Method for producing L-leucine and L-isoleucine by using halophilic bacteria

Abstract

The invention relates to a recombinant halomonas, which comprises an ilvC (acetohydroxy acid isoreductase, AHAIR) gene, an ilvD (dihydroxy acid dehydratase, DHAD) gene and an optional ilvE (transaminase, TA) gene, wherein the ilvC (acetohydroxy acid isoreductase, AHAIR) gene and the ilvD (dihydroxy acid dehydratase, DHAD) gene are externally introduced. According to the recombinant halomonas disclosed by the invention, the L-leucine and / or the L-isoleucine can be efficiently produced without sterilization, in an energy-saving and water-saving manner through open type fermentation, and the industrial production of the recombinant halomonas is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of metabolic engineering biotechnology, and more specifically, to a method for producing L-leucine and L-isoleucine by utilizing halophilic bacteria. Background Art

[0002] L-leucine is an essential amino acid. Adult males need 2.2g / day. It is necessary for the normal growth of infants and the maintenance of normal nitrogen balance in adults. This product is used as a nutritional supplement, to prepare amino acid infusion and comprehensive amino acid preparations, hypoglycemic agents, and plant growth promoters. According to my country's GB 2760-86 regulations, this product can be used as a spice. L-leucine can be used as amino acid infusion and comprehensive amino acid preparations for the diagnosis and treatment of idiopathic hyperglycemia in young children, and is suitable for glucose metabolism disorders, liver diseases with decreased bile secretion, anemia, poisoning, muscular atrophy, sequelae of poliomyelitis, neuritis and mental illness.

[0003] As an essential amino acid, L-isoleucine has multiple physiological functions and plays an important regulatory role in human and animal health.

[0004] Can produce L-leucine and L-isoleucine by microbial fermentation, but have deficiencies.For example, Corynebacterium glutamicum (Corynebacterium glutamicum) is the main strain of L-isoleucine fermentation production, mainly obtains by screening and random mutagenesis, its genetic background is unclear and is difficult to further improve output by mutagenesis, thus produces L-isoleucine by metabolic engineering Corynebacterium glutamicum and becomes research hotspot in recent years.But Corynebacterium glutamicum needs strict sterilization before fermentation, is easy to contaminate bacteria in fermentation process.In addition, be difficult to realize the metabolic balance between upstream and downstream flux, cause strain growth slow, L-leucine and / or L-isoleucine output is low.

[0005] The halophilic microorganism Halomonas bluephagenesis TD01 (preservation number CGMCC No. 4353) screened from Xinjiang Aidin Lake disclosed in CN102120973A is a PHA super-producing bacterium. It can grow and accumulate PHA under high salt and high pH conditions, thereby achieving continuous fermentation and continuous and efficient production of PHA under non-sterilized conditions. Halomonas bluephagenesis TD1.0 is a strain obtained by inserting the lacI gene (facilitating IPTG-induced target gene expression) on the basis of Halomonas bluephagenesis TD01 (see Zhao, H. et al. (2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140).

[0006] CN111607551A discloses a method for producing aspartic acid and glutamic acid using recombinant halophilic bacteria, wherein experimental results show that the isoleucine production of wild-type Halomonas bluephagenesis TD01 and Halomonas aydingkolgenesis M1 is 0 g / L.

[0007] Therefore, how to transform halophilic bacteria into chassis bacteria that produce L-leucine and L-isoleucine in high yields is very challenging and economically valuable. Summary of the invention

[0008] In view of the above problems, the inventors conducted in-depth research, utilized the next generation industrial technology, used the extreme microorganism Halomonas as the chassis strain, constructed the L-leucine and / or L-isoleucine biosynthesis pathway from scratch, solved the above problems, and finally completed the present invention. For specific metabolic pathways, see Figure 1 .

[0009] Therefore, according to one aspect of the present invention, a recombinant Halomonas is provided, which comprises exogenously introduced ilvC (acetohydroxyacid isomeroreductase, AHAIR) gene and ilvD (dihydroxyacid dehydratase, DHAD) gene.

[0010] In one embodiment, the Halomonas is Halomonas bluephagenesis, Halomonasaydingkolgenesis or Halomonas campaniensis, preferably Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD01 (culture collection number CGMCC No.4353), Halomonasaydingkolgenesis M1 (culture collection number CGMCC No.19880), Halomonas campaniensis LS21 (culture collection number CGMCC No.6593).

