WO2021110993A1 - Système de vecteur navette efficace pour l'expression de protéines hétérologues et homologues pour le genre zymomonas - Google Patents

Système de vecteur navette efficace pour l'expression de protéines hétérologues et homologues pour le genre zymomonas Download PDF

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WO2021110993A1
WO2021110993A1 PCT/EP2020/084774 EP2020084774W WO2021110993A1 WO 2021110993 A1 WO2021110993 A1 WO 2021110993A1 EP 2020084774 W EP2020084774 W EP 2020084774W WO 2021110993 A1 WO2021110993 A1 WO 2021110993A1
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zymomonas
promoter
gene
shuttle vector
site
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PCT/EP2020/084774
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English (en)
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Yannika NEUKRANZ
Jakob MACIEJKO
Patrick Schmitt
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Synbionik Gmbh
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

  • the invention belongs to the technical field of genetic engineering of new and efficient shuttle vectors for the expression of heterologous and homologous proteins for the genus Zymomonas.
  • Each vector comprises one multiple-cloning site located downstream of a promoter sequence and upstream of a terminator sequence.
  • the multiple cloning site contains one or more ribosome-binding sites. This facilitates the incorporation of multiple genes downstream of one promoter resulting in the transcription of a polycistronic mRNA.
  • this invention can be considered as a tool for the genetic modification of organisms; therefore, placing the invention in the biotechnological, microbiological and bacteriological field.
  • Zymomonas Bacteria from the genus Zymomonas are facultative anaerobic and are gram-negative bacteria. It is a natural ethanologen with a high tolerance to ethanol (up to 16%). Zymomonas uses the Entner-Doudoroff (ED) pathway for glycolysis instead of the classical Embden-Meyerhof- Pamas (EMP) pathway such as in the model organisms Saccharomyces cerevisiae and Escherichia coli. In the ED pathway only 1 mole of ATP is yielded per mole of glucose, but it requires less enzymatic proteins to sustain the same flux (Flamholz, Avi, et al. "Glycolytic strategy as a tradeoff between energy yield and protein cost. " Proceedings of the National
  • the highly specific cell surface of Zymomonas enables it to consume glucose in high amounts. It has a relatively small genome, which depending on the strain ranges from 2.01 to 2.22 Mb with two (2) to eight (8) additional native plasmids.
  • Routine cloning vectors such as pBR322 cannot be stably maintained in Zymomonas as the vectors need an additional origin of replication derived from the genome or replication-related segments of the native plasmids ( Sprenger , Carbohydrate metabolism in Zymomonas mobilis: a catabolic highway with some scenic routes, FEMS Microbiology Letters . Volume 145, Issue
  • shuttle vectors have been designed for heterologous protein expression, which were stably maintained in Zymomonas. These include strong native Zymomonas promoters such as upstream of the genes pyruvate decarboxylase, glyceraldehyde-3 -phosphate dehydrogenase and phosphopyruvate hydratase (So etal, 2014 doi: 10.1186/1471-2180-14-68; Yam et al.
  • the microorganism Zymomonas has attracted significant interest for its use in industrial scale production of bioethanol (Doelle, Horst W., et al. "Zymomonas mobilis science and De Ley. "The biology of Zymomonas. " Bacteriolosical reviews 41.1 (1977): 1. ; Rosters, P. L ⁇ , et al. "Zymomonas mobilis for fuel ethanol and hisher value products. " Biofuels. Sprinser, Berlin, Heidelbers. 2007. 263-288.; Buchholz, Steven E., and Douslas E. Eveleish. "Genetic modification of Zymomonas mobilis.” Biotechnology advances 8.3 (1990): 547-581).
  • the shuttle and expression vector system of the invention allows for an improved, innovative and more efficient technique for the expression of more than one gene in Zymomonas.
  • the presence of multiple cloning sites located downstream of each of the promoters includes additional ribosome-binding sites incorporated. This improvement enables a simple insertion of genes each separated by a ribosome-binding site and as such forming a polycistronic mRNA.
  • the possibility to change the Zymomonas derived origins of replication as well as the resistance cassettes allow for the transformation of more than one plasmid in the same cell.
  • the system facilitates the metabolic engineering of Zymomonas enabling a more efficient biotechnological production of biosynthetic compounds.
  • the present invention provides a new method to express more than one homologous or heterologous proteins in Zymomonas. It is based on a highly variable shuttle vector system which is stably maintained in bacteria from the genus Zymomonas. Each vector comprises one or more strong promoters each followed by a highly variable multiple-cloning site. Generally, each promoter of the vector of the invention has a ribosome-binding site (RBS) that is already incorporated into the sequence. However, the plasmid contains one or more additional ribosome-binding sites in the multiple cloning sites of the plasmid.
  • RBS ribosome-binding site
  • This ribosome-binding site facilitates the insertion of more than one gene into the plasmid at the 3’ end of the promoter resulting in the transcription of a polycistronic mRNA.
  • Another advantage of the invention is the origin of replication introductory site present on the plasmid. Different origins of replication can be introduced into the plasmid allowing the transformation of more than one pZM-DUET shuttle vectors into Zymomonas. Also, the resistance cassette can be replaced by other resistance cassettes with standard PCR methods. This results in a highly variable shuttle vector system where plasmids are constructed with different combinations of origins of replication and resistance cassettes allowing them to be transformed together into Zymomonas.
  • the insertion of fluorescent proteins into one of the open reading frames enables an easy detection method for successful transfection of the plasmid into Zymomonas.
  • Zymomonas exhibits multiple antibiotic resistances (Buchholz, Steven E., and Douglas E. Eveleigh. "Genetic modification of Zymomonas mobilis. "Biotechnology advances 8.3 (1990): 547-581). Therefore, some strains are not sensitive enough for antibiotic selection after transformation.
  • the insertion of a fluorescent protein into the plasmid enables a simple way to detect successfully transformed cells while still enabling the expression of other genes.
