase stability" or "enzyme stability" or "stability" refers to how well a biocatalyst tolerates AN and AMD under certain reaction conditions (e.g., temperature, solvent, etc.). That is, good stability under certain reaction conditions means that a nitrile hydratase has a lower deactivation rate compared to another nitrile hydratase under the same conditions under certain reaction conditions (as both AN and AMD are known to deactivate endogenous nitrile hydratase biocatalysts).

[0046] In the context of the present invention, "nitrile hydratase activity" or "enzyme activity" or "activity" refers to the time it takes for an enzyme biocatalyst to initiate the conversion of AN to AMD. That is, if the conversion is initiated very rapidly, the enzyme is said to have high activity, i.e., biocatalytic activity refers to the initial reaction rate. [Mode for carrying out the invention]

[0047] Development of new biocatalysts The present inventors have generated a novel nitrile hydratase enzyme and the DNA sequence encoding the enzyme and transferred it into an industrial production microorganism. This biocatalyst can be used in the industrial production of acrylamide (AMD) from acrylonitrile (AN).

[0048] Technically, the inventors aimed to obtain a novel nitrile hydratase biocatalyst that has high activity and can be used to produce high concentrations (i.e., 54% AMD). As mentioned above, many natural nitrile hydratase enzymes have been sequenced and used for AMD synthesis, but typically endogenous nitrile hydratases lack one or both of the required characteristics (activity and stability).

[0049] The development of the biocatalyst of the present invention involves screening of multiple nitrile hydratases and host cells. The host cell, or industrial production microorganism, is a microbe species into which the nitrile hydratase gene is preferably inserted and used for biocatalyst production via fermentation. The development of novel variant nitrile hydratases and operons involves the introduction of genetic modifications into multiple nitrile hydratase genes and evaluation of the performance (stability / activity) of the variants. Nitrile hydratases from several different microbial species were studied. The methods used by the inventors to improve the biocatalyst included in vivo recombination, site-directed and random mutagenesis, chaperone co-expression, operon optimization, expression plasmid optimization, codon usage optimization, and fermentation optimization.

[0050] As disclosed in the Examples, the amino acid sequences of both the alpha and beta nitrile hydratase subunits of the nitrile hydratase endogenously produced by Pseudonocardia thermophila strains were mutated with the aim of obtaining variant nitrile hydratases with enhanced stability and / or activity compared to the parent nitrile hydratase endogenously produced by Pseudonocardia thermophila strains.

[0051] The sequences of the variant nitrile hydratase genes and operons (both the nitrile hydratase genes and operons) were derived from specific Pseudonocardia thermophila strains and were extensively modified relative to the wild-type nitrile hydratase genes and operons endogenously contained in Pseudonocardia thermophila. The alpha and beta nitrile hydratase subunits containing the specific mutations contained in SEQ ID NO:2 and SEQ ID NO:1 (this combination was selected after screening a large number of different mutation combinations) were found to provide the best combination of enhanced stability and / or activity compared to the parent nitrile hydratase produced endogenously by Pseudonocardia thermophila strains. (See also the sequence alignment of the wild-type and variant alpha and beta nitrile hydratase subunits contained in Figures 1 and 2).

[0052] As shown in the examples, the variant nitrile hydratase with specific mutations in the alpha subunit contained in SEQ ID NO:2 (L6T, A19V, F126Y) and specific mutations in the beta subunit contained in SEQ ID NO:1 (E108D, A200E) showed the best stability / activity, and the variant nitrile hydratase with the same mutations in the alpha subunit contained in SEQ ID NO:2 (L6T, A19V, F126Y) and E108R / A200E / S212Y mutations in the beta subunit showed the second best stability / activity when compared to the amide synthesis experiments disclosed in the examples.

[0053] As further described below in the Examples, the nitrile hydratase operon from a Pseudonocardia thermophila strain was further engineered to enhance the stability and / or activity of the nitrile hydratase enzyme and to facilitate expression in a selected exemplary industrial strain (e.g., Corynebacterium glutamicum, optionally C. glutamicum ATCC 13032 strain or its derivative MB001(DE3)). This parent C. glutamicum MB001(DE3) strain has been deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071. A detailed description of this strain has been published in Kortmann M. et al. (2015), “A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level”, Microb Biotechnol. 8:253-65.

[0054] These operon modifications included, among other changes, codon optimization of the parent Pseudonocardia thermophila strain nhhA, nhhB, and nhhG coding sequences, introduction of intergenic ribosome binding sites to eliminate the overlap of the TGA stop codon of nhhB with the ATG start codon of nhhA and the overlap of the TGA stop codon of nhhA with the GTG start codon of nhhG in the variant operon, introduction of NdeI / XhoI restriction sites used for cloning, and deletion of the amidase gene that is part of this operon upstream of nhhB. These changes can further be seen in FIG. 3, which includes an alignment of the modified operon with the parent operon of the Pseudonocardia thermophila strain used to derive the modified operon.

