WO2018099719A1 - Alcool-acétyl transférases pour la production d'alcanoate d'alkyle - Google Patents

Alcool-acétyl transférases pour la production d'alcanoate d'alkyle Download PDF

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WO2018099719A1
WO2018099719A1 PCT/EP2017/079111 EP2017079111W WO2018099719A1 WO 2018099719 A1 WO2018099719 A1 WO 2018099719A1 EP 2017079111 W EP2017079111 W EP 2017079111W WO 2018099719 A1 WO2018099719 A1 WO 2018099719A1
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seq
amino acid
identity
nucleotide sequence
acid sequence
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Aleksander Johannes KRUIS
Mark LEVISSON
Astrid Elisabeth MARS
Servatius Wilhelmus Maria KENGEN
John Van Der Oost
Johan Pieter Marinus Sanders
Ruud Alexander Weusthuis
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Wageningen Universiteit
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01084—Alcohol O-acetyltransferase (2.3.1.84)

Definitions

  • the invention relates to process for the production of alkyl alkanoates from alcohol and Acyl coenzyme A using polypeptides having alcohol acetyl transferase activity (hereinafter referred to as AAT).
  • AAT alcohol acetyl transferase activity
  • the process is further directed to the use of a recombinant expression vector or plasmid or host cell comprising the nucleotide sequence encoding the polypeptide.
  • Alcohol acetyl transferase catalyzes the transfer of the acetyl group of acetyl-coenzyme A to an alcohol (EC2.3.1 .84) and produces Coenzyme A and the corresponding ester.
  • Ethyl acetate is an environmentally friendly solvent with many industrial applications. Also, the higher alkyl alkanoates are interesting compounds for the industry. The production of ethyl acetate currently proceeds by energy-intensive petrochemical processes which are based on natural gas and crude oil without exception. The following methods play a larger role in industrial-scale production (Lin et al. 1998; Colley et al.2004; Arpe 2007);
  • Fischer esterification reaction of ethanol with acetic acid in the presence of strong acids as a catalyst, in part combined with removal of water for shifting the equilibrium toward the ester
  • Tischtschenko reaction addition of two equivalents acetaldehyde by disproportionation under the action of alkoxides, especially aluminum triethoxide, as a catalyst.
  • ethanol is dehydrogenated to acetaldehyde, the aldehyde is then added to ethanol to form a hemiacetal which in turn is dehydrogenated to generate ethyl acetate
  • Avada process developed by BP Chemicals Avada abbreviates Advanced acetates by direct addition" and means synthesis of ethyl acetate by catalytic addition of ethylene and acetic acid in the gas phase
  • Ethanol as a precursor for chemical synthesis of ethyl acetate could be produced from sugar by fermentation (Silveira et al. 2005; Aziz et al. 2009; Guimaraes et al. 2010; Rodrussamee et al. 201 1 ), however the direct microbial production of alkyl esters such as ethyl acetate would provide more advantages. Microbial synthesis of alkyl alkanoate could become an interesting alternative. Although the ability of yeasts for producing larger amounts of this ester is known for a long time, these native microorganisms are not able to produce acetate esters in yields that are suitable for industrial bulk production.
  • Hemiacetal dehydrogenase reaction wherein alcohol and acetaldehyde form hemiacetal, which is formed into alkyl acetate, and
  • AAT Alcohol acetyl transferase
  • Eht1 and Eeb1 produce medium chain ethyl esters in S. cerevisiae. They do not resemble Atf1 and Atf2 on a protein level and contain an alpha/beta hydrolase fold.
  • the reaction typically associated with alpha/beta hydrolases is hydrolysis, but Eht1 and Eeb1 show AAT, thioesterase and esterase activities.
  • Eht1 and Eeb1 are not known to produce ethyl acetate, but since no combined gene deletions of Eht1 and Eeb1 and aft 1 and aft2 have been reported, their involvement in the remaining ethyl acetate synthesis is unconfirmed.
  • An object of this invention is to provide a process for the production of alkyl alkanoates using a polypeptide with alcohol acetyl transferase activity to convert:
  • the alkanoate may be an acetate, but also higher carboxyl acid residues up to decanoyi may be formed, thus, the term "C3-C10 acyl".
  • C3-C10 alkanol it is indicated that propanol up to decanol, i.e. C10 alcohol, may be converted.
  • Both the alcohols and the acyls may concern linear as well as branched compounds.
  • the genes encoding these polypeptides when expressed in a suitable host, are able to produce alkyl alkanoate in a yield suitable for industrial scale.