[0011] In another embodiment, the ilvC gene and the ilvD gene are expressed by an inducible promoter or a constitutive promoter, preferably the inducible promoter is selected from lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof, preferably the constitutive promoter is selected from wild-type Pporin promoter (SEQ ID No. 1) or a mutant thereof, wherein the mutant is selected from Pporin42 (SEQ ID No. 2), Pporin51 (SEQ ID No. 3), Pporin58 (SEQ ID No. 4), Pporin68 (SEQ ID No. 5), Pporin140 (SEQ ID No. 6), Pporin141 (SEQ ID No. 7), Pporin194 (SEQ ID No. 8), Pporin221 (SEQ ID No. 9), Pporin226 (SEQ ID No. 10) and Pporin278 (SEQ ID No. 11), preferably Pporin42 (SEQ ID No. 2), Pporin58 (SEQ ID No. 3), Pporin58 (SEQ ID No. 4), Pporin68 (SEQ ID No. 5), Pporin140 (SEQ ID No. 6), Pporin141 (SEQ ID No. 7), Pporin194 (SEQ ID No. 8), Pporin221 (SEQ ID No. 9), Pporin226 (SEQ ID No. 10) and Pporin278 (SEQ ID No. 11). ID No.4) and Pporin68 (SEQ ID No.5), more preferably Pporin58 (SEQ ID No.4).

[0012] In another preferred embodiment, the ilvC gene and the ilvD gene are derived from Escherichia coli, preferably the amino acid sequence encoded by the ilvC gene is shown in SEQ ID No.13, more preferably the nucleotide sequence of the ilvC gene is shown in SEQ ID No.12; and / or preferably the amino acid sequence encoded by the ilvD gene is shown in SEQ ID No.15, more preferably the nucleotide sequence of the ilvD gene is shown in SEQ ID No.14.

[0013] In another preferred embodiment, the recombinant Halomonas further comprises an exogenously introduced ilvE (transaminase, TA) gene.

[0014] In another preferred embodiment, the ilvE gene is derived from Escherichia coli, and preferably the amino acid sequence encoded by the ilvE gene is shown as SEQ ID No.17, and more preferably the nucleotide sequence of the ilvE gene is shown as SEQ ID No.16.

[0015] In another preferred embodiment, the ilvC gene, ilvD gene and optionally ilvE gene are expressed on one or more plasmids or integrated into the genome of the recombinant Halomonas.

[0016] In another preferred embodiment, the gene is introduced into Halomonas by conjugative transformation.

[0017] In another preferred embodiment, the recombinant Halomonas overexpresses the ilvC gene, the ilvD gene and the optional ilvE gene. The ilvC gene, the ilvD gene and the optional ilvE gene or a combination thereof are operably linked to the above-mentioned promoter, respectively.

[0018] According to another aspect of the present invention, a method for producing L-leucine is provided, the method comprising fermenting and culturing the recombinant Halomonas comprising the exogenously introduced ilvC gene and ilvD gene according to the present invention. In a preferred embodiment, the culturing is carried out with or without the addition of an inducer such as IPTG.

[0019] According to another aspect of the present invention, there is provided a method for producing L-isoleucine, the method comprising fermenting and culturing the recombinant Halomonas comprising the exogenously introduced ilvC gene, ilvD gene and ilvE gene according to the present invention. In a preferred embodiment, the culturing is carried out with or without the addition of an inducer such as IPTG.

[0020] The present invention designs a substrate scaffold and improves substrate utilization by expressing a specially selected gene combination (i.e., a combination of the ilvC gene and the ilvD gene and an optional ilvE gene) of the synthesis pathway of L-leucine / L-isoleucine in Halomonas, thereby realizing efficient production of L-leucine and / or L-isoleucine by open fermentation without sterilization, energy saving and water saving, and facilitating industrial production thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0022] Figure 1A metabolic pathway for the synthesis of L-leucine and L-isoleucine by recombinant Halomonas; and

[0023] Figure 2 This is the pI01 plasmid map. DETAILED DESCRIPTION

[0024] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in the art, it is particularly recommended that the technician refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0025] In the present invention, the singular articles "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety.

[0026] In the present invention, the term "halophiles" refers to a type of archaea living in a high salinity environment. In a preferred embodiment of the present invention, the halophiles include but are not limited to the genus Halomonas. Preferably, the genus Halomonas includes but is not limited to Halomonas bluephagenesis, Halomonasaydingkolgenesis or Halomonas campaniensis or its derivatives, more preferably Halomonasbluephagenesis TD1.0, Halomonas bluephagenesis TD01 (strain collection number CGMCC No.4353), Halomonas aydingkolgenesis M1 (strain collection number CGMCC No.19880), Halomonascampaniensis LS21 (strain collection number CGMCC No.6593).

[0027] In the present invention, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables a specific gene to be transcribed. The promoter can be recognized by RNA polymerase and begin to transcribe and synthesize RNA. In ribonucleic acid (RNA) synthesis, the promoter can interact with the transcription factor that regulates gene transcription to control the start time and degree of gene expression (transcription). The promoter includes a core promoter region and a regulatory region, which is located in the regulatory sequence that controls gene expression and upstream of the gene transcription start site (5' direction of the DNA antisense strand), and has no compilation function itself. According to its mode of action and function, it is divided into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue specificity or developmental period specificity) and inducible promoters (regulated by external chemical or physical signals).