[0055] In regard to the above, in the exemplary embodiments described in the Examples below, plasmids containing these variant nitrile hydratase sequences have been introduced into the host strain Corynebacterium glutamicum. As further shown in the Examples, the exemplary Corynebacterium glutamicum strains expressing the variant nitrile hydratase sequences of the invention have been shown to provide enhanced expression and stability when used to produce acrylamide from acrylonitrile, as compared to the parent strains as well as other comparative controls (e.g., other microbial strains engineered to contain nitrile hydratases containing different mutations).

[0056] Although the exemplary Corynebacterium glutamicum strain expressing the exemplary variant nitrile hydratase provides the best activity / stability combination, it is anticipated that the variant nitrile hydratase sequences of the present invention may be expressed in other microbia, preferably other industrial microbes that are preferred for use in industrial processes, examples of which include Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacteri microorganisms selected from the group consisting of bacteria of the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or more specifically bacteria of the genera Rhodococcus, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas and Pyrococcus, or more specifically bacteria of the genera Rhodococcus, Pseudomonas, Escherichia and Geobacillus, or bacteria of the following species: Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillussubtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp.RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putid, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candidaguilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp. 163, optionally comprising a bacterium of the species Corynebacterium glutamicum, further optionally comprising the C. glutamicum strain ATCC13032 or its derivative MB001(DE3) deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071.

[0057] Novel biocatalyst forms that can be used for amide synthesis The biocatalyst of the invention, e.g., the exemplary Corynebacterium glutamicum strain expressing the exemplary variant nitrile hydratase, can be fresh (i.e., directly from fermentation), stored (e.g., stored frozen (wet frozen)), or dried (e.g., in lyophilized form). In an exemplary embodiment during the use of the microbial biocatalyst of the invention as a biocatalyst, the inventors make a slurry by mixing the microbial biocatalyst with water.

[0058] After fermentation, the harvested biomass or harvested cells may optionally be washed or otherwise processed, for example by freezing or drying.

[0059] Also, the biocatalyst capable of converting acrylonitrile to acrylamide can be a microorganism encoding a variant nitrile hydratase or any part of the microorganism having nitrile hydratase activity. The biocatalyst can be selected from the microorganism, lysed cells of the microorganism, cell lysate of the microorganism, or any combination thereof. In a very particular embodiment, the biocatalyst is a nitrile hydratase.

[0060] In one embodiment, the amount of biocatalyst is between 0.1 kg dry cells / m3 and 5 kg dry cells / m3 of reaction mixture.

[0061] In another embodiment, the amount of biocatalyst is from 0.1 g dry cells / kg 100% AMD to 3 g dry cells / kg 100% AMD, more specifically from 0.2 g dry cells / kg 100% AMD to 3 g dry cells / kg 100% AMD, more specifically from 0.2 g dry cells / kg 100% AMD to 2.5 g dry cells / kg 100% AMD, based on the final AMD amount. In another embodiment, the amount of biocatalyst is from 0.5 g dry cells / kg 100% AMD to 2 g dry cells / kg 100% AMD, or more specifically from 1.1 g dry cells / kg 100% AMD to 1.5 g dry cells / kg 100% AMD.

[0062] In yet another embodiment, the amount of biocatalyst is from 0.5 g dry cells / kg 50% AMD to 1 g dry cells / kg 50% AMD, more specifically from 1.6 g dry cells / kg 50% AMD to 1.8 g dry cells / kg 50% AMD, based on the final AMD amount.

[0063] In yet another embodiment, the amount of biocatalyst is between 0.1 kg dry cells / m3 and 1.5 kg dry cells / m3 of reaction mixture at the end of maturation of the reaction mixture.In another embodiment, the amount of biocatalyst is between 0.1 kg dry cells / m3 and 1.0 kg dry cells / m3 of reaction mixture.

[0064] During the process, for example, if acrylonitrile begins to accumulate in the reactor, more biocatalyst may be added. The biocatalyst may be added, for example, as a homogenous slurry in water.

[0065] Exemplary Amide Reaction Conditions The reaction is typically carried out at atmospheric pressure, more specifically at 1 bar.

[0066] The slurry, i.e., the aqueous mixture containing the biocatalyst, may be produced by any method known in the art, such as by mixing water and the biocatalyst in a receptacle or reactor. Optionally, the slurry is homogenous and not strongly aggregated.

[0067] The reaction of acrylonitrile in an aqueous solution to acrylamide in the presence of a biocatalyst having nitrile hydratase activity is initiated when acrylonitrile is fed to a reactor containing the slurry. In an exemplary embodiment, acrylonitrile may be fed to a reactor containing the slurry to provide a reaction mixture containing water, acrylamide, acrylonitrile, and a biocatalyst. In an exemplary embodiment, the biocatalyst slurry may be added to the reactor along with additional water, and the feeding of AN may be initiated after such addition. The amount of additional water and biocatalyst slurry in an exemplary embodiment may be about 5000 kg for 100 kg of slurry, and may range from about 25000 kg for 100 kg of slurry to about 1000 kg for 100 kg of slurry.