  • the group of polypeptides that can be used in the process according to the invention was found to represent enzymes that have alcohol acetyl transferase activity and whose three-dimensional structure contains an alpha-beta hydrolase fold and an active site comprising a serine, histidine and optionally aspartic acid, forming a dyad of serine- histidine or a triad of serine-aspartic acid- histidine wherein the histidine of the serine-histidine dyad or serine-apartic acid -histidine triad in the polypeptide according to the invention, is present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment, a glutamic acid-asparagine-proline (ENP) fragment, or glutamic-acid- methionine- proline (EMP) fragment as the 5 th , 6 th and 7 th amino acid from the histidine on the C-termin
  • amino acid sequence of the polypeptide comprises an amino acid sequence that has:
  • amino acid sequence according to SEQ ID NO: 13 or an amino acid sequence that has at least 70% identity with the sequence as shown in SEQ ID NO: 13 and/or,
  • nucleotide sequence encoding for the polypeptide according to the invention comprises,
  • nucleotide sequence according to SEQ ID NO:2, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 2 and/or,
  • nucleotide sequence according to SEQ ID NO:4, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 4 and/or,
  • nucleotide sequence according to SEQ ID NO:6, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 6,
  • nucleotide sequence according to SEQ ID NO:8, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 8 and/or,
  • nucleotide sequence according to SEQ ID NO:10, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 10 and/or,
  • nucleotide sequence according to SEQ ID NO:12, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 12 and/or,
  • nucleotide sequence according to SEQ ID NO:14, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 14 and/or,
  • nucleotide sequence according to SEQ ID NO:16, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 and/or,
  • nucleotide sequence according to SEQ ID NO:18 or a nucleotide sequence having at least 70% identity with SEQ ID NO: 18 and/or, a nucleotide sequence according to SEQ ID NO:20, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 20.
  • the invention is further directed to the process using a recombinant vector, plasmid or a host cell comprising the nucleotide sequence encoding for the polypeptide used in the process invention.
  • Said host cell may be selected from bacteria, yeasts and filamentous fungi.
  • Preferred bacteria are selected from Escherichia species and Bacillus species.
  • a preferred Escherichia species is preferably E. coli and a preferred Bacillus species is Bacillus subtilis.
  • Other suitable bacteria are the ones that are used in industrial production such as
  • thermophilic species since they allow production at high temperatures. Examples thereof are Geobacillus thermoglucosidasius, Caldicellulosiruptor bescii, Clostridium thermocellum, Thermoanaerobacterium aotearoense,
  • Preferred yeasts are the ones from which the polypeptides according to the invention originate such as Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Kluyveromyces marxianus Kluyveromyces lactis, Cyberlindnera jadinii, Hanseniaspora u varum, Eremothecium cymbalarie, and Saccharomyces cerevisiae, but also yeasts that are often used in the industry as production yeasts such as Saccharomyces cerevisiae species, Pichia species such as P. pastoris and Schizosaccharomyces species.
  • Preferred filamentous fungi are selected from Aspergillus species, Trichoderma species or Penicillium species; Preferred Aspergillus species are preferably A. niger, A. oryzae, and A. nidulans. Also Monascus ruber is suitable.
  • the process may be conducted at a temperature of between 10 and 130 °C.
  • the polypetides used in the process are produced in a host cell.
  • the alcohol and the acetyl coenzyme A, or Acyl coenzyme A may be produced in a host cell.
  • the alkyl alkanoate formed may be isolated from the host cell and optionally purified.
  • the invention is further directed to the use of the hosts cells according to the invention in the preparation of alkyl alkanoates.
  • a sugar compound may be used as a growth substrate for the host cell.
  • SEQ ID NO:1 sets out the amino acid sequence of AAT1 obtained from Wickerhamomyces anomalus DSM 6766)
  • SEQ ID NO:2 sets out the nucleotide sequence encoding for AAT1.
  • SEQ ID NO:3 sets out the amino acid sequence of AAT 2 obtained from Wickerhamomyces ciferrii CBS 111
  • SEQ ID NO:4 sets out the nucleotide sequence encoding for AAT2.
  • SEQ ID NO:5 sets out the amino acid sequence of AAT 3 obtained from Kluyveromyces marxianus DSM 5422.
  • SEQ ID NO:6 sets out the nucleotide sequence encoding for AAT3.
  • SEQ ID NO:7 sets out the amino acid sequence of AAT4 obtained from Kluyveromyces lactis CBS 2359
  • SEQ ID NO:8 sets out the nucleotide sequence encoding for AAT4.
  • SEQ ID NO:9 sets out the amino acid sequence of AAT 5 obtained from Cyberlindnera jadinii DSM 2361
  • SEQ ID NO:10 sets out the nucleotide sequence encoding for AAT5.
  • SEQ ID NO:1 1 sets out the amino acid sequence of AAT 6 obtained from Cyberlindnera fabianii CBS 5640
  • SEQ ID NO:12 sets out the nucleotide sequence encoding for AAT6.
  • SEQ ID NO:13 sets out the amino acid sequence of AAT7 obtained from Hanseniaspora uvarum CECT 11105
  • SEQ ID NO:14 sets out the nucleotide sequence encoding for AAT7.