[0028] There is no particular restriction on the type of promoter for the expression or overexpression of the exogenously introduced ilvC gene, ilvD gene and / or ilvE gene of the present invention, which may be an inducible promoter or a constitutive promoter. For example, the promoter may be a promoter for IPTG-induced expression. In a specific embodiment, the inducible promoter is selected from lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof. In a specific embodiment, the constitutive promoter is selected from wild-type Pporin promoter or a mutant thereof, wherein the mutant is selected from Pporin42, Pporin51, Pporin58, Pporin68, Pporin140, Pporin141, Pporin194, Pporin221, Pporin226 and Pporin278, preferably Pporin42, Pporin58 and Pporin68, more preferably Pporin58.

[0029] The nucleotide sequence of an exemplary promoter used in the embodiments of the present invention is as follows:

[0030] Wild-type Pporin:

[0031] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatagagtTT GACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACA GCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag (SEQ ID No. 1)

[0032] Pporin42:

[0033] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCACTAtagagtTT GACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACA GCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.2)

[0034] Pporin51:

[0035] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCATTTtagagtTT GACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACA GCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.3)

[0036] Pporin58:

[0037] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAATAtagagtTT GACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.4)

[0038] Pporin68:

[0039] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAGACtagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.5)

[0040] Pporin140:

[0041] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatAAGAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.6)

[0042] Pporin141:

[0043] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatATACtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.7)

[0044] Pporin194:

[0045] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatCTAAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.8)

[0046] Pporin221:

[0047] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatGCTAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag(SEQ ID No.9)

[0048] Pporin226:

[0049] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatAAAGtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACA GCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag (SEQ ID No. 10)

[0050] Pporin278:

[0051] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatTAAAtTT GACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACA GCCTCTACAAATAATTTTGTTTAAgagttatactagagaaagaggagaaatactag (SEQ ID No. 11)

[0052] In the present invention, the term "operably connected" generally refers to placing the regulatory sequence necessary for the expression of the coding sequence in a suitable position relative to the coding sequence so as to achieve the expression of the coding sequence. For example, when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. In some embodiments, the arrangement of the coding sequence and the transcription control element in the expression vector can be represented. The control element can include a promoter, an enhancer and a termination element. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably connected to the coding sequence. In some embodiments, "operably connected" can also refer to that the target gene is connected to the vector so that the transcription and translation control sequences in the vector play their intended functions of regulating the transcription and translation of the target gene.

[0053] In the present invention, the term "overexpression" means that a gene is transcribed at a high rate, resulting in a large amount of mRNA production. For the overexpression of the ilvC gene, ilvD gene and ilvE gene of the present invention, it means that the DNA or RNA expression level thereof is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300% higher than that of a control (a negative plasmid control or a host strain not containing these genes), or even 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 times or more the expression level of the ilvC gene, ilvD gene and ilvE gene in the control.

[0054] There is no particular limitation on the source type of the exogenously introduced ilvC gene, ilvD gene and / or ilvE gene used in the present invention. In a specific embodiment, the ilvC gene, ilvD gene and / or ilvE gene are derived from Escherichia coli. Exemplary ilvC gene, ilvD gene and ilvE gene derived from Escherichia coli and the amino acid sequences encoded by them used in the embodiments of the present invention are as follows:

[0055] Nucleotide sequence of the ilvC gene from Escherichia coli (SEQ ID No. 12)

[0056]

[0057] Amino acid sequence encoded by the ilvC gene from E. coli (SEQ ID No. 13)

[0058] MANYFNTLNLRQQLAQLGKCRFMGRDEFADGASYLQGKKVVIVGCGAQGLNQGLNMRDSGLDISYALRKEAIAEKRASWRKATENGFKVGTYEELIPQADLVINLTPDKQHSDVVRTVQPLM KDGAALGYSHGFNIVEVGEQIRKDITVVMVAPKCPGTEVREEYKRGFGVPTLIAVHPENDPKGEGMAIAKAWAAATGGHRAGVLESSFVAEVKSDLMGEQTILCGMLQAGSLLCFDKLVEEGT DPAYAEKLIQFGWETITEALKQGGITLMMDRLSNPAKLRAYALSEQLKEIMAPLFQKHMDDIISGEFSSGMMADWANDDKKLLTWREETGKTAFETAPQYEGKIGEQEYFDKGVLMIAMVKAG VELAFETMVDSGIIEESAYYESLHELPLIANTIARKRLYEMNVVISDTAEYGNYLFSYACVPPLKPFMAELQPGDLGKAIPEGAVDNGQLRDVNEAIRSHAIEQVGKKLRGYMTDMKRIAVAG