[0068] Aqueous solutions of acrylamide at high concentrations (e.g., at least 35 wt%, or at least 40 wt%, or at least 45 wt%, or at least 50 wt%), at least 55 wt%, at least 56%, or at least about 57%, or about 55-60% AMD may be produced with controlled acrylonitrile feed and process temperature profiles. In the foregoing, the maximum AMD concentration may be constrained by precipitation concerns; i.e., at high AMD concentrations, AMD may begin to precipitate.

[0069] Cooling of the reactor is typically required to keep the reaction mixture at the desired reaction temperature. The temperature and acrylonitrile feed rate at the start of the reaction are relatively high, respectively, to achieve a fast reaction rate and short synthesis time. The reactor begins to cool, for example, 60 minutes after the start of the reaction, because the deactivation of the biocatalyst from the accumulation of acrylamide is significantly lower at lower temperatures compared to the reaction mixture at higher temperatures. The feed rate of acrylonitrile is relatively low for the last few hours to avoid the accumulation of acrylonitrile in the reactor.

[0070] The reaction is exothermic and cooling capacity and safety concerns may be the limiting factor determining the AN feed rate. In some exemplary embodiments, the amount of AN in the reactor is maintained at a maximum of about 3%.

[0071] In some exemplary embodiments, the reactor containing the biocatalyst is at about 15° C. before the AN feed is added, and the AN feed is started and the temperature is maintained at about 23° C. until the end of the reaction to minimize deactivation of the biocatalyst.

[0072] The acrylonitrile feed may be continued throughout the process, more specifically, until the maturation stage. The feed rate of acrylonitrile may vary during the process. The feed rate of acrylonitrile may be continuous or intermittent. The feed rate of acrylonitrile depends on the reaction rate of acrylonitrile to acrylamide and the deactivation rate of the biocatalyst. In one embodiment, the feed of acrylonitrile is continued throughout the process until the maturation stage.

[0073] In one embodiment, the feed rate of acrylonitrile is adjusted during the process to avoid accumulation of acrylonitrile in the reaction mixture. Acrylonitrile is fed during the process at a rate such that acrylonitrile is converted to acrylamide. More specifically, the amount of acrylonitrile in the reaction mixture is maintained as less than 3 wt%, or less than 2 wt%, more specifically less than 1 wt%, and even more specifically less than 0.5 wt%, based on the total amount of the reaction mixture. However, in some embodiments, the reaction can be carried out at much higher AN concentrations (e.g., 5%, 10%, or even higher) when all of the AN is added at once.

[0074] In another embodiment of the process, 38%-48% of the total amount of acrylonitrile fed to the reactor is fed from 0 to 60 minutes from the start of the process, 22%-30% of the total amount of acrylonitrile is fed from 60 to 120 minutes of the process, 12%-18% of the total amount of acrylonitrile is fed from 120 to 180 minutes of the process, and 8%-12% of the total amount of acrylonitrile is fed from 180 to 240 minutes of the process. The remaining acrylonitrile is fed during the process prior to the maturation stage in order to reach a balance of 100% of the acrylonitrile fed.

[0075] In the maturation stage, substantially no acrylonitrile, more specifically no acrylonitrile, is fed to the reactor. During maturation, acrylonitrile monomer still present in the reaction mixture reacts to acrylamide. The reaction mixture is maturated until the desired characteristics are reached.

[0076] Generally, the temperature of the reaction mixture is monitored. The monitoring and measuring can be done by any suitable means and methods in the art.

[0077] In an exemplary embodiment, the temperature of the reaction mixture is maintained between 15-25° C. In one embodiment, the temperature is maintained between 19-25° C., more specifically between 20-22° C., and even more specifically at 22° C. In one embodiment, the temperature is maintained in the desired range by measuring the temperature of the reaction mixture and cooling or heating the mixture such that the temperature remains in the desired range. Cooling and / or heating the reaction mixture may be accomplished by methods known in the art.

[0078] Once cooling of the reaction mixture has begun, the temperature of the reaction mixture may be the same as, higher than, or lower than the temperature of the reaction mixture at the start of the process.

[0079] In some embodiments, once the acrylamide concentration reaches 37% to 55% by weight, cooling of the reaction mixture is continued such that the temperature of the reaction mixture is within the range of 10° C. to 18° C. or 10° C. to 21° C. In other words, the period during which the reaction mixture is cooled to a temperature of 10° C. to 18° C. or 10° C. to 21° C. is a period during which an acrylamide concentration of at least 27% by weight (more specifically, 27% to 38% by weight) increases to an acrylamide concentration of 37% to 55% by weight (more specifically, 40% to 50% by weight).

[0080] In one embodiment, once the acrylamide concentration reaches 37% to 55% by weight, cooling of the reaction mixture continues such that the temperature is in the range of 10° C. to 16° C., more specifically 13° C. to 16° C., and even more specifically the temperature is 15° C. In one embodiment, cooling is initiated, for example, after the reaction mixture is maintained at 15° C. to 25° C. In one embodiment, the reaction mixture is cooled by at least 10° C., more specifically at least 5° C., and even more specifically at least 4° C. Cooling can be done linearly or stepwise, typically linearly.