  • the Zymomonas starting vector can be ordered to commercial providers or synthesized e.g. by GeneArt (Thermo Fischer) (see Figure 1) (identified as SEQ ID NO 1).
  • the present disclosure describes a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a. at least one selectable marker cassette, b. at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication, c. a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d.
  • each of the multiple cloning sites of the vector of the invention comprises at least one ribosome-binding site.
  • the ribosome binding site is derived from Zymomonas having a sequence selected from SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11.
  • the multiple cloning sites comprise at least 2 different restriction sites
  • the restriction sites are selected from XmaESall, Xbal, Ncol, BamHI, Hindlll, Pstl, Ndel, Bglll, KpNI and/or Xhol. More preferably, the restriction sites are located between e. the 3’ end of the promoter sequence and the 5’ end of the ribosome-binding site. f. the 3’ end of the ribosome-binding site and the 5’ end of the terminator; and/or. g. each ribosome-binding site within a multiple cloning site.
  • the invention concerns a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a. at least one selectable marker cassette, b. at least two prokaryotic origins of replication wherein at least one is a
  • Zymomonas origin of replication c. a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d.
  • the invention concerns a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a. at least one selectable marker cassette, b. at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication, c. a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d.
  • the selectable marker cassette is selected from a. an ampicillin cassette, b. a kanamycin cassette, c. a chloramphenicol cassette, d. a tetracycline cassette, or e. a spectinomycin cassette.
  • the vector comprises at least one selectable marker cassette.
  • the cassette includes nucleic acid sequence encoding an amino acid sequence to be expressed linked operably to at least one genetic control element which enables or regulates its expression (i.e. transcription and / or translation).
  • a selectable marker cassette typically includes the sequence of the marker to be expressed, and sequences necessary for expression of the sequence to be expresseda promoter.
  • the selectable marker cassette is an ampicillin resistance cassette an ampicillin cassette, a kanamycin cassette, a chloramphenicol cassette, a tetracycline cassette, and/or a spectinomycin cassette.
  • the invention concerns a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a) at least one selectable marker cassette, b) at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication, c) a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d) at least a second gene promoter derived from the genus Zymomonas and at least a corresponding second terminator sequence derived from the genus Zymomonas and which can be operably linked to the at least second promoter; wherein at least one multiple cloning site is located between the 3 'end of each of the promoter and the 5 'end of the corresponding terminator sequence.; and wherein
  • the selectable marker cassette is obtained by amplification via PCR
  • the selectable marker cassette comprises a promoter, the selectable marker gene and a terminator
  • a Sail restriction site is contained on the forward primer (SEQ ID NO 2) and a Xbal restriction site on the reverse primer (SEQ ID NO 3), 4. the selectable marker cassette is cloned into the Sail and Xbal restriction sites of the vector.
  • the invention concerns a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a) at least one selectable marker cassette, b) at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication, c) a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d) at least a second gene promoter derived from the genus Zymomonas and at least a corresponding second terminator sequence derived from the genus Zymomonas and which can be operably linked to the at least second promoter; wherein at least one multiple cloning site is located between the 3 'end of each of the promoter and the 5 'end of the corresponding terminator sequence.; and wherein
  • the selectable marker cassette is obtained by amplification via PCR
  • the selectable marker cassette comprises a promoter, the selectable marker gene and a terminator
  • a Xmal restriction site is contained on the forward primer (SEQ ID NO 4) and a Xbal restriction site on the reverse primer (SEQ ID NO 5),
  • the selectable marker cassette is cloned into the Sail and Xbal restriction sites of the vector.
  • the invention concerns a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a) at least one selectable marker cassette, b) at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication, c) a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d) at least a second gene promoter derived from the genus Zymomonas and at least a corresponding second terminator sequence derived from the genus Zymomonas and which can be operably linked to the at least second promoter; wherein at least one multiple cloning site is located between the 3 'end of each of the promoter and the 5 'end of the corresponding terminator sequence.; and wherein one of the origins
  • the invention concerns a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a) at least one selectable marker cassette, b) at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication, c) a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d) at least a second gene promoter derived from the genus Zymomonas and at least a corresponding second terminator sequence derived from the genus Zymomonas and which can be operably linked to the at least second promoter; wherein at least one multiple cloning site is located between the 3 'end of each of the promoter and the 5 'end of the corresponding terminator sequence.; and wherein a) the Z
  • one of the origins of replication is a Zymomonas mobilis (ZM4) derived origin of replication selected from the genome or the native plasmids pSUZMl, pSUZM2, pSUZM3, pPTZl, pPTZ3, pPTZ4, pZZM401, pZZM402, pZZM403, pZZM404 and pZZM405
  • ZM405 Zymomonas mobilis
  • the gene promoters derived from Zymomonas are inducible and/or constitutive.
  • the constitutive gene promoters derived from Zymomonas are the promoters of a) pyruvate decarboxylase (Ppdc) (Gene ID ZMO1360), b) glyceraldehyde-3 -phosphate dehydrogenase (Pgap) (Gene ID ZMO0177), c) phosphopyruvate hydratase (Peno) (Gene ID ZMO1608).
  • the terminator sequence of the vector of the invention is derived from Zymomonas and is SEQ ID NO 8.
  • At least two proteins are enzymes from the same enzymatic pathway.
  • the proteins expressed by the vector of the invention are homologous proteins of Zymomonas and/or heterologous proteins.
  • the at least two proteins are enzymes from the same enzymatic pathway.