[0059] Nucleotide sequence of the ilvD gene from Escherichia coli (SEQ ID No. 14)

[0060]

[0061] Amino acid sequence encoded by the ilvD gene from E. coli (SEQ ID No. 15)

[0062] MPKYRSATTTHGRNMAGARALWRATGMTDADFGKPIIAVVNSFTQFVPGHVHLRDLGKLVAEQIEAAGGVAKEFNTIAVDDGIAMGHGGMLYSLPSRELIADSVEYMVNAHCADAMVCISNCDKITPGMLMASLRLNIPVIFVSGGPMEAGKTK LSDQIIKLDLVDAMIQGADPKVSDSQSDQVERSACPTCGSCSGMFTANSMNCLTEALGLSQPGNGSLLATHADRKQLFLNAGKRIVELTKRYYEQNDESALPRNIASKAAFENAMTLDIAMGGSTNTVLHLLAAAQEAEIDFTMSDIDKLSRKV PQLCKVAPSTQKYHMEDVHRAGGVIGILGELDRAGLLNRDVKNVLGLTLPQTLEQYDVMLTQDDAVKNMFRAGPAGIRTTQAFSQDCRWDTLDDDRANGCIRSLEHAYSKDGGLAVLYGNFAENGCIVKTAGVDDSILKFTGPAKVYESQDDAV EAILGGKVVAGDVVVIRYEGPKGGPGMQEMLYPTSFLKSMGLGKACALITDGRFSGGTSGLSIGHVSPEAASGGSIGLIEDGDLIAIDIPNRGIQLQVSDAELAARREAQDARGDKAWTPKNRERQVSFALRAYASLATSADKGAVRDKSKLGG

[0063] Nucleotide sequence of the ilvE gene from Escherichia coli (SEQ ID No. 16)

[0064] TTATTGATTAACTTGATCTAACCAGCCCCATTTATCTTCGGTTTCGCCAGTGAAGAGGCCGAAGAAGGCTTGCTGAATGCGTTTGGTAACCGGGCCACAACGGCCTTCGCCAACCTGAATACCGTCTACGCTGCGCACTGGCGTGATTTCTGCCGCCGTACCGGACATAAACACTTCATCCGCCAGGTACAGGGATTCGCGCGACAGCACCTGCTCACGTACTTCAATTCCCAGCTCTTTCGCCAGTTTGATGATGGCATCACGGGTAATACCCGGCAGCGCGGAGGAGGTGAACGGTGGGGTGAACAGCACACCATCTTTCACTTCAAACAGGTTTTCGCCTGCGCCTTCAGAGATATAACCGTTCACATCCAGCGCGATACCTTCCTGATAACCGTGGCGGCGCGCTTCGCTACCCACCAGCAGGGAAGAGAGGTAGTTACCACCGGCTTTTGCCGCCGTCGGGATGGTGTTTGGTGCTGCGCGGTTCCAGGAGGAAACCATCGCATCGATCCCCTGCTCCAGCGCTTCTGCGCCCAGATACGCTCCCCACGGGAAAGCAGCGATAATCACGTCGGTTGAGTATCCCGCTGGCGGGTTTACTCCCATGCCAACATCACCGACGAAGATCAGCGGACGGATATAGGCGCTGGTGAGATTGTTTTTGCGGATCACGTCACGACAAGCTTCCATCAGCTCATCAATGCTCTGCGAAACCGGGAAGCGATAGATTTTGGCGGAGTCATGCAGACGCTGCATATGCTCACGATGGCGGAATACAACCGGTCCTTTGTGCGAGTCGTAGCAACGGATGCCTTCAAAAACCGAAGTGCCATAGTGCAGCGCGTGCGACATCACATGCACCTTCGCGTCTTCCCAGCGAACCATCTCCCCATTGAACCAAATGTAATCAGCTTTCTTCGTGGTCAT

[0065] Amino acid sequence encoded by the ilvE gene from Escherichia coli E. coli (SEQ ID No. 17)

[0066] MTTKKADYIWFNGEMVRWEDAKVHVMSHALHYGTSVFEGIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFPVSQSIDELMEACRDVIRKNNLTSAYIRPLIFVGDVGMGVNPPAGYSTDVIIAAFPWGAYLGAEALEQGIDAMVSSWNRAAPNT IPTAAKAGGNYLSSLLVGSEARRHGYQEGIALDVNGYISEGAGENLFEVKDGVLFTPPFTSSALPGITRDAIIKLAKELGIEVREQVLSRESLYLADEVFMSGTAAEITPVRSVDGIQVGEGRCGPVTKRIQQAFFGLFTGETEDKWGWLDQVNQ

[0067] Methods for introducing exogenous ilvC gene, ilvD gene and / or ilvE gene into the Halomonas to be transformed are well known to those skilled in the art. In a specific embodiment, the gene is introduced into the Halomonas by conjugation transformation. For example, the ilvC gene, ilvD gene and / or ilvE gene can be expressed on one or more plasmids or integrated into the genome of the recombinant Halomonas. The plasmid can be an expression plasmid or a gene editing plasmid, preferably a CRISPR-Cas9 gene editing plasmid.