[0081] In one embodiment, the reaction mixture is aged at a temperature in the range of 10° C. to 18° C. or 10° C. to 21° C. once the acrylamide concentration reaches 37% to 55% by weight.

[0082] During the aging, substantially no acrylonitrile, more specifically, no acrylonitrile, is fed to the reactor. During the aging, unreacted acrylonitrile in the reactor reacts to acrylamide. The aging starts after the reaction mixture is cooled and the temperature of the reaction mixture is within the range of 10°C to 18°C ​​or 10°C to 21°C, and / or after the feeding of acrylonitrile to the reactor is terminated. More specifically, the aging continues until the final concentration of acrylonitrile in the reaction mixture is at most 1000 ppm, at most 500 ppm, at most 250 ppm, at most 100 ppm, at most 50 ppm, at most 10 ppm, or at most 0 ppm.

[0083] In one embodiment of the process, the temperature of the reaction mixture is maintained at 15°C to 25°C for a period of 30 minutes to 90 minutes (e.g., 45 minutes to 60 minutes) and cooling of the reaction mixture to a temperature of 10°C to 18°C ​​or 10°C to 21°C is performed over a period of 45 minutes to 120 minutes (e.g., 60 minutes to 120 minutes).

[0084] After the reaction is complete, the biocatalyst may be separated from the aqueous AMD solution by any known separation method (e.g., centrifugation, flotation, filter press, or filtration). The aqueous acrylamide solution produced by the process is then typically used to manufacture polyacrylamide.

[0085] Separation or recovery of biomass cells after fermentation may in some exemplary embodiments be facilitated by the addition of a flocculant to help harvest the cells, which may be performed prior to the separation method (e.g., prior to centrifugation / filtration). In exemplary embodiments, salts or other stabilizers will be added prior to freezing the biomass. For example, after the cells are harvested, a suitable preservative salt, such as an ammonium, calcium, iron, magnesium, potassium, or sodium salt, may be added to the biomass / cell mass.

[0086] In some exemplary embodiments, the separation or recovery of biomass cells after fermentation can be performed after first adding a cationic solution followed by an anionic solution. The amount of cationic solution added can be, for example, in the range of 2-10% relative to the mass to be flocculated, while the anionic solution can be, for example, in the range of 0.5-3.5%. In some exemplary embodiments, the cationic solution is in the range of 4-7% and the anionic solution is in the range of 1-2%. The suspension may also be stirred during this process. [In this process, a low molecular weight cationic flocculant acts as a flocculant, resulting in the formation of a microflocculant, after which an anionic solution is added, and the microflocculant acts as a substrate to provide flocculation for the high molecular weight anionic flocculant].

[0087] After addition of the anionic solution, for example, about 5-30 minutes, 5-15 minutes, or 10 minutes, a flocculant is formed which can be separated by any desired means, such as centrifugation, flotation, pressure filtration, or filtration. For example, separation can be accomplished by use of a filter having pores <0.45 pm in diameter. If desired, the filtered biomass can be optionally resuspended in demineralized water and the filtration repeated to provide further removal of broth culture residues from the biomass.

[0088] As mentioned above, if the resulting recovered biomass is stored as a paste, the biomass is typically mixed with a stabilizer or preservative (e.g., ammonium, calcium, iron, magnesium, potassium, or sodium salts).

[0089] After the present invention has been described in detail, the present invention will be further illustrated in the following examples. EXAMPLES

[0090] The following examples are offered for illustrative purposes only and are not intended to be limiting.

[0091] Example 1: Production of variant nitrile hydratases and variant nitrile hydratase operons The development of the biocatalyst of the present invention included screening multiple nitrile hydratases and host cells, introducing various genetic modifications into multiple nitrile hydratase genes, and evaluating the performance (stability / activity) of the variants. Nitrile hydratases from several different microbial species were studied. The methods used by the inventors to improve the biocatalyst included in vivo recombination, site-directed and random mutagenesis, chaperone co-expression, operon optimization, expression plasmid optimization, codon usage optimization, and fermentation optimization.

[0092] These efforts resulted in the isolation of variant nitrile hydratase alpha and beta subunits comprising SEQ ID NOs: 2 and 1, respectively. Both of these variants were derived from the alpha and beta subunits of nitrile hydratase produced by a Pseudonocardia thermophila strain (Pseudonocardia thermophila strain available under deposit DSM 43832). The specific combinations of mutations contained in these variants are further shown in the sequence alignments in Figures 1 and 2. When co-expressed, these variants were found to result in enzymes that exhibit the best combination of enhanced stability and / or activity compared to the parent nitrile hydratase produced endogenously by Pseudonocardia thermophila strains.

[0093] The inventors also observed that a variant nitrile hydratase comprising an alpha unit comprising L6T, A19V, F126Y, and a beta subunit comprising E108R / A200E / S212Y mutations possesses the second best combination of enhanced stability and / or activity compared to the parent nitrile hydratase.