  • SEQ ID NO:15 sets out the amino acid sequence of AAT 8 obtained from Hanseniaspora uvarum CECT 11105
  • SEQ ID NO:16 sets out the nucleotide sequence encoding for AAT8.
  • SEQ ID NO:17 sets out the amino acid sequence of AAT 9 obtained from Eremothecum cymbalariae).
  • SEQ ID NO:18 sets out the nucleotide sequence encoding for AAT 9.
  • SEQ ID NO:19 sets out the amino acid sequence of AAT 10 obtained from Saccharomyces cerevisiae NCYC 2629
  • SEQ ID NO:20 sets out the nucleotide sequence encoding for AAT10.
  • a preferred polypeptide to be used in the process according to the invention was encoded by a gene isolated from the genome of W. anomalus, hereinafter referred to as AAT 1 .
  • the amino sequence of AAT 1 is given under SEQ ID No:1
  • the nucleotide sequence encoding for AAT 1 is given in SEQ ID NO: 2.
  • homologs of AAT 1 Upon investigating homologs of AAT 1 according to the invention it was found that these homologs contain a highly conserved nucleophilic elbow (GYSLG) at Ser 121 . Moreover, Asp 145, Asp 178, and His 295 were also highly conserved. It was found that homologs of AAT 1 had the same highly conserved serine, aspartic acid and histidine. Upon visualization with a three-dimensional model of the first AAT found (AAT 1 ), Ser 121 , Asp 145 and His 295 showed to be in the correct spatial proximity to form a catalytic Ser-Asp-His triad that occurs in alpha/beta hydrolases. Some homologs however did not have the aspartic acid at a
  • a novel group of polypeptides was found to be enzymes that have alcohol acetyl transferase activity and have in their three-dimensional structure an alpha-beta hydrolase fold and an active site comprising a serine, histidine dyad or a serine, aspartic acid, histidine triad.
  • This triad and alpha/beta hydrolase fold is not present in the known aftl and aft2 enzymes of S. cerevisiae.
  • Non-producers it was found that in the producers, the histidine of the serine-aspartic acid-histidine triad or serine-glycine-histidine triad was present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment a glutamic acid-asparagine - proline (ENP) fragment or glutamic-acid- methionine- proline (EMP) fragment as the 5 th , 6 th and 7 th amino acid from the histidine on the C-terminal side of the polypeptide.
  • EMP glutamic acid-arginine- proline
  • EMP glutamic acid-asparagine - proline
  • EMP glutamic-acid- methionine- proline
  • polypeptides with AAT activity and the structural features as described above had a good yield in alkyl alkanoate production when expressed in a proper host cell.
  • Other preferred polypeptides to be used according to the invention were genes isolated from Kluyveromyces marxianus, Kluyveromyces lactis, Wickerhamomyces ciferrii, Cyberlindnera jadinii, Hanseniaspora uvarum, Eremothecium cymbalarie and Saccharomyces cerevisiae. These genes showed roughly 50% identity to AAT 1 , but some of them showed even less identity with AAT 1 .
  • these genes Upon expression in a host, these genes also have alkyl C3-10 alkanoate producing AAT activity, similar to AAT 1. These new enzymes are referred to as AAT 2 to AAT 10.
  • the amino sequence of these AAT 2-10 are given under SEQ ID No:3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No:1 1 , SEQ ID No:13, SEQ ID No:15, SEQ ID No:17 and SEQ ID No: 19, respectively.
  • nucleotide sequences encoding for AAT 2-10 are given in SEQ ID NO: 4, SEQ ID No: 6, SEQ ID NO: 8, SEQ ID No: 10, SEQ ID NO: 12, SEQ ID No: 14, SEQ ID NO: 16, SEQ ID No: 18, and SEQ ID NO: 20, respectively.
  • Non-producers Upon further investigating the sequences of the polypeptides that proved to have AAT activity (such as in AAT 1 to AAT 10, hereinafter referred to as Producers) and sequences of polypeptides that did not prove to have AAT activity (hereinafter referred to as Non-producers), it was found that in the producers, the histidine of the serine-aspartic acid-histidine triad or serine- histidine dyad was present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment or glutamic-acid- methionine- proline (EMP) fragment as the 5 th , 6 th and 7 th amino acid from the histidine on the C-terminal side of the polypeptide.
  • the polypeptides that turned out to be a non-producer did not have such a ERP or EMP fragment.
  • the invention is further directed to the use of polypeptides in the process according to the invention which have:
  • polypeptides have the structural features as described above.
  • sequence identity in order to determine the degree of sequence identity shared by two amino acid sequences or by two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). Such alignment may be carried out over the full lengths of the sequences being compared. Alternatively, the alignment may be carried out over a shorter comparison length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences being compared are of the same or substantially the same length.