[0068] The present invention also provides a method for producing L-leucine or L-isoleucine, which comprises fermenting and culturing the recombinant Halomonas according to the present invention. In the present invention, the fermentation can be "aerobic fermentation" or "anaerobic fermentation". In the present invention, the term "aerobic fermentation" refers to growth in the presence of oxygen. In the present invention, the term "anaerobic fermentation" refers to growth in the absence of oxygen.

[0069] The present invention is further described in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The chemical substances used in the following reactions are all commercially available products unless otherwise indicated.

[0070] Escherichia coli was grown in LB medium, and the medium composition contained: 10 g / L sodium chloride, 10 g / L peptone (OXID Company, UK, product catalog number LP0042), and 5 g / L yeast extract (OXID Company, UK, product catalog number LP0021).

[0071] Halomonas bluephagenesis TD1.0 was cultured on LB60 medium, which had the same ingredients as LB except that the concentration of sodium chloride was changed to 60 g / L.

[0072] Halomonas bluephagenesis TD1.0 See Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD1.0 (Tan et al., Bioresource Technology, 2011 Volume 102, Issue 17, September 2011, Pages 8130-8136).

[0073] For the CRISPR genome editing method, please refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al., Metabolic Engineering, 2018 May: 47: 219-229).

[0074] For the genome editing sites described in this patent, refer to the document CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al., Metabolic Engineering, 2018, May: 47: 219-229).

[0075] L-Leucine

[0076] Culture medium:

[0077] 60LB medium: 60g / L sodium chloride, 5g / L yeast extract, 10g / L tryptone

[0078] Basic culture medium: 20-40g / L glucose, 55-70g / L sodium chloride, 1-10g / L yeast extract, 3-6g / L urea, 1.5-5.2g / L potassium dihydrogen phosphate, 0.2-0.4g / L magnesium sulfate, 8.5-10g / L sodium dihydrogen phosphate, 7-15ml / L component III, 1-5ml / L component IV.

[0079] Component III: 5g / L ammonium ferric citrate, 2g / L calcium chloride dihydrate, 41.7ml concentrated hydrochloric acid (12mol / L), add water to make up to 1000ml.

[0080] Component IV: 100 mg / L zinc sulfate heptahydrate, 30 mg / L manganese chloride tetrahydrate, 300 mg / L boric acid, 200 mg / L cobalt chloride hexahydrate, 10 mg / L anhydrous copper sulfate, 20 mg / L nickel chloride hexahydrate, 30 mg / L sodium molybdate dihydrate.

[0081] 1 g / L L-phenylalanine and 2 g / L sodium malonate were added during the fermentation process.

[0082] The above culture media can be prepared using standard preparation methods.

[0083] L-Leucine detection method:

[0084] After the fermentation is completed, the sample is first subjected to a derivatization reaction (DEEMM (Diethylethoxymethylenemalonate) derivatization method), and then further detected using an HPLC ultraviolet detection method (ultraviolet detection wave of 284 nm) to detect whether L-leucine is synthesized (reference: Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases, Aug 21; 23(9):2102).

[0085] Specific derivatization reaction (DEEMM derivatization method):

[0086] 540 μL of borate buffer, 200 μL of methanol, 10 μL of DEEMM, and 250 μL of bacterial supernatant were mixed evenly and reacted at 70°C for 2 h.

[0087] HPLC was performed on a HPLC with a C18 column (Agilent PN990967-902SUNH096763), mobile phase A was sodium acetate (80%), mobile phase B was acetonitrile (20%), and L-leucine was detected.

[0088] The specific sodium acetate ratio is 4.1 g of sodium acetate, which is added to 2 L of water, and then 675 μL of acetic acid is added to obtain a sodium acetate mobile phase.

[0089] L-Isoleucine detection method:

[0090] After the fermentation is completed, the sample is first subjected to a derivatization reaction (DEEMM derivatization method), and then further detected using an HPLC ultraviolet detection method (ultraviolet detection wave of 284 nm) to determine whether L-isoleucine is synthesized.

[0091] Specific derivatization reaction (DEEMM derivatization method):

[0092] 540 μL of borate buffer, 200 μL of methanol, 10 μL of DEEMM, and 250 μL of bacterial supernatant were mixed evenly and reacted at 70°C for 2 h.

[0093] HPLC was performed on a HPLC with a C18 column (Agilent PN990967-902SUNH096763), mobile phase A was sodium acetate (80%), mobile phase B was acetonitrile (20%), and L-isoleucine was detected.