[0094] Additionally, to enhance the expression of nitrile hydratase and the stability of nitrile hydratase during amide synthesis (when introduced into an exemplary industrial host strain), the endogenous nitrile hydratase gene containing operon of Pseudonocardia thermophila DSM43832 was further extensively modified compared to the wild-type nitrile hydratase gene and operon content. Specifically, as shown in FIG. 3, the codon usage of the variant nhhA and nhhB genes, as well as the nhhG gene, was optimized for C. glutamicum (Eikmanns, B. (1998), Identification, Sequence Analysis, and Expression of a Corynebacterium glutamicum Gene Cluster Encoding the Three Glycolytic Enzymes Glyceraldehyde-3-Phosphate Dehydrogenase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase, J Bac The nhhBAG operon was modified to include NdeI / XhoI restriction sites used for cloning the nhhBAG operon in strain DSM43832 (based on the codon usage of highly expressed genes in C. glutamicum, as published in J. Med. Soc. 174, 6076-6086), the operon was modified to remove the amidase gene present in the endogenous operon upstream of nhhB, and to include an intergenic ribosome binding site to eliminate the overlap, since in the wild-type operon sequence, the TGA stop codon of nhhB overlaps with the ATG start codon of nhhA, which in turn overlaps with the GTG start codon of nhhG. A variant operon containing these modifications is included in SEQ ID NO:7.

[0095] Example 2: Development of novel nitrile hydratases and operons The development of the biocatalytic Corynebacterium glutamicum strain of the present invention involves the introduction of a plasmid containing the operon described in the previous example into a selected industrial strain. As mentioned previously, the inventors selected C. glutamicum MB001(DE3) as the industrial parent strain, which has been deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071. A detailed description of this strain has also been published in Kortmann M.et al.(2015), “A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level”, Microb Biotechnol.8:253-65.

[0096] The parent strain was selected because it is used in other industrial processes and because it does not contain a nitrile hydratase gene. An exemplary production strain contains a nitrile hydratase operon present on a plasmid at about 30 copies per cell. However, it is expected that equivalent or even enhanced results can be achieved by instead engineering the C. glutamicum MB001(DE3) strain so that one or more copies of the nitrile hydratase operon are integrated into the C. glutamicum cell genome (i.e., to improve strain stability, avoid the use of antibiotic resistance genes, and further maximize enzyme yield).

[0097] Additionally, the use of encapsulated nitrile hydratase from Pseudonocardia thermophila has been described (see, e.g., Martinez et al. (2014), “Acrylamide production using encapsulated nitrile hydratase from Pseudonocardia thermophila in a sol-gel matrix”, J Molecular Catalysis B:Enzymatic, 100, 19-24) and reported to result in improved thermostability compared to the enzyme in solution; alternatively, variant nitrile hydratase enzymes may be encapsulated in sol-gel matrices or lipid vesicles and used in this form as biocatalysts in amide synthesis processes).

[0098] Example 3: Use of a novel nitrile hydratase biocatalyst for the synthesis of AMD The performance of exemplary biocatalysts including different variant nitrile hydratases expressed in different bacteria, including the C. glutamicum nitrile hydratase producing strain described in the previous example, was compared for their stability / activity when used in the synthesis of acrylamide (AMD) from acrylonitrile (AN), as described below. These experiments and results are further summarized in the following table.

[0099] In these experiments, demineralized water and biocatalyst were mixed and the AN feed was started. As shown, in these methods, the total AN feed was fed for either 3 hours or 1 hour. In these syntheses, the target AMD concentration was either 52 or 54%. As a result of sampling during the synthesis, the final AMD concentration was usually a little lower. If the synthesis was successful, no AN was detected in the solution at the end of the test.

[0100] Biocatalyst X1 has a variant nitrile hydratase enzyme derived from Pseudonocardia and subsequently engineered to contain specific mutations, and the host is E. coli. At a dosage of 5.7 g dry cells / kg 100% AMD, it converted all AN into AMD. However, separation of the biocatalyst from the ready AMD by centrifugation was not successful, so E. coli as the host cell was discarded.

[0101] As mentioned above, different biocatalysts including different variant nitrile hydratases (also derived from Pseudonocardia and subsequently modified to contain specific mutations) were expressed in different bacteria including the C. glutamicum nitrile hydratase producing strain described in the previous example and their stability / activity was compared when used during the synthesis of acrylamide (AMD) from acrylonitrile (AN).

[0102] The experiments and the results obtained thereby are summarized below in Tables 1 and 2. Additionally, the different biocatalysts identified in the tables and their comparative results when utilized in the synthesis of AMD are briefly summarized below:

[0103] Biocatalyst X2 contains a nitrile hydratase enzyme from Rhodococcus and the host is Corynebacterium glutamicum. It converted only about one-third of the added AN, resulting in a final AMD concentration of 18% at a dose of 6.0 g dry cells / kg 100% AMD. Therefore, development of this combination was discontinued.

[0104] Biocatalysts X3, X4, and X5 contain the nitrile hydratase enzyme from Pseudonocardia and the host is Corynebacterium glutamicum. They are all biocatalysts from different batches and at different stages of development. In experiments, all three versions converted all AN to AMD.