  • the proteins are selected from: a) Prenyltransferases b) Enzymes from the mevalonate pathway such as MvaE (Acetyl-CoA acetyltransferase, MvaS (Hydroxy-methylglutaryl-CoA synthase, MvaE (Acetyl-CoA acetyltransf erase, Erg 12 (Mevalonate kinase), Erg8 (Phophomevalonate kinase and MVD1 (Diphophomevalonate decarboxylase) c) GPP synthase d) IDP isom erase e) Enzymes from the olivetolic acid pathway such as olivetolic acid cyclase, triox
  • TPI, G3PD, PGK, PGM, Enolase, Pyruvate Kinase and DOXP-Synthase g) Enzymes from the DOXP Pathway such as DOXIP reductoisomerase, MEPC transferase, DPME-Kinase, MEC- Synthase, HMBD- Synthase, IPP/DMAPP synthase, IDP-Isomerase, GPP-Synthase.
  • CBDA synthase or THCA synthase i) Enzymes from the Malonyl-CoA pathway such as Acetyl-CoA carboxylase j) Enzymes from the Hexanoyl-CoA Synthase such as hydroxyacyl-CoA dehydrogenase, enolyl-CoA hydratase, Enoyl-CoA reductase, AcCoA-Acetyl transferase, Hydroxyl-CoA dehydrogenase, Enoyl-CoA Hydratase, Enoyl-CoA reductase
  • the invention concerns a host cell comprising the vector of the invention and any of the preferred embodiments described above wherein the host cell is from a Zymomonas genus, preferably Zymomonas mobilis.
  • the vector is present in the host cell of the invention in a single plasmid or inserted into a genome of the host cell at a single locus.
  • the host cell comprises more than one vector of the invention. More preferably, when the host cell comprises more than one vector, the vectors have different selectable marker cassettes.
  • a shuttle vector for the expression of at least two proteins in a strain of the genus Zymomonas comprising a. at least one selectable marker cassette, b. at least two prokaryotic origins of replication wherein at least one is a Zymomonas origin of replication , c. a first gene promoter derived from the genus Zymomonas and a corresponding first terminator sequence derived from the genus Zymomonas and which can be operably linked to the first promoter; d.
  • each of the multiple cloning sites comprises at least one ribosome-binding site 3.
  • the ribosome-binding site is derived from Zymomonas having a sequence selected from SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11.
  • restriction sites are selected from Xmal/Sall, Xbal, Ncol, BamHI, Hindlll, Pstl, Ndel, Bglll, KpNI and/or Xhol
  • a shuttle vector according to claim 6 wherein one gene encoding one of the at least two proteins is inserted into a multiple cloning site between one of the promoter sequence and the corresponding terminator between one restriction site located between the 5’ end of a ribosome-binding site and the 3’ end of one of the promoter and another restriction site between the 3’ end of the ribosome-binding site and the 5’ end of the corresponding terminator.
  • a shuttle vector according to claim 1, wherein the selectable marker cassette is a. an ampicillin cassette, b. a kanamycin cassette, c. a chloramphenicol cassette, d. a tetracycline cassette, or e. a spectinomycin cassette.
  • the selectable marker cassette is cloned into the Sail and Xbal restriction sites of the vector.
  • a shuttle vector according to claim 1 or 10 wherein a. the selectable marker cassette is obtained by amplification via PCR, b. the selectable marker cassette comprises a promoter, the selectable marker gene and a terminator, c. a Xmal restriction site is contained on the forward primer (SEQ ID NO 4) and a Xbal restriction site on the reverse primer (SEQ ID NO 5), d. the selectable marker cassette is cloned into the Sail and Xbal restriction sites of the vector.
  • ZM4 Zymomonas mobilis
  • a shuttle vector according to claim 1 wherein the terminator sequence derived from Zymomonas is SEQ ID NO 8.
  • the proteins are homologous proteins of Zymomonas and/or heterologous proteins.
  • a shuttle vector according to claim 19 wherein the at least two proteins are enzymes from the same enzymatic pathway.
  • a shuttle vector according to claim 19 wherein the proteins are selected from: a) Prenyltransferases b) Enzymes from the mevalonate pathway such as MvaE (Acetyl-CoA acetyltransferase, MvaS (Hydroxy-methylglutaryl-CoA synthase, MvaE (Acetyl-CoA acetyltransf erase, Erg 12 (Mevalonate kinase), Erg8 (Phophomevalonate kinase and MVD1 (Diphophomevalonate decarboxylase) c) GPP synthase d) IDP isom erase e) Enzymes from the olivetolic acid pathway such as olivetolic acid cyclase, trioxododecanoyl-CoA synthase f) Enzymes from the Glycolysis pathway such as hexokinase, GPI, PFK1, Adolase, TPI, G
  • IDP-Isomerase GPP-Synthase.
  • CBDA synthase or THCA synthase i) Enzymes from the Malonyl-CoA pathway such as Acetyl-CoA carboxylase j) Enzymes from the Hexanoyl-CoA Synthase such as hydroxyacyl-CoA dehydrogenase, enolyl-CoA hydratase, Enoyl-CoA reductase, AcCoA-Acetyl transferase, Hydroxyl-CoA dehydrogenase, Enoyl-CoA Hydratase, Enoyl-CoA reductase.
  • a host cell comprising any one of the vector according to claims 1-21 wherein the host cell is .from a Zymomonas genus, preferably Zymomonas mobilis 23.
  • the vector of the invention facilitates the metabolic engineering of Zymomonas enabling a more efficient biotechnological production of biosynthetic compounds of interest.
  • each step of a metabolic pathway that produces the compound of interests occurs in a modified Zymomonas strain as described herein.
  • at least one step of the metabolic pathway occurs in a modified recombinant strain described herein, and at least one step of the metabolic pathway occurs extracellularly, e.g., in microorganism media or within a co-cultured modified recombinant cell.
  • the compounds produced at each step of the metabolic pathway may be referred to as “intermediates” or “intermediate compounds” or summoncompound intermediates”.
  • any of the terms “comprising”, “consisting essentially of and “consisting of may be replaced with either of the other two terms.
  • some embodiments may encompass a host cell “comprising” a number of components, other embodiments would encompass a host cell “consisting essentially of the same components, and still other embodiments would encompass a host cell “consisting of the same components.