  • the skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/qcq/), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the polypeptide comprises a substantially homologous amino acid sequence that has of at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any of the amino acid sequences shown in SEQ ID NO: 1 and/or SEQ ID NO:3, and/or SEQ ID NO 5, and/or SEQ ID NO: 7 and/or SEQ ID NO:9, and/or SEQ ID NO 1 1 , and/or SEQ ID NO: 13 and/or SEQ ID NO:15, and/or SEQ ID NO 17, and or SEQ ID NO 19.
  • the enzyme may be a polypeptide derived from amino acid SEQ ID NO: 1 to 19 by substitution, deletion or addition of one or several amino acid residues in any of the amino acid sequences of SEQ ID NO: 1 and/or SEQ ID NO:3, and/or SEQ ID NO 5, and/or SEQ ID NO: 7 and/or SEQ ID NO:9, and/or SEQ ID NO 1 1 , and/or SEQ ID NO: 13 and/or SEQ ID NO:15, and/or SEQ ID NO 17, and or SEQ ID NO 19 and having the same activity as any of the amino acid residue sequences of SEQ ID NO: 1 -19.
  • polypeptide according to the invention comprises:
  • nucleotide sequence according to SEQ ID NO:2, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 2 and/or,
  • nucleotide sequence according to SEQ ID NO:4, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 4 and/or,
  • nucleotide sequence according to SEQ ID NO:6, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 6,
  • nucleotide sequence according to SEQ ID NO:8, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 8 and/or,
  • nucleotide sequence according to SEQ ID NO:10, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 10 and/or,
  • nucleotide sequence according to SEQ ID NO:12, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 12 and/or,
  • nucleotide sequence according to SEQ ID NO:14, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 14 and/or,
  • nucleotide sequence according to SEQ ID NO:16, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 and/or,
  • nucleotide sequence according to SEQ ID NO:18, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 18, and/or,
  • nucleic acid molecule comprises a nucleotide sequence encoding a protein, comprising a substantially homologous nucleotide sequence of at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the nucleotide sequences shown in nucleotide SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ ID NO: 6, SEQ ID NO: 8 and/or SEQ ID NO: 10 and/or SEQ ID NO: 12, SEQ ID NO: 14 and/or SEQ ID NO: 16 and/or SEQ ID NO: 18, SEQ ID NO: 20.
  • the present invention provides a gene, which hybridizes selectively under stringent conditions to all or part of the DNA as shown in any of nucleotide SEQ ID NO: 2-20 to all or part of a DNA complementary to the sequence as shown in any of nucleotide SEQ ID NO: 2-20 and which encodes a protein having the activity of alcohol acetyl transferase.
  • hybridizes selectively are intended to describe conditions for hybridization and washing under which nucleotide sequences at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other.
  • hybridizing sequences may share at least 45%, at least 50%, at least 55%, at least 60%, at least 65, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% sequence identity.
  • a preferred, non-limiting example of such stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 q C, followed by one or more washes in 1 X SSC, 0.1 % SDS at about 50 ⁇ €, preferably at about 55 ⁇ €, preferably at about 60 °C and even more preferably at about 65 °C.
  • Highly stringent conditions include, for example, hybridization at about 68 °C in 5x SSC/5x Denhardt's solution / 1 .0% SDS and washing in 0.2x SSC/0.1 % SDS at room temperature. Alternatively, washing may be performed at 42 °C.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double- stranded cDNA clone).
  • Homologous gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.
  • the template for the reaction can be total chromosomal DNA from the strain known or suspected to express a polynucleotide according to the invention.
  • the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new oxidoreductase nucleic acid sequence, or a functional equivalent thereof.
  • the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention.
  • the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new oxidoreductase nucleic acid sequence, or a functional equivalent thereof.
  • the PCR fragment can then be used to isolate a full-length cDNA clone by a variety of known methods.
  • the amplified fragment can be labelled and used to screen a
  • the labelled fragment can be used to screen a genomic library.
  • RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source.
  • a reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • RNA/DNA hybrid can then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer).
  • second strand synthesis can then be primed (e.g., with a poly-C primer).
  • vectors including cloning and expression vectors, comprising a polynucleotide of the invention encoding alcohol acetyl transferase protein or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of the invention occurs.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • nucleotide sequence present in a host organism, microorganism or cell refers to a nucleotide sequence which is not naturally present in said organisms, microorganism or cell. This includes nucleotide sequences which are foreign to said organisms, microorganism or cell and nucleotide sequences which are introduced at a position other than their natural position in the genome and endogenous gene sequences which have been modified.
  • heterologous is used herein with reference to a nucleotide sequence present in a host organism, microorganism or cell refers to a nucleotide sequence that has been derived from a different organism.