[0105] Biocatalyst X4 was dried after fermentation and rewetted before use in AMD synthesis. The rewetted biocatalyst was able to convert all AN to AMD.

[0106] Biocatalyst X5, which comprises the final version, showed substantially higher activity per cell dry mass as shown in the table. A dose of only 1.7 g dry cells / kg 100% AMD converted all AN to AMD.

[0107] Biocatalyst R1 contains nitrile hydratase from Rhodococcus and the host is Rhodococcus. As shown in the table, in experiments where all AN feed was added in 1 hour, only about two-thirds of the added AN was converted to AMD at a high dose of 6.9 kg dry cells / kg 100% AMD. The biocatalyst is deactivated by high levels of AN.

[0108] Biocatalyst X6 contains the nitrile hydratase enzyme from Pseudonocardia and the host is Corynebacterium glutamicum. In experiments where all AN was added at 1 hour, it converted all added AN to AMD at a dose of 4.6 kg dry cells / kg 100% AMD in a 21 hour experiment. Thus, this biocatalyst tolerates high AN and AMD concentrations.

[0109] The following table summarizes the results observed from these experiments. [Table 1]

[0110] Based on the results in Table 1 above, it can be seen that the biocatalyst of the present invention has enhanced stability and activity over an extended period of time (at least 3 hours) when utilized in AMD synthesis. The results also indicate that different forms (e.g., dried forms) of the biocatalyst of the present invention can be used.

[0111] Exemplary sequences used in the examples are included in the sequence listing on the following page. Sequence Listing Variant DSM43832 nhhB beta subunit: Amino acid sequence: SEQ ID NO:1 MNGVYDVGGTDGLGPINRPADEPVFRAEWEKVAFAMFPATFRAGFMGLDEFRFGIEQMNPAEYLESPYYWHWIRTYIHHGVRTGKIDLEELERRTQYYRENPDAPLPDHEQKPELIE FVNQAVYGGLPASREVDRPPKFKEGDVVRFSTASPKGHARRARYVRGKTGTVVKHHGAYIYPDTAGNGLGECPEHLYTVRFTEQELWGPEGDPNSSVYYDCWEPYIELVDTKAAAA* Variant DSM43832 nhhA alpha subunit: Amino acid sequence: SEQ ID NO:2 MTENITRKSDEEIQKEITRVRVKALESMLIEQGILTTSMIDRMAEIYENEVGPHLGAKVVVKAWTDPEFKKRLLADGTEACKELGIGGLQGEDMMWVENTDEVHHVVVCTLCSCYPWPVLGLPPNWYKEPQYRSRVVREPRQLLKEEFGFEVPPSKEIKVWDSSSEMRFVVLPQRPAGTDGWSEEELATLVTRESMIGVEPAKAVA* DSM43832 nhhG activator protein: amino acid sequence: SEQ ID NO:3 MSAEAKVRLKHCPTAEDRAAADALLAQLPGGDRALDRGFDEPWQLRAFALAVAACRAGRFEWKQLQQALISSIGEWERTHDLDDPSWSYYEHFVAALESVLGEEGIVEPEALDERTAEVLANPPNKDHHGPHLEPVAVHPAVRS* Variant DSM43832 nhhB: beta subunit: nucleic acid coding sequence SEQ ID NO:4 ATGAACGGCGTTTACGACGTTGGCGGCACCGACGGCCTGGGTCCAATCAACCGCCCAGCAGACGAGCCAGTTTTCCGCGCTGAGTGGGAGAAGGTTGCATTCGCTATGTTCCCAGCAACCTTCCGCGCTGGCTTCATGGGCCTGGACGAGTTCCGCTTCGGCATCGAGCAGATGAACCCAGCAGAGTACCTGGAGTCCCCATACTACTGGCACTGGATCCGCACCTACATCCACCACGGCGTTCGCACCGGCAAGATCGACCTGGAGGAGCTGGAGCGTCGCACCCAGTACTACCGCGAGAACCCAGACGCTCCACTGCCAGACCACGAGCAGAAGCCAGAGCTGATCGAGTTCGTTAACCAGGCAGTTTACGGCGGCCTGCCAGCTTCCCGCGAGGTTGACCGCCCACCAAAGTTCAAGGAAGGCGACGTTGTTCGCTTCTCCACCGCATCCCCAAAGGGTCACGCACGCCGCGCTCGCTACGTTCGCGGCAAGACCGGCACCGTTGTTAAGCACCACGGCGCATACATCTACCCAGACACCGCTGGTAACGGCCTGGGCGAGTGCCCAGAGCACCTGTACACCGTTCGCTTCACCGAGCAGGAGCTGTGGGGTCCAGAGGGCGACCCAAACTCCTCCGTTTACTACGACTGCTGGGAGCCATACATCGAGCTGGTTGACACCAAGGCAGCTGCAGCTTGA Variant DSM43832 nhhA alpha subunit: nucleic acid coding sequence: SEQ ID NO: 5 ATGACCGAGAACATCACCCGCAAGTCCGACGAGGAGATCCAGAAGGAGATCACCGTTCGCGTTAAGGCTCTGGAGTCCATGCTGATCGAGCAGGGCATCCTGACCACCTCTATGATCGACCGCATGGCAGAGATCTACGAGAACGAGGTTGGCCCACACCTGGCGTAAGGTTGTTGTTAAGGCATGGACCGACCCAGAGTTCAAGAAGCGCCTGCTGGCTGACGGTACCGAGGCATGCAAGGAGCTGGGTATCGGCGGCCTGCAGGAGGACATGATGTGGGTTGAGACACCGACGAGGTTCAC CACGTTGTTGTTTGCACCTGTGCTCCTGCTACCCATGGCCAGTTCTGGGCCTGCCACAAACTGGTACAAGGAGCCACAGTACCGCTCCCGCGTGTTCGCGAGCCAGCCCAGCTGCTGAAGGAAGAGTTCGGCTTCGAGGTTCCACCATCCAAGGAGATCAAGGTTTGGGACTCCTCCTCCGAGATGCGCTTCGTTGTTCTGCCCACAGGCCCAGCTGGTACCGACGGTTGGTCGAAGGAGCTGGCAACCCTGGTTACCCGCGAGTCCATGATCGGCGTTGAGCCAGCAAAGGCTGTTGCCTGA. バレリアントDSM43832 nhhG フィックックックショートローミン: nucleic acid code sequence: sequence number6 ATGTCCGCTGAGGCAAAGGTTCGCCTGAAGCACTGCCCAACCGCAGAGGACCGCGCAGCTGCAGACGCACTGCTGGCTCAGCTGCCAGGCGGCGACCGCGCACTGGACCGCGGCTTCGACGAGCCATGGCAGCTGCGCGCTTTCGCACTGGCTGTTGCAGCATGCCGCGCAGGCCGCTTCGAGTGGAAGCAGCTGCAGCAGGCTCTGATCTCCTCCA TCGGCGAGTGGGAGCGCACCCACGACCTGGACGACCCATCCTGGTCCTACTACGAGCACTTCGTTGCTGCACTGGAGTCCGTTCTGGGCGAGGAAGGCATCGTTGAGCCAGAGGCACTGGACGAGCGCACCGCAGAGGTTCTGGCTAACCCACCAAACAAGGACCACCACGGCCCACACCTGGAGCCAGTTGCAGTTCACCCAGCTGTTCGCTCCTAA Variant DSM43832 nhhBAG operon (containing NdeI / XhoI restriction sites used for cloning of the DSM43832 strain nhhBAG operon and lacking the amidase gene present in the endogenous operon upstream of nhhB): SEQ ID NO: 7 Wild-type DSM43832 nhhBAG operon (lacking the amidase gene present in the endogenous operon upstream of nhhB): SEQ ID NO:8