[0094] The specific sodium acetate ratio is 4.1 g of sodium acetate, which is added to 2 L of water, and then 675 uL of acetic acid is added to obtain a sodium acetate mobile phase.

[0095] Example 1

[0096] Construction of a recombinant halophilic strain TDIA01 producing L-leucine

[0097] Construction of recombinant bacterium TDIA01

[0098] The E. coli ilvC gene and ilvD gene were inserted into the plasmid pSEVA321 (see the literature for plasmid: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41.D1 (2012):

[0099] D666-D675) expression frame, and obtain plasmid pSEVA321-mmp1-ilvC-ilvD, i.e., pI01 (see the plasmid map for details). Figure 2 ).

[0100] Plasmid pI01 was transformed into Halomonas bluephagenesis TD1.0 by conjugation. PCR (primer sequences for ilvC and ilvD genes are shown below) and sequencing were used to verify that ilvC and ilvD genes were expressed in Halomonas bluephagenesis TD1.0, and recombinant strain TDIA01 was obtained.

[0101] L1 gtgacggcagagagacaatcaaatcTTAACCCGCAACAGCAATACGTTTC(SEQ ID No.18)

[0102] L2 cgccagggttttcccagtcacgacATGCCTAAGTACCGTTCCGCCACCA(SEQ ID No.19)

[0103] L3 TGGTGGCGGAACGGTACTTAGGCATgtcgtgactgggaaaaccctggcg(SEQ ID No.20)

[0104] L4 GAAACGTATTGCTGTTGCGGGTTAAgatttgattgtctctctgccgtcac (SEQ ID No. 21)

[0105] Example 2

[0106] Shake flask verification of L-leucine accumulation by recombinant strain

[0107] The recombinant bacteria TDIA01 expressing ilvC and ilvD were inoculated into 20 mL of LB60 medium. After culturing for 12-16 hours, they were transferred to a new 20 mL of LB60 medium at a volume ratio of 1%, and continued to be cultured for 8-12 hours. Take 2.5 mL of seed bacterial liquid and inoculate it into a 500 mL conical flask containing 47.5 mL of basal medium. The IPTG concentrations were 0 mg / L, 10 mg / L, and 100 mg / L, respectively. Perform a shake flask experiment. The shaker temperature was 37 ° C and the speed was 200 rpm. After culturing for 48 hours, the content of L-leucine was detected. Three parallel experiments were set for each group of experiments, and the results were averaged.

[0108] The shake flask results showed that L-leucine was not synthesized at 0 mg / L IPTG, while L-leucine could be synthesized at 10 mg / L and 100 mg / L IPTG with yields of 15 g / L and 32 g / L, respectively.

[0109] Example 3

[0110] Constitutive expression system for L-leucine synthesis

[0111] Based on the plasmid pSEVA321, Pporin68, Pporin42 and Pporin58 promoters (reference: Promoter Engineering for Enhanced P(3HB-co-4HB)Production by Halomonasbluephagenesis) were used to express the ilvC gene and ilvD gene, respectively, to construct the induction system-related plasmids pSEV321-Pporin68-ilvC-ilvD, pSEV321-Pporin42-ilvC-ilvD, pSEV321-Pporin58-ilvC-ilvD, and named them pIA02, pIA03 and pIA04, respectively.

[0112] Example 4

[0113] Recombinant halophilic strains TDIA02, TDIA03 and TDIA04 producing L-leucine were constructed.

[0114] Plasmid pIA02, plasmid pIA03 and plasmid pIA04 were transformed into Halomonas bluephagenesis TD1.0 by conjugation transformation. PCR and sequencing were used to verify that ilvC and ilvD genes were expressed in Halomonas bluephagenesis TD1.0, and recombinant strains TDIA02, TDIA03 and TDIA04 were obtained respectively.

[0115] The verification primers for plasmid pIA02, plasmid pIA03 and plasmid pIA04 are:

[0116] L1 gtgacggcagagagacaatcaaatcTTAACCCGCAACAGCAATACGTTTC(SEQ ID No.18)

[0117] L2 cgccagggttttcccagtcacgacATGCCTAAGTACCGTTCCGCCACCA(SEQ ID No.19)

[0118] L3 TGGTGGCGGAACGGTACTTAGGCATgtcgtgactgggaaaaccctggcg(SEQ ID No.20)

[0119] L4 GAAACGTATTGCTGTTGCGGGTTAAgatttgattgtctctctgccgtcac (SEQ ID No. 21)

[0120] Example 5

[0121] Shake flask verification of L-leucine accumulation by recombinant strain

[0122] The recombinant bacteria TDIA02, TDIA03, and TDIA04 expressing ilvC and ilvD were inoculated into 20 mL of LB60 medium, respectively. After culturing for 12-16 hours, they were transferred to a new 20 mL of LB60 medium at a volume ratio of 1%, and continued to be cultured for 8-12 hours. Take 2.5 mL of seed bacterial liquid and inoculate it into a 500 mL conical flask containing 47.5 mL of basal medium. Perform a shake flask experiment. The shaker temperature is 37 ° C and the speed is 200 rpm. After 48 hours of culture, the L-leucine content was detected. Three parallel experiments were set for each group of experiments, and the results were averaged.