  • homologous is used herein with reference to a nucleotide sequence present in a host organism, microorganism or cell refers to a nucleotide sequence that has been derived from the same organism as the host organism.
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell. Suitable host cells are described below.
  • the vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced.
  • a vector to be used in the process according to the invention may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e. g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably herein as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.
  • Vectors to be used in the process according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • a vector to be used in the process of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression.
  • the recombinant expression vectors used in the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell), i.e.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide according to the invention.
  • the present invention also provides a vector or a plasmid containing the nucleotide sequence encoding for the polypeptide for use in the process according to the invention.
  • Said plasmid can be introduced into proper host cells.
  • Said host cell may contain a homologous, or a heterologous gene, which is capable of generating an amino acid sequence that has at least 70% identity with the sequence shown in any of the amino acid SEQ ID NO: 1 -19.
  • this gene may be incorporated into the host cell in such a way so as to increase the alkyl alkanoate yield compared to the alkyl alkanoate yield that can be obtained via the gene in its natural position in the genome and with endogenous gene sequences.
  • the present invention further provides proper host cells for use on the process according to the invention, which comprises a nucleotide sequence exhibiting at least 70% identity with that shown in any of the nucleotide SEQ ID NO: 2-20.
  • Suitable host organisms are selected from bacteria, yeasts, or filamentous fungi.
  • Preferred bacteria are selected from Escherichia species and Bacillus species.
  • a preferred Escherichia species is preferably E. coli and a preferred Bacillus species is Bacillus subtilis.
  • Other suitable bacteria are the ones that are used in industrial production such as
  • thermophilic species since they allow production at high temperatures. Examples thereof are Geobacillus thermoglucosidasius, Caldicellulosiruptor bescii, Clostridium thermocellum, Thermoanaerobacterium aotearoense,
  • Preferred yeasts are the ones from which the polypeptides according to the invention originate such as Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Kluyveromyces marxianus, Kluyveromyces lactis, Cyberlindnera jadinii, Hanseniaspora u varum, Eremothecium
  • yeasts that are often used in the industry as production yeasts such as Saccharomyces cerevisiae species, Pichia species such as P. pastoris and
  • Preferred filamentous fungi are selected from Aspergillus species, Trichoderma species or Penicillium species; Preferred Aspergillus species are preferably A. niger, A. oryzae, and A. nidulans. Also Monascus ruber is suitable.
  • the process of the present invention is directed to the use of host cells comprising the nucleotide sequence encoding for the polypeptide with of alcohol acetyl transferase activity according to this invention.
  • host cells comprising the nucleotide sequence encoding for the polypeptide with of alcohol acetyl transferase activity according to this invention.
  • alcohol and acetyl coenzyme A or its higher carboxyl acid counterpart is converted in the presence of the polypeptide with AAT activity to form alkyl alkanoate.
  • the acetyl coenzyme A or its higher carboxyl acid counterpart may be produced by the host cell.
  • the alcohol can either be added to the reaction or the conditions are arranged such that the alcohol is also produced in the host cell.
  • suitable host cells were transformed with said DNA by conventional methods in the field, and the recombinant enzyme produced by the recombinant cells after transformation convert alcohol and acetyl coenzyme A or its higher carboxyl acid counterpart to alkyl alkanoate in the host cell.
  • the invention is further directed to a process for the production of alkyl alkanoate wherein the polypeptide with AAT activity according to the invention converts alcohol and acyl coenzyme A into alkyl alkanoate.
  • the polypeptide according to the invention may be produced in a host cell.
  • the formed polypeptide may be used after isolation from the host cell, or the process may be a microbial process, taking place in the host cell.
  • the process may also be arranged so that the alcohol needed to form alkyl alkanoate is formed by fermentation of sugar compounds either separately from the conversion to alkyl alkanoate or simultaneously.
  • the process is conducted as a full microbial process wherein both alcohol and acyl coenzyme A are formed within the host cell and converted into alkyl alkanoate.
  • the process according to the invention may be conducted at a temperature of between 10 and 130 °C. This has clear advantages over any known chemical process for the production of ethyl acetate and higher alkyl alkanoates, where much higher temperatures, higher amounts of energy and increased pressures are needed.
  • Thermophilic hosts allow the process to be conducted at relatively high temperature such as between 45 and 130 °C, preferably between 50 and 100 °C. It goes without saying that the polypeptide to be incorporated in a thermophilic host should be able to undergo the growth- and production temperatures used.
  • Preferred sugar compounds to be used as the growth substrates are selected from compounds comprising both hexose and pentose sugars.
  • the sugar compound may be a hexose sugar such as glucose, fructose, galactose, glycan or other polymers of glucose, hexose oligomers such as sucrose, lactose, maltose, maltotriose and isomaltotriose, panose, and fructose oligomers.
  • the micro-organism is modified to have the ability to ferment pentose sugars, and the medium includes a pentose sugar such as xylose, xylan or other oligomer of xylose.