  • the terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
  • polynucleotides provided by the invention can either be isolated from their natural genomic environment, modified after their isolation or produced artificially from pure sequence information.
  • invention or "present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.
  • singular forms “a”, “and,” and “the” include plural referents unless the context clearly dictates otherwise.
  • genomic DNA is referring to the heritable genetic information of a host organism.
  • Said genomic DNA comprises the entire genetic material of a cell or an organism.
  • genome or genomic DNA refers to the total amount of DNA of a cell, including the DNA of the nucleus (chromosomal DNA), extrachromosomal DNA, and organellar DNA (e.g. of mitochondria).
  • chromosomal DNA chromosomal DNA
  • extrachromosomal DNA e.g. of mitochondria
  • organellar DNA e.g. of mitochondria
  • the terms genome or genomic DNA when used in context of eukaryotic organisms, is referring to the chromosomal DNA of the nucleus.
  • nucleic acid refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • the monomer is typically referred to as a nucleotide.
  • Nucleic acids can include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.
  • the terms crizo and “around” indicate a close range around a numerical value when used to modify that specific value. If ‘X” were the value, for example, “about X” or courtaround X” would indicate a value from 0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X.
  • any reference to “about X” or possibly around X” specifically indicates at least the values X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, and values within this range.
  • nucleic acid sequence encoding or a “nucleic acid coding sequence” refers to a nucleic acid which directs the expression of a specific protein or peptide.
  • nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA, and the RNA sequence that is translated into protein.
  • the nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences.
  • the nucleotide sequence is codon-optimized to reflect the typical codon usage of the host cell without altering the polypeptide encoded by the nucleotide sequence.
  • the term "codon optimization” or “codon- optimized” refers to modifying the codon content of a nucleic acid sequence without modifying the sequence of the polypeptide encoded by the nucleic acid to enhance expression in a particular host cell.
  • the term is meant to encompass modifying the codon content of a nucleic acid sequence as a means to control the level of expression of a polypeptide (e.g., either increase or decrease the level of expression).
  • described are nucleic sequences encoding the enzymes involved in the engineered metabolic pathways.
  • a metabolically engineered cell may express one or more polypeptide having an enzymatic activity necessary to perform the steps described below.
  • a particular cell may comprises one, two, three, four, five or more than five nucleic acid sequences, each one encoding the polypeptide(s) necessary to produce compound of interest, or compound intermediate described herein.
  • a single nucleic acid molecule can encode one, or more than one, polypeptide.
  • a single nucleic acid molecule can contain nucleic acid sequences that encode two, three, four or even five different polypeptides.
  • Nucleic acid sequences useful for the invention described herein may be obtained from a variety of sources such as, for example, amplification of cDNA sequences, DNA libraries, de novo synthesis, excision of genomic segment.
  • nucleic acid sequences may then be modified using standard molecular biology and/or recombinant DNA technology to produce nucleic sequences having desired modifications.
  • exemplary methods for modification of nucleic acid sequences include, for example, site directed mutagenesis, PCR mutagenesis, deletion, insertion, substitution, swapping portions of the sequence using restriction enzymes, optionally in combination with ligation, homologous recombination, site specific recombination or various combination thereof.
  • the nucleic acid sequences may be a synthetic nucleic acid sequence. Synthetic polynucleotide sequences may be produced using a variety of methods described in U.S. Patent No. 7,323,320, as well as U.S. Pat. Appl. Pub. Nos. 2006/0160138 and 2007/0269870.
  • promoter refers to a polynucleotide or a nucleic acid fragment which directs/controls the transcription of a structural gene to produce mRNA.
  • a promoter is located in the 5' region of a gene, proximal to the start codon of the coding region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent or by the induced release of a suppressor. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter.
  • a polynucleotide is "heterologous to" an organism or a second polynucleotide if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e. g. a genetically engineered coding sequence or an allele from a different ecotype, variety or strain).
  • a promoter will be associated to a corresponding or operably linkable “terminator sequence” located in 3’ of the promoter sequences.
  • the term “terminator sequence”, “termination sequence”, “transcription terminator”, “termination sequences”, or “terminator” refer to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht, I.
  • a polynucleotide sequence with "terminator activity” refers to a polynucleotide sequence that, when operably linked to the 3' end of a second polynucleotide sequence that is to be expressed, is capable of terminating transcription from the second polynucleotide sequence.
  • Transcription termination is the process by which RNA synthesis by RNA polymerase is stopped and both the RNA and the enzyme are released from the DNA template. Improper termination of an RNA transcript can affect the stability of the RNA, and hence can affect protein expression. Variability of transgene expression is sometimes attributed to variability of termination efficiency (Bieri et al (2002) Molecular Breeding 10: 107-1 17).
  • restriction endonuclease and “restriction enzyme” refer to enzymes (e.g. bacterial enzymes), each of which cut double-stranded DNA at or near a specific nucleotide sequence (a cognate restriction site). Examples include, but are not limited to, BamHI, EcoRV, Hindlll, Hindi, Ncol, Sail, and Noth
  • restriction means cleavage of DNA by a restriction enzyme at its cognate restriction site.
  • restriction site also named herein “digestion sites” refers to a particular DNA sequence recognized by its cognate restriction endonuclease.
  • Transgene refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man.
  • a trans genie expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al, Molecular Cloning- A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc.
  • a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature.
  • restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide.
  • polynucleotides can be manipulated in many ways and are not limited to the examples above.
  • the plasmid contains, one or more resistance cassettes (selected from ampicillin resistance (AmpR), kanamycin resistance (KanaR), chloramphenicol resistance (CamR), spectinomycin resistance (SpecR), tetracycline resistance (TetR)) and an Zymomonas mobilis Origin of Replication (OR).