[0123] The results of the shake flasks showed that the contents of L-leucine synthesized by the recombinant bacteria TDIA02, TDAI03, and TDIA04 were 8 g / L, 19 g / L, and 38 g / L, respectively (no inducer needed to be added, thus reducing production costs).

[0124] Example 6

[0125] Construction of a recombinant halophilic strain TDIA05 producing L-isoleucine

[0126] The ilvC gene, ilvD gene and ilvE gene of Escherichia coli were inserted into the expression frame of plasmid pSEVA321 to obtain plasmid pSEVA321-mmp1-ilvC-ilvD-ilvE, i.e., pIA05.

[0127] The recombinant strain TDIA05 was obtained by transforming the ilvC, ilvD and ilvE genes into Halomonas bluephagenesis TD1.0 through conjugation. The expression of ilvC, ilvD and ilvE genes in Halomonas bluephagenesis TD1.0 was verified by PCR and sequencing.

[0128] The PCR verification primer sequences are as follows:

[0129] L1 gtgacggcagagagacaatcaaatcTTAACCCGCAACAGCAATACGTTTC(SEQ ID No.18)

[0130] L5 gccagggttttcccagtcacgacATGACCACGAAGAAAGCTGATTACA(SEQ ID No.22)

[0131] L4 GAAACGTATTGCTGTTGCGGGTTAAgatttgattgtctctctgccgtcac (SEQ ID No. 21)

[0132] L6 TGTAATCAGCTTTCTTCGTGGTCATgtcgtgactgggaaaaccctggc(SEQ ID No.23)

[0133] Example 7

[0134] Shake flask verification of L-isoleucine accumulation by recombinant strain

[0135] The recombinant bacteria TDIA05 expressing ilvC, ilvD and ilvE were inoculated into 20mL of LB60 medium, and after culturing for 12-16h, they were transferred to a new 20mL of LB60 medium at a volume ratio of 1%, and continued to be cultured for 8-12h. Take 2.5mL of seed bacterial liquid and inoculate it into a 500mL conical flask containing 47.5mL of basal medium, and IPTG was 0mg / L, 10mg / L, and 100mg / L respectively. Perform a shake flask experiment. The shaker temperature was 37°C and the speed was 200rpm. After culturing for 48h, the content of L-isoleucine was detected. Three parallel experiments were set for each group of experiments, and the results were averaged.

[0136] The shake flask results showed that L-isoleucine was not synthesized at 0 mg / L IPTG, while L-isoleucine could be synthesized at 10 mg / L and 100 mg / L IPTG with yields of 10 g / L and 22 g / L, respectively.

[0137] Example 8

[0138] Constitutive expression system for L-isoleucine synthesis

[0139] The inducible system-related plasmids pSEV321-Pporin68-ilvC-ilvD-ilvE, pSEV321-Pporin42-ilvC-ilvD-ilvE, and pSEV321-Pporin58-ilvC-ilvD-ilvE were constructed and named pI06, pI07, and pI08, respectively.

[0140] like Figure 1 As shown, these three genes convert a-acetyl-a-hydroxybutyrate into a,b-dihydroxyaminovaleric acid through ilvC, then into b-methyl-a-aminovaleric acid through ilvD, and finally into L-isoleucine through ilvE.

[0141] Example 9

[0142] Construction of a recombinant halophilic strain TDI02 producing L-isoleucine

[0143] Plasmid pI06, plasmid pI07 and plasmid pI08 were transformed into Halomonas bluephagenesis TD1.0 by conjugation transformation, respectively. PCR and sequencing were used to verify that ilvC, ilvD and ilvE genes were expressed in Halomonas bluephagenesis TD1.0, and TDI06, TDI07 and TDI08 were obtained.