  • the organisms are cultivated on combinations of hexose and pentose sugars.
  • the sugars can be hydrolysates of a hemicellulose, an amylose or cellulose-containing biomass.
  • the micro-organism is modified to ensure degradation of the biomass to monomers (e.g. expression of cellulase genes).
  • the substrate comprises a sugar oligomer or polymer such as cellulose, hemicellulose or pectin.
  • enzymes can be added to the fermentation medium to ensure degradation of the substrate into fermentable monomers.
  • yields were obtained of up to 35% on mole to mole glucose basis in shake flask tests. In these tests no process optimalization has taken place yet. With the AATs used in the process according to the invention yields may be reached of up to 60 % alkyl alkanoate on mole to mole glucose basis. In general, the
  • polypeptides according to the invention are able to have a yield ranging from 10 to 55% mole alkyl alkanoate per mole glucose. In most cases, a yield of 45 to 55 % mole of alkyl alkanoate per mole of glucose may be reached.
  • the invention provides methods for producing a alkyl alkanoate which, in addition to the steps detailed above further comprise the step of recovering the C3-C10 alkyl alkanoate of interest. This comprises recovery of the C3-C10 alkyl alkanoate from the host cell mixture and optionally additional purification.
  • Suitable purification can be carried out by methods known to the person skilled in the art such as by using distillation, extraction, ion exchange resins, electrodialysis, nanofiltration, etc
  • pYES2 derived plasmids were constructed by inserting genes into the multiple cloning site, either by using appropriate restriction enzymes, or by in vivo yeast assembly (Saccharomyces cerevisiae. Bio-protocol 5, (2015) pCUP1 derived plasmids were constructed by replacing the GAL1 promoter of pYES2 with the S. cerevisiae NCYC 2629 CUP1 promoter and inserting a gene of interest with in vivo yeast recombination, using S.
  • ⁇ DE3 ⁇ sBamHIo AEcoRI-B int::(lacl::PlacUV5::T7gene1 ) i21 ⁇
  • pCUP1:AAT 11 Non- S. cerevisiae, CUP1 W. anomalus DSM 6766
  • pCUP1:AAT 12 Non- S. cerevisiae, CUP1 K. marxianus DSM 5422
  • pCUP1:AAT 14 Non- S. cerevisiae, CUP1 C. fabianii CBS 5640 (CDR40570.1) producer
  • pCUP1 :AAT 15 Non- S. cerevisiae, CUP1 S. cerevisiae NCYC 2629 IM032 producer) (YGR015C)
  • Wild type yeast strains were routinely cultured and propagated in YPD medium (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract).
  • Uracil auxotrophic yeast strains were routinely cultured and propagated in YS (yeast synthetic) medium (6.7 g/L Yeast nitrogen base without amino acids, 1.92 g/L Medium Supplements without uracil) with 20 g/L glucose
  • E. coli strains were routinely cultured and propagated in LB or M9 medium supplemented with 50 ⁇ g/mL ampicillin or kanamycin.
  • Yeast and E. coli strains were grown at 30 °C, the plasmids were cloned at 37 °C, respectively, unless stated otherwise. S.
  • pyridoxamine-HCI pyridoxal-HCI
  • Flasks were inoculated with 1 mL overnight YPD cultures and shaken at 150 rpm.
  • All batch fermentations were operated at 30 °C.
  • the S. cerevisiae and £. co// ' pre-cultures were made by inoculating 50 mL YS medium with 20 g/L glucose or 50 mL M9 medium, respectively in a 250 mL Erlenmeyer flask to an initial OD of 0.5.
  • the cultures were grown at 250 rpm and 30°C until an OD of 3.
  • the cells were centrifuged at 4700 rpm for 5 minutes, resuspended in 50 mL sterile water and transferred to the fermenter.
  • E. coli cultures were induced with 0.2 mM IPTG after 4 hours of growth.
  • the dilution rate was 0.1 h _1 .
  • Ethyl acetate production was controlled by aeration. Producing (aerobic) and non-producing (oxygen limited) conditions were achieved by sparging the fermenter with 3 L/h air or a 0.3 L/h air, 2.7 L/h N 2 mixture, respectively. Steady state was achieved after 50 h. 50 mL medium was withdrawn, centrifuged and the pellet frozen at -80 °C.
  • the pellets from the continous cultures were kept on ice as much as possible during the protocol.
  • the pellets were resuspended in 0.5 mL cold TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA) and divided into two 2 mL screw capped tubes containing 0.5 g zirconium beads, 30 ⁇ 10 %SDS, 30 ⁇ 3 M sodium acetate (pH 5.2) and 500 ⁇ Roti-® Phenol (pH 4.5-5.0, Roth).