  • AmpR ampicillin resistance
  • KanaR kanamycin resistance
  • CamR chloramphenicol resistance
  • SpecR spectinomycin resistance
  • TetR tetracycline resistance
  • OR Zymomonas mobilis Origin of Replication
  • recombinant is used to specify an organism or cell, e.g. a microorganism, it is used to express that the organism or cell comprises at least one "transgene", "transgenic” or “recombinant” polynucleotide, which is usually specified later on.
  • Recombinant refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism.
  • a recombinant polynucleotide or a copy or complement of a recombinant polynucleotide
  • a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994- 1998)).
  • a recombinant expression cassette (or expression vector) typically comprises polynucleotides in combinations that are not found in nature.
  • recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant ceils, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).
  • flanking regions refers to regions or sequences located upstream and/or downstream of a nucleic acid coding sequence in a recombinant expression cassette which is involved in double homologous recombination (e.g., integration) of a portion of the cassette with a host cell's genome.
  • double homologous recombination refers to the ability of nucleic acid sequences to exchange, wherein a nucleic acid stably integrates into the genome of a host cell's DNA sequence to make a new combination of DNA sequence.
  • complementarity refers to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide.
  • sequence A-G-T is complementary to the sequence T-C-A.
  • Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, where ail the nucleic acids match according to base pairing.
  • protein protein
  • peptide and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutaniate, and O-phosphoserme.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • non-naturally occurring amino acid and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides likewise, may be referred to by their commonly accepted single letter codes.
  • a “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and/or size of the side group chain is maintained.
  • Illustrative sets of amino acids that may be substituted for one another include (i) positively- charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic amino acids Gly, Ala, Val, Leu and He; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro; (viii) small hydroxyl amino acids Ser and Thr; and sulfur-containing amino acids Cys and Met.
  • Reference to the charge of an amino acid in this paragraph refers to the charge at pH 7 0
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Conservatively modified variants can include polymorphic variants, interspecies homologs (orthologs), intraspecies homologs (paralogs), and allelic variants.
  • % identity and its derivatives are used interchangeably herein with the term “% homology” and its derivatives to refer to the level of a nucleic acid or an amino acid sequence’s identity between another nucleic acid sequence or any other polypeptides, or the polypeptide's amino acid sequence, where the sequences are aligned using a sequence alignment program.
  • % homology and its derivatives to refer to the level of a nucleic acid or an amino acid sequence
  • nucleic acids or proteins refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g.
  • sequences are then said to be "substantially identical.”
  • This definition also refers to, and can be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • Optimal alignment of such sequences can be carried out by any of the publically available algorithms or programs for determining sequence identity and alignment; e.g., BLAST. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage.
  • expression refers to the transcription and stable accumulation of coding (mRNA) or functional RNA derived from a gene. Expression may also refer to translation of mRNA into a polypeptide. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
  • cassette or "expression cassette” means those constructs in which the nucleic acid sequence encoding an amino acid sequence to be expressed is linked operably to at least one genetic control element which enables or regulates its expression (i.e. transcription and / or translation).
  • An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed.
  • the sequence to be expressed can be a coding sequence or a non-coding sequence (e.g., an inhibitory sequence).
  • an expression cassette is inserted into an expression vector (e.g., a plasmid) to be introduced into a host cell.
  • the expression may be, for example, stable or transient, constitutive or inducible.
  • Expression cassettes may also comprise the coding regions for two or more polypeptides and lead to the transcription of polycistronic RNAs.
  • RBS ribosome binding site
  • an RBS in a bacterial (e.g., Zymomonas) cell is selected to bind to bacterial (Zymomonas) ribosomes (e.g., the 16S rRNA)
  • an RBS in a cyanobacterial cell e.g., Synechocystis
  • the cell or expression system can be manipulated to include heterologous ribosomes that bind to a particular RBS.
  • the term "origin of replication” refers to a nucleic acid from which nucleic acid replication is initiated.
  • the origin of replication helps to control the number of copies of the vector in the host cell.
  • sequences are well known for a variety of plasmids, bacteria, yeast and viruses.
  • a low copy origin of replication yields from 1 to about 10 copies of the vector per cell.
  • a high copy origin of replication yields from about 20 to about 200 copies of the vector per cell.
  • multiple cloning site refers to nucleotide sequences that may be cleaved by a single or a series of restriction endonuclease enzymes (herein referred to as “restriction enzymes”), each of which has a distinct endonuclease restriction site and cleavage pattern.
  • restriction enzymes restriction endonuclease enzymes
  • restriction enzymes as few as 4-6 nucleotides are sufficient to provide a restriction site, while some restriction enzymes require a restriction site of 8 or more nucleotides.
  • transfection and “transformation” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods.
  • the nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 18.1- 18.88.
  • an "intron” is an intervening sequence in a gene that is transcribed into RNA and then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences.
  • An "exon” is a portion of the sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, and is not necessarily a part of the sequence that encodes the final gene product.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
  • a polynucleotide or polypeptide sequence is "heterologous to" an organism or a second sequence if it originates from a different species, or, if from the same species, it is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
  • a heterologous expression cassette includes sequence) that are from a different species than the cell into which the expression cassette is introduced, or if from the same species, is genetically modified.
  • Yeast or other eukaryotic species may be introduced on high-level expression plasmid vectors or through genomic integration using methods well known to those skilled in the art. Such methods may involve CRISPR Cas-9 technology, yeast artificial chromosomes (YACs) or the use of retrotransposons.
  • YACs yeast artificial chromosomes
  • retrotransposons retrotransposons
  • Plasmid and "vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
  • Vectors may be, for example, "cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors” which are designed for expression of a nucleotide sequence in a host cell.
  • replication means duplication of a vector.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed.
  • An operably linkage does not necessarily require a direct linkage in the chemical sense.
  • Genetic control sequences such as, for example, enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked.
  • Preferred arrangements are those in which the nucleic acid sequence to be expressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another.