[0112] After describing exemplary embodiments of the invention, the invention is further described in the following claims.

Claims

1. 1. A variant nitrile hydratase comprising: (i) an alpha subunit (nhhA) having an amino acid sequence having at least 98, 99, or 100% sequence identity to SEQ ID NO:2; and (ii) a beta subunit (nhhB) comprising an amino acid sequence having at least 98, 99, or 100% sequence identity to SEQ ID NO:1, wherein the alpha subunit comprises one, two, or three of the following mutations: L6T, A19V, and F126Y; and the beta subunit comprises one or both of the following mutations: E108D and A200E, or all three of the following mutations: E108R, A200E, and S212Y; A variant nitrile hydratase, wherein said variant nitrile hydratase has enhanced stability and / or activity.

2. (i) the alpha subunit contains the following mutations: L6T, A19V, and F126Y, and the beta subunit contains the following mutations: E108D and A200E, or E108R, A200E, and S212Y; or (ii) the enzyme subunit is expressed together with an nhhG activator protein, optionally an nhhG activator protein comprising an amino acid sequence that is at least 98, 99, or 100% sequence identical to SEQ ID NO:3; (iii) the nitrile hydratase comprises a soluble enzyme or a cell that produces the nitrile hydratase; (iv) the nitrile hydratase is immobilized on a solid support; (v) the nitrile hydratase is optionally encapsulated, e.g., in a vesicle, sol-gel matrix, or other material that provides improved thermal stability compared to the enzyme in solution; or (vi) The nitrile hydratase according to claim 1, which is any combination of (i) to (v).

3. One or more nucleic acids encoding a nitrile hydratase comprising an alpha subunit and a beta subunit according to claim 1 or 2 or 3, and optionally an activator protein, wherein the nucleic acids encoding one or more of the alpha subunit, the beta subunit, and optionally the activator protein are codon-optimized to increase expression in a desired microorganism, optionally a yeast, a fungus, or a bacterium, or further optionally, the nucleic acids encoding one or more of the alpha subunit, the beta subunit, and optionally the activator protein are codon-optimized to increase expression in C. glutamicum ATCC13032 or its derivative MB001(DE3) deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under strain number 102071.