  • the cells were disrupted with a FastPrep apparatus (MP biomedials) at speed 6 for 40 seconds and centrifuged at 4 °C and 14.000 rpm for 5 minutes.
  • RNAseq analysis was performed by BaseClear and the reads mapped to the annotated genome of 1/1/. anomalus DSM 6766, See FEMS Yeast Res. 12, 382-386 (2012).
  • Homologs of AAT 1 were identified by performing standard protein BLAST searches against the non-redundant protein sequences database. Phylogenetic analysis was performed by making a structural alignment of wan5543 and close homologues using the Tcoffee (Espresso) server (http://tcoffee.crg. cat/apps/tcoffee/do:expresso) [1]. A three dimensional structure of Wan5543 was modelled using the PHYRE2 Protein Fold Recognition Server
  • AAT 1 was purified from E. coli BL21 (DE3) pET26b:harmAAT 1 -His. Three 1 L Erienmeyers with 250 mL M9 medium were inoculated with 2 mL overnight preculture and cultivated at 37 °C and 200 rpm. After 4 hours the cultures were chilled on ice for 15 minutes and induced with 0.2 mM IPTG. The cultures were then incubated at 20 °C and 200 rpm. After 18 hours, the cells were combined, harvested by centrifugation at 5000 rpm for 5 minutes, washed with 50 mL 50 mM phosphate buffer (KPi, pH 7.5) and stored at -20 °C.
  • KPi 50 mM phosphate buffer
  • the cells were resuspended in 20 mL buffer HA (50 mM KPi, 300 mM NaCI, pH 8) and passed twice through a chilled French Press Cell (Thermo Scientific) at 20000 psi. 25 mg DNAsel was added to the lysate, which was centrifuged at 4 °C and 18000 rpm for 20 minutes. The supernatant was filtered through 0.45 ⁇ filter and was used for protein purification. An AKTA Purifier system was used to purify AAT 1 . The cell free extract was loaded on a 1 ml_ HisTrap HP column (GE Healthcare Life Sciences) that was equilibrated with buffer HisA.
  • buffer HA 50 mM KPi, 300 mM NaCI, pH 8
  • 25 mg DNAsel was added to the lysate, which was centrifuged at 4 °C and 18000 rpm for 20 minutes. The supernatant was filtered through 0.45 ⁇ filter and was used for protein purification
  • the protein was eluted with a gradient of buffer HB (50 mM KPi, 300 mM NaCI, 500 mM imidazole, pH 8).
  • the fractions containing the protein were desalted over three connected 5 ml_ HiTrap Desalting columns (GE Healthcare Life Sciences), equilibrated with buffer CA (50 mM KPi, pH 7).
  • the desalted protein fractions were loaded on a 1 mL HiTrap HP SP column (GE Healthcare Life Sciences).
  • AAT 1 was eluted with a gradient of buffer CB (50 mM KPi, pH 7, 1 M NaCI). The fractions with the highest content of protein were combined and used for further analyses.
  • Glucose and organic acids were analysed by HPLC using an ICS5000 HPLC system (Thermo Scientific) equipped with a Dionex DP pump, Dionex AS-AP autosampler, Dionex VWD UV detector operated at 210 nm and Shodex Rl detector operated at 35 °C.
  • An Aminex HPX-87H cation-exchange column was used with a mobile phase of 0.16 N H 2 S0 4 and was operated at 0.8 mL/min and 60 °C. 10 mM dimethylsulfoxide in 0.04 N H 2 S0 4 was used as internal standard. Volatile compounds were analysed on a Shimadzu 2010 gas chromatograph equipped with a 20i-s autosampler.
  • the sequenced and annotated genome of ethyl acetate producing Wickerhamomyces anomalus DSM 6766 contains five putative Atf1 or Atf2 homologs and one Eht1 homolog. To see if they are involved in ethyl acetate production they were expressed in S. cerevisiae INVSc (See Table I). The transformants 0.005 g/L ethyl acetate at most. However, they did produce 3 - 15 fold more isoamyl acetate from the endogenously produced isoamyl alcohol. Overexpression of the S. cerevisiae atfl gave similar results, but S. cerevisiae INVSd expressing atf2 and ehtl showed poor growth and did not produce esters.
  • the known homologs of Atf1 , Atf2 and Eht1 did not show a significant change in expression levels.
  • the overexpressed genes that were not upregulated due to the metabolic shift from an aerobic to fermentative metabolism during ethyl acetate production code for two hypothetical proteins with an alpha/beta hydrolase fold. This fold has been observed in esterases, as well as some AATs, see J. Biol. Chem. 281 , 4446-4456 (2006). Both types of enzymes are involved in ester metabolism in yeast, making AAT 1 and WANOMALA 7754 potential candidates for explaining ethyl acetate formation in W. anomalus. Their protein products are 99% identical and only AAT 1 was studied further.