  • the distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
  • selectable marker means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.
  • transformation refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance.
  • the transferred nucleic acid may be in the form of a plasmid maintained in the host cell, or some transferred nucleic acid may be integrated into the genome of the host cell.
  • Host organisms containing the transferred nucleic acid fragments are referred to as "transgenic” or “recombinant” or “transformed” organisms or “transformants”.
  • the term “introduced” means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell, means “transfection". The terms “culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,”
  • expand when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside its normal environment under controlled conditions, e.g., under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, stasis, differentiation or division. The term does not imply that all cells in the culture survive, grow, or divide, as some may naturally die or senesce. Cells are typically cultured in media, which can be changed during the course of the culture.
  • media and “culture solution” refer to the cell culture milieu.
  • Media is typically an isotonic solution, and can be liquid, gelatinous, or semi-solid, e.g., to provide a matrix for ceil adhesion or support.
  • Media as used herein, can include the components for nutritional, chemical, and structural support necessary for culturing a cell
  • media includes a carbon source for biosynthesis and metabolism. In the case of plant or other photosynthetic cell cultures, the carbon source is typically C02.
  • a "control,” e.g. a control cell, control sample, or control value, refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample or condition.
  • a test sample can include cells exposed to a test condition or a test agent, while the control is not exposed to the test condition or agent (e.g., negative control).
  • the control can also be a positive control, e.g., a known cell exposed to known conditions or agents, for the sake of comparison to the test condition.
  • a positive control can include a cell with a known level of production of the product of interest.
  • a control can also represent an average value gathered from a plurality of samples, e.g., to obtain an average value.
  • a control value can also be obtained from the same cell or population of cells, e.g., from an earlier- obtained sample, prior to the disorder or deficiency, or prior to treatment.
  • controls can be designed for assessment of any number of parameters.
  • One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data.
  • Host cells are generally cultured in the presence of starting materials, such as hexanoic acid, prenol, isoprenol, or the like, for periods of time ranging from a few hours to a day or longer (e.g, 24 hours, 30 hours, 36 hours, or 48 hours) at temperatures ranging from about 20 °C to about 40 °C depending on the particular host cells employed.
  • starting materials such as hexanoic acid, prenol, isoprenol, or the like
  • temperatures ranging from about 20 °C to about 40 °C depending on the particular host cells employed.
  • host cells transformed or genomically integrated with plasmids or vectors containing at least one or more than one expression cassette.
  • the genetically modified Zymomonas cells do not occur in nature.
  • Suitable cells are capable of expressing a nucleic acid construct (expression cassette) encoding biosynthetic enzymes, as described herein.
  • the cell naturally produces at least some biosynthetic precursors, e.g., Acetyl- CoA.
  • biosynthetic precursors e.g., Acetyl- CoA.
  • genes encoding desired enzymes can be heterologous to the cell, or native to the cell but operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the cell.
  • Any Zymomonas strains can be used in the present method so long as it remains viable after being transformed with the vector comprising the heterologous genes.
  • nucleic acid constructs described herein can be operably linked to a promoter and/or terminator so that the desired transcript(s) and protein(s) are expressed in a cell cultured under suitable conditions.
  • Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.
  • Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning.
  • direct chemical synthesis oligonucleotides of up to about 40 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence.
  • commercial services are available that can supply synthetic genes of the desired sequence.
  • the desired sequences may be isolated from natural sources using well known cloning methodology, e.g., employing PGR to amplify the desired sequences and join the amplified regions.
  • Zymomonas expression vectors include, without limitation: plasmids, such as pZZM401, pZZM402, pZZM403, pZZM404, pZZM405, pSUZMl, pSUZM2, pSUZM3, pPTZl, pPTZ3, pPTZ4 (Cao, 2016, Reynen 1990).
  • Homologous recombination can occur between, the expression vector and the homologous region in one or more genomic copies present in the host cell.
  • a selectable marker present on the expression vector is used to isolate transformant cells having undergone double homologous recombination by a selection method, such as antibiotic resistance or drug resistance.
  • the vector of the invention contains one or more specific promoters, one or more multiple cloning sites for the insertion of single genes or gene clusters, one or more terminators, one or more resistance cassettes (selected from ampicillin resistance (AmpR), kanamycin resistance (KanaR), chloramphenicol resistance (CamR), spectinomycin resistance (SpecR), tetracycline resistance (TetR)) and an Zymomonas mobilis Origin of Replication (OR). Detection of the successful transformation of vectors can be accomplished via PCR and sequencing. The gene transcription and enzyme translation and activity are determined by transcriptome, proteome and metabolome analysis.
  • the cell is cultured and typically allowed to grow.
  • this process entails culturing the cells in a suitable medium. It is important that the culture medium contain an excess carbon source, such as a sugar when an intermediate is not introduced. In this way, cellular production of acetyl-CoA, a starting material for IPP and DMAPP synthesis is ensured.
  • the intermediate is present in an excess amount in the culture medium.
  • Cell culture techniques are commonly known in the art and described, e.g., in Sambrook, et al. (1989) Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Cells are typically cultured in isotonic media that includes a carbon source, and in some cases, selection factors to select for recombinant cells (e.g., those with antibiotic resistance).
  • the Zymomonas host cell is preferably cultured at a temperature between 22° C and 37° C. While commercial biosynthesis in host cells such as E. coli can be limited by the temperature at which overexpressed and/or foreign enzymes (e.g., enzymes derived from plants) are stable, recombinant enzymes (including the terpenoid synthase) may be engineered to allow for cultures to be maintained at higher temperatures, resulting in higher yields and higher overall productivity.
  • foreign enzymes e.g., enzymes derived from plants
  • recombinant enzymes including the terpenoid synthase
  • the host cell (bacterial or yeast host cell) is cultured at about 22° C or greater, about 23° C or greater, about 24° C or greater, about 25° C or greater, about 26° C or greater, about 27° C or greater, about 28° C or greater, about 29° C or greater, about 30° C or greater, about 31° C or greater, about 32° C or greater, about 33° C or greater, about 34° C or greater, about 35° C or greater, about 36° C or greater, or about 37° C.