4. (i) The desired microorganism is selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, , Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Se rratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium erium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobas idium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyverom or the microorganism may be selected from bacteria of the genera Rhodococcus, Pseudomonas, Escherichia, and Geobacillus, or from the following species Rhodococcus: rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderiacenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp. vvificola、Mrrozobiumm ciceri、rerrzzabiumopportunistumm、Mbrrzzobium sp F28、Moraxella、Pantoea endophytica、Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperial, Corynebacterium nitrophilus, Corynebacterium pseudodiphthericum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFM-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1、Hanseniaspoora、Kluyveromycesthermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp. 163, optionally wherein the microorganism comprises a bacterium of the species Corynebacterium glutamicum, further optionally comprising C. glutamicum strain ATCC13032 or its derivative MB001(DE3); (ii) the beta subunit is encoded by a nucleic acid of SEQ ID NO:4; (iii) the alpha subunit is encoded by a nucleic acid of SEQ ID NO:5; (iv) the activator protein is encoded by a nucleic acid of SEQ ID NO:6; or (v) One or more nucleic acids according to claim 3, which is any combination of (i) to (iv).

5. 5. A nitrile hydratase operon comprising a nucleic acid encoding the alpha (nhhA) and beta (nhhB) subunits of a variant nitrile hydratase of claim 1 or 2 and optionally an activator protein (nhhG), optionally wherein the nucleic acid is a nucleic acid of claim 3 or 4.

6. 6. The operon of claim 5, derived from a yeast, fungus, or bacterium expressing a nitrile hydratase, optionally a Pseudomonas bacterium, and further optionally from a Pseudonocardia thermophila bacterium.

7. 7. The operon of claim 5 or 6, comprising SEQ ID NO:

7.

8. An extrachromosomal sequence, optionally a plasmid, comprising a nucleic acid or an operon according to any one of claims 3 to 7.

9. A microorganism, optionally a yeast, a fungus or a bacterium, further optionally an industrial microorganism, optionally not endogenously expressing a nitrile hydratase or endogenously expressing a nitrile hydratase, wherein the microorganism has been engineered to contain a nucleic acid encoding the nitrile hydratase according to claim 1 or 2, or optionally a nucleic acid encoding said nitrile hydratase according to any one of claims 3 to 8, or an operon or extrachromosomal sequence comprising said nucleic acid, further optionally one or more of said nucleic acids are contained in one or more extrachromosomal sequences (plasmids) or integrated into the chromosomal DNA of said microorganism.

10. Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Brevibacterium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhi Rhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium "m、]mmmemmmmemmememmememmememme idiom、Candida、Cry~acoccus、Débaryomyces、Geotrichumm、Hanseniaspo{a、Kluyver omyces、Pichia、Rhōdototorula、Comoomonas、-Py｡ococcus ccus, Pseudomonas, Escherichia, and Geobacillus are selected from bacteria, or the following species rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opaque, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus、Bacillus smithii、Bacillus sp BR449、Brady.-:LOOOOOOOOOOOOOOOOOOOOOOO Japanesegladioli, Escherichia coli, Geobacillus sp. ciceri、Mesorhizobiumoppreabiumm、Mesorhizabium sp F28、Moraxella、Pantoea endophytica、Pantoea agglomerans、Pseudomonas Chlorophagus, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperialis, Corynebacterium nitrophilus, Corynebacterium pseudodiphthericum, Corynebacterium glutamicum, Corynebacterium glutamicum, Corynebacterium hoffmannii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFM-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1、Hanseniaspoora、Kluyveromyces10. The microorganism according to claim 9, wherein the microorganism is selected from P. thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosterone, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, or Nocardia sp. 163, and optionally a bacterium of the species Corynebacterium glutamicum, optionally the C. glutamicum strain ATCC13032 or its derivative MB001(DE3).

11. 11. The microorganism according to claim 9 or 10, which is an industrial microorganism that endogenously expresses a nitrile hydratase, and said nucleic acid or operon replaces said endogenous nitrile hydratase gene or an operon containing said endogenous nitrile hydratase gene.

12. A method for producing an amide compound from a nitrile compound, the method comprising contacting the nitrile compound with the nitrile hydratase according to any one of claims 1 to 11 or a microorganism expressing the nitrile hydratase.

13. 13. The method of claim 12, wherein the amide compound is selected from the group consisting of acrylamide, methacrylamide, acetamide, and nicotinamide, preferably acrylamide, and the nitrile compound is selected from the group consisting of acrylonitrile, methacrylonitrile, acetonitrile, and 3-cyanopyridine, preferably acrylonitrile.

14. 14. The method according to claim 12 or 13, which uses one or more of soluble nitrile hydratase, encapsulated nitrile hydratase, immobilized nitrile hydratase, or whole cell or lysed microbial cell biocatalysts containing said nitrile hydratase.

15. 15. The method of any one of claims 12, 13 or 14, using an intact microbial cell biocatalyst, which may optionally be fresh (i.e. directly from fermentation), stored (e.g. stored frozen (wet frozen)), or dried, e.g. a lyophilisate or spray dried form thereof.

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