  • the enzyme was expressed in S. cerevisiae INVSd pYES2-AAT 1 , which produced 0.13 ⁇ 0.01 g/L ethyl acetate. This is 26- fold higher than the best W. anomalus Atf1 , Atf2 or Eht1 homolog.
  • S. cerevisiae INVSd was grown on galactose, which also served as the inducer for gene expression.
  • AAT 1 makes ethyl acetate either as an AAT or as a reversed esterase, and excluded HADH.
  • AAT 1 was purified from £. coli BL21 (DE3) pET26b:harm AAT 1 -His by Ni/NTA affinity chromatography, followed by cation exchange. The Purified AAT1 protein fraction was subjected to a GC assay for AAT activity. To this end both ethanol and acetyl coenzyme A were contacted with AAT1 . When omitting either ethanol and/or acetyl coenzyme A no ethyl acetate was formed. This confirms that AAT 1 produces ethyl acetate as an AAT, and not as a reversed esterase.
  • Example 4A Activity measurements with ethanol
  • the AAT 1 homologs used to compile the multiple sequence alignment originate from other ethyl acetate producing yeast species. When the homologs were expressed in S. cerevisiae
  • CEN.PK2-1 D several of the transformants produced ethyl acetate upon expression in a host.
  • the growth and product production profiles of the transformants resembled that of CEN.PK2-1 D pCUP1 :AAT 1 , but the final titer of ethyl acetate varied per homolog.
  • the amino acid sequences and the nucleotide sequences of the homologs that proved to have ethyl actetate production activity are compiled in sequence listings. At least one ethyl acetate producing homolog of AAT 1 was present in each ethyl acetate producing yeast.
  • the homologs found which proved to have ethyl acetate producing activity were genes isolated from Kluyveromyces marxianus,
  • AAT 2 to AAT 10.
  • the amino sequence of these AAT 2-10 are given under SEQ ID No:3, SEQ ID No:5, SEQ ID No:7, SEQ ID No:9, SEQ ID No:1 1 , SEQ ID No:13, SEQ ID No:15, SEQ ID No:17 and SEQ ID No: 19, respectively.
  • nucleotide sequences encoding for AAT 2-10 are given in SEQ ID NO: 4, SEQ ID No: 6, SEQ ID NO: 8, SEQ ID No: 10, SEQ ID NO: 12, SEQ ID No: 14, SEQ ID NO: 16, SEQ ID No: 18, and SEQ ID NO: 20, respectively.
  • Producers sequences of polypeptides that did not prove to have ethyl acetate production activity
  • Non-producers it was found that in the producers, the histidine of the serine-aspartic acid-histidine triad or serine-glycine-histidine triad was present in a polypeptide fragment that has a glutamic acid-arginine- proline (ERP) fragment a glutamic acid-asparagine - proline (ENP) fragment or glutamic-acid- methionine- proline (EMP) fragment as the 5 th , 6 th and 7 th amino acid from the histidine on the C-terminal side of the polypeptide.
  • EMP glutamic acid-arginine- proline
  • EMP glutamic acid-asparagine - proline
  • EMP glutamic-acid- methionine- proline
  • AAT 1 homologs were also present in Cyberlindnera fabianii CBS 5640 and Wickerhamomyces ciferrii CBS 1 1 1 (AAT 6 and AAT 2, respectively). These strains have, to our knowledge, not been studied for ethyl acetate production before. Therefore, their potential for ethyl acetate production was tested in shake flasks in YM medium. C. fabianii and W. ciferrii produced 4.1 and 5.7 g/L ethyl acetate from 50 g/L glucose, respectively.

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Abstract

La présente invention concerne un procédé de production d'alcanoate d'alkyle, un polypeptide qui présente une activité d'alcool-acétyl transférase (AAT) et qui présente, dans sa structure tridimensionnelle, un pli d'alpha-bêta-hydrolase et un site actif comprenant une sérine, une histidine et éventuellement de l'acide aspartique, étant utilisé pour convertir : - un C3-C10-alcanol et une acétyl-coenzyme A ou - un C1-C10-alcanol avec une C3-C10 acyl-coenzyme A ou - du méthanol avec une acétyl-coenzyme A, en un alcanoate d'alkyle. Le procédé concerne en outre l'utilisation d'un vecteur d'expression recombinant ou d'un plasmide ou d'une cellule hôte comprenant la séquence nucléotidique codant pour le polypeptide. Les polypeptides selon l'invention sont des gènes isolés de Kluyveromyces marxianus, Kluyveromyces lactis, Wickerhamomyces ciferrii, Cyberlindnera jadinii, Hanseniaspora uvarum, Eremothecium cymbalarie et Saccharomyces cerevisiae.
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CN114262695B (zh) * 2021-12-10 2023-02-17 杭州恩和生物科技有限公司 一种生产cbga前体的酿酒酵母工程菌及其构建方法和应用

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