  • Product of interest can be extracted from media and/or whole cells, and recovered.
  • the product of interest are recovered and optionally enriched by fractionation (e.g. fractional distillation).
  • fractionation e.g. fractional distillation
  • the product can be recovered by any suitable process, including partitioning the desired product into an organic phase.
  • Identification and quantification of the product of interest or intermediates can be performed by several methods combining chromatographic separation; for example LC, HPLC, UHPLC or GC, and mass sensitive or photo optical detection.
  • Chromatographic methods can include liquid (MeOH, ACN, hexan, water, acetic acid and others) or gaseous mobile phases (3 ⁇ 4, He, N2, Ar) and liquid or solid stationary phases including silica gel, polydimethylsiloxane or reversed phase materials.
  • Detection of scutellarein and related compounds can be achieved by using MS or MS/MS including sector mass spectrometry, time-of-flight mass spectrometry, the use of quadrupole mass analyzer, three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, orbitrap or fourier transform ion cyclotron resonance and the use of an andequat detector, including electron multiplier systems, faraday cups, ion-to-photon detectors, microchannel plate detectors or inductive detectors.
  • UV-ab sorption fluorescence, charged aerosol detector, evaporative light scattering detector, flame ionization detector, flame photometric detector, nitrogen phosphorus detector, atomic-emission detector, refractive index detector, radio flow detector, conductivity monitor, thermal conductivity detector, electron capture detector and photoionization detectors or combination of those principles can be applied.
  • Scutellarein can also be detected by using chemical reactions, the use of appropriate stains like iodine vapor, iodoplatinate, marquis reagent, nihydrin, HNO3 -atmosphere, NNCD- reagent, PDAB-TS, TACOT, TCBI, vanillin reagents, Van Urk reagent or xanthydrol and the use of an authentic reference substance (Barker et al. 2012 DOI 10.1002/dta.422; Mulga et al. 2012).
  • the following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
  • Figure 1 shows the ZM-DUET starting vector synthesized by Gene Art (Thermo Fischer)(identified as SEQ ID NO 1)
  • Figure 2 shows the pZM-DETET vector with the Zymomonas mobilis (ZM4) origin of replication from the native plasmid ZM402 cloned into the plasmid using the restriction sites Notl and EcoRI.
  • ZM4 Zymomonas mobilis
  • Figure 3 shows the pZM-DUET vector with the Zymomonas mobilis (ZM4) origin of replication from the native plasmid ZM402, wherein the ampicillin resistance cassette is exchanged for the chloramphenicol resistance cassette by PCR and a subsequent cloning step.
  • the terminator sequence is derived from Zymomonas mobilis (identified as SEQ ID NO 8)
  • the ribosome-binding site is derived from Zymomonas mobilis (having a sequence selected from SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11).
  • Figure 4 shows a picture of ZM4 wildtype grown on a rich medium (RM) plate.
  • Figure 5 shows a picture of ZM4 wildtype grown on a RM plate with the addition of 100 pg/ml spectinomycin.
  • Figure 6 shows a picture of ZM4 wildtype grown on a RM plate with the addition of 100 pg/ml chloramphenicol.
  • Figure 7 shows a picture of ZM4 transformed with pZMDuet_pZZM402_Spec grown on a RM plate with the addition of 100 pg/ml spectinomycin.
  • Figure 8 shows a picture of ZM4 transformed with pZMDuet_pZZM401_Spec grown on a RM plate with the addition of 100 pg/ml spectinomycin.
  • Figure 9 shows a picture of ZM4 transformed withpZMDuet_pZZ402_Cam grown on a RM plate with the addition of 100 pg/ml chloramphenicol.
  • Figure 10 shows the results of FACS analysis of ZM4 cells expressing emGFP using pZMDuet. emGFP fluorescence [arbitrary units, a.U.] of different ZM4 strains against normalized cell count. Cells and samples were treated and measured as described above.
  • the plasmids from Table 2 were transformed in ZM4 as indicated by GFP 1.2, GFP 2.1 and GFP_2.2.
  • ZM4 wt plasmid ZM4 wild type
  • Rich medium (RM) media is defined as rich media and its characteristics are described below.
  • E. coli cells are grown in Lysogeny Broth (LB) medium at 37°C.
  • the Z. mobilis strains are grown in RM medium (20 g/1 Glucose, 10 g/1 yeast extract, 2 g/1 KH2PO4) with the addition of 15g/l agar for plates at 30°C. If transformed, the medium is supplemented with 100pg/ml ampicillin, 30pg/ml kanamycin, 33pg/ml chloramphenicol, 15pg/ml tetracycline and 50pg/ml spectinomycin for E.
  • E. coli cells are made chemically competent and transformed using the standard heat shock method.
  • Z. mobilis competent cells are prepared according to Yang et al, and transformed by electroporation using the Bio-Rad Gene Pulser, 0,1 cm gap cuvettes at 1.6 kV (Yans et al, 2016).
  • the pZM-DUET starting vector is synthesized by GeneArt (Thermo Fischer) (see Figure 1) (identified as SEQ ID NO 1).
  • the vector comprises an Ampicillin resistance cassette, an E. coli origin of replication (colEl origin), Notl and EcoRI restriction sites for the insertion of a Zymomonas origin of replication, the native Zymomonas mobilis promoter sequence of pyruvate decarboxylase, a multiple-cloning site (MSC1) with a ribosome-binding site in the center, a Zymomonas terminator, the native Zymomonas mobilis promoter sequence of glyceraldehyde-3 -phosphate dehydrogenase, a second multiple cloning site (MSC2) with a ribosome-binding site in the center and another Zymomonas Terminator.