[0144] The PCR verification primers for plasmid pI06, plasmid pI07 and plasmid pI08 are:

[0145] L1 gtgacggcagagagacaatcaaatcTTAACCCGCAACAGCAATACGTTTC(SEQ ID No.18)

[0146] L5 gccagggttttcccagtcacgacATGACCACGAAGAAAGCTGATTACA(SEQ ID No.22)

[0147] L4 GAAACGTATTGCTGTTGCGGGTTAAgatttgattgtctctctgccgtcac (SEQ ID No. 21)

[0148] L6 TGTAATCAGCTTTCTTCGTGGTCATgtcgtgactgggaaaaccctggc(SEQ ID No.23)

[0149] Example 10

[0150] Shake flask verification of L-isoleucine accumulation by recombinant strain

[0151] The recombinant bacteria TDI06, TDI07 and TDI08 expressing ilvC, ilvD and ilvE were inoculated into 20 mL of LB60 medium respectively. After culturing for 12-16 hours, they were transferred to new 20 mL of LB60 medium at a volume ratio of 1% and continued to be cultured for 8-12 hours. Take 2.5 mL of seed bacterial liquid and inoculate it into a 500 mL conical flask containing 47.5 mL of basal medium. Perform a shake flask experiment. The shaker temperature is 37 ° C and the speed is 200 rpm. After culturing for 48 hours, the content of L-isoleucine was detected. Three parallel experiments were set for each group of experiments, and the results were averaged.

[0152] The shake flask results showed that the contents of L-isoleucine synthesized by the recombinant bacteria TDI06, TDI07 and TDI08 were 5 g / L, 15 g / L and 31 g / L, respectively (no inducer was required, thus reducing production costs).

[0153] Embodiment 11

[0154] Shake flask verification of L-leucine and L-isoleucine accumulation in Halomonas campaniensis and Hialomonas aydlingholgenesis

[0155] The plasmid pIA04 which is superior to Halomonas bluephagenesis in synthesizing L-leucine was selected. According to the experimental method of the above example, pIA04 (pSEV321-Pporin58-ilvC-ilvD) was overexpressed in Halomonas campaniensis LS21, and the test results showed that the content of synthesized L-leucine was 12 g / L.

[0156] The plasmid pI08 which is better for synthesizing L-isoleucine by Halomonas bluephagenesis was selected. According to the experimental method of the above embodiment, the strain Hialomonas aydlingholgenesis M1 was overexpressed (pSEV321-Pporin58-ilvC-ilvD-ilvE), and the test results showed that the content of synthesized L-isoleucine was 17g / L.

[0157] Those skilled in the art should understand that, although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, without departing from the spirit of the present invention, those skilled in the art can make appropriate modifications or improvements, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. A recombinant Halomonas sp. comprising an exogenously introduced ilvC (acetohydroxyacid isomeroreductase, AHAIR) gene and an ilvD (dihydroxyacid dehydratase, DHAD) gene.

2. The recombinant Halomonas according to claim 1, wherein the Halomonas is Halomonas bluephagenesis, Halomonas aydingkolgenesis or Halomonas campaniensis, preferably Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD01 (culture collection number CGMCC No.4353), Halomonas aydingkolgenesis M1 (culture collection number CGMCC No.19880), Halomonas campaniensis LS21 (culture collection number CGMCC No.6593).

3. The recombinant salt mononas according to claim 1 or 2, wherein the ilvC gene and the ilvD gene are expressed by an inducible promoter or a constitutive promoter, preferably the inducible promoter is selected from lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof, preferably the constitutive promoter is selected from wild-type Pporin promoter or a mutant thereof, wherein the mutant is selected from Pporin42, Pporin51, Pporin58, Pporin68, Pporin140, Pporin141, Pporin194, Pporin221, Pporin226 and Pporin278, preferably Pporin42, Pporin58 and Pporin68, more preferably Pporin58.

4. The recombinant salt mononas according to any one of claims 1 to 3, wherein the ilvC gene and the ilvD gene are derived from Escherichia coli, preferably the amino acid sequence encoded by the ilvC gene is shown in SEQ ID No.13, more preferably the nucleotide sequence of the ilvC gene is shown in SEQ ID No.12; and / or preferably the amino acid sequence encoded by the ilvD gene is shown in SEQ ID No.15, more preferably the nucleotide sequence of the ilvD gene is shown in SEQ ID No.

14.

5. The recombinant Halomonas according to any one of claims 1 to 4, wherein the ilvC gene and the ilvD gene are expressed on one or more plasmids or integrated into the genome of the recombinant Halomonas. The recombinant Halomonas according to claim 5 , wherein the gene is introduced into Halomonas by conjugation transformation.

7. The recombinant Halomonas according to any one of claims 1 to 5, further comprising an exogenously introduced ilvE (transaminase, TA) gene.

8. The recombinant Halomonas according to claim 7, wherein the ilvE gene is derived from Escherichia coli, preferably the amino acid sequence encoded by the ilvE gene is as shown in SEQ ID No. 17, and more preferably the nucleotide sequence of the ilvE gene is as shown in SEQ ID No.

16.

9. A method for producing L-leucine, comprising fermenting and culturing the recombinant Halomonas according to any one of claims 1 to 8.

10. A method for producing L-isoleucine, the method comprising fermenting and culturing the recombinant Halomonas according to any one of claims 1 to 8, preferably fermenting and culturing the recombinant Halomonas according to claim 7 or 8.

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