  • MSC1 multiple-cloning site
  • MSC2 second multiple cloning site
  • the origins of replication are amplified from the genomic DNA or the native plasmids using primers with a Notl and EcoRI overhang.
  • the amplified PCR product is cloned into the pZM- DUET starting vector via restriction digest. If the origin of replication contains a Notl or EcoRI restriction site, then other overhangs are chosen for the PCR amplification (e.g. Sail and Xbal). In that case the Notl and EcoRI restriction sites in the pZM-DUET must be changed, which is also done by overhang PCR amplification before followed by the cloning procedure.
  • Figure 2 shows the pZM-DUET vector with the Zymomonas mobilis (ZM4) origin of replication from the native plasmid ZM402 cloned into the plasmid using the restriction sites Notl and EcoRI.
  • a Sail restriction site is contained on the forward primer (identified as SEQ ID NO 2) and Xbal restriction on the reverse primer (identified as SEQ ID NO 3).
  • a Xmal restriction site is contained on the forward primer (identified as SEQ ID NO 4) and Xbal restriction site on the reverse primer (identified as SEQ ID NO 5).
  • a Sail restriction site is contained on the forward primer (identified as SEQ ID NO 6) and a Xbal restriction site on the reverse primer (identified as SEQ ID NO 7).
  • the pZM-DUET starting vector is amplified by PCR with primer overhangs to generate restriction sites.
  • the forward primer binds the 5’ end of the colEI E.coli origin of replication and the reverse primer binds to the 3’ end of the second terminator and the 5‘ end of the Ampicillin resistance cassette and are identified as SEQ ID NO 12, 13 and 14.
  • the amplification then leads to the generation of Xmal/Sall restriction sites at one end and Xbal at the other end.
  • the resistance cassettes are amplified with primers that have the corresponding overhangs. After the restriction digest, the cassettes are then cloned into the pZM-DUET generating new shuttle vectors.
  • Figure 3 shows the pZM-DUET vector with the Zymomonas mobilis (ZM4) origin of replication from the native plasmid ZM402, the ampicillin resistance cassette is exchanged for the chloramphenicol resistance cassette by PCR and a subsequent cloning step
  • the terminator sequence derived from Zymomonas mobilis identified as SEQ ID NO 8.
  • the ribosome-binding site is derived from Zymomonas mobilis having a sequence selected from SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11.
  • Ncol, BamHI, Hindlll and Pstl restriction sites are in the first multiple cloning site (MCS1).
  • BamHI and Nod restriction sites are between the 3’ end of the pyruvate decarboxylase promoter and the 5’ end of the ribosome-binding site.
  • Hindlll and Pstl restriction sites are between the ribosome-binding site and the terminator.
  • only one gene is cloned into MCS1 of pZM-DUET.
  • the gene is amplified by PCR using a forward primer with an overlap containing the BamHI restriction site and a reverse primer with an overlap containing either the Hindlll or the Pstl restriction site.
  • the PCR product is cloned into MCS1 using BamHI and Hindlll or Pstl, cutting out the ribosome-binding site present in the multiple cloning site.
  • the pZM-DUET shuttle vector codes for a fluorescent protein selected from the green fluorescent protein (GFP), the red fluorescent protein (RFP), the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • CFP cyan fluorescent protein
  • YFP yellow fluorescent protein
  • Ndel, Bglll, KpNI and Xhol restriction sites are in the second multiple cloning site (MCS2).
  • Ndel and Bglll restriction sites are between the 3’ end of the pyruvate decarboxylase promoter and the 5’ end of the ribosome-binding site.
  • KpNI and Xhol restriction sites are located between the ribosome binding site and the terminator.
  • one of the genes cloned into the MCS2 if the pZM-DUET shuttle vector codes for a fluorescent protein selected from the green fluorescent protein (GFP), the red fluorescent protein (RFP), the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • CFP cyan fluorescent protein
  • YFP yellow fluorescent protein
  • ZM4 cells were transformed with pZMDuet_ pZZM402 _Spec, pZMDuet_ pZZM401_Spec and pZMDuet_ pZZM402_Cam. After incubation for 2 - 5 days at 30°C, plates were photographed (see Figures 8-14). As an additional control, ZM4 wt cells were plated out on RM + 2 % glucose agar plate ( Figure 8). No growth could be observed of ZM4 wt on RM + 2 % glucose agar plates containing 100 pg/ml spectinomycin ( Figure 9) and 100 pg/ml chloramphenicol ( Figure 10), .
  • GFP was inserted in MCS1 and MSC2. FACS analysis was used to show that GFP was expressed and correctly folded after the transformation of the pZMDuet GFP Vectors.
  • the gene coding for emGFP was cloned into pZMDuet with spectinomycin cassette and harboring the pZZM402 zymomonas origin of replication.
  • GFP was cloned into MCS1 and MCS2.
  • MCS1 is under the control of the native promoter from PDC and MCS2 is under the control of the native GAP promoter.
  • GFP was cloned either in front of the synthetic Ribosome binding site (using BamHI and Ncol restriction sites for MCS1) of pZMDuet or behind it (using Hindlll and Pstl restriction sites for MCS1).
  • Table 2 Plasmids used in this experiment.
  • Results Results are shown in Figure 10. Compared to the ZM4 wild type (wt) as negative control, all tested strains show fluorescence at least 100 times higher, thus demonstrating that emGFP expression is functional.
  • ZM4 wild type did not produce any CBGA at all, CBGA could be detected in SS and TS samples from ZM4 bearing pZMDuet NphB. This proves that pZMDuet can also be used as a shuttle vector expression system for enzymes following the conversion of substrates to a specific metabolic product.
  • a shuttle vector identified as pZM-DUET for the genus Zymomonas comprising a. one selectable marker gene, b. two prokaryotic origins of replication, c. two or more constitutive gene promoters derived from the genus Zymomonas , d. two or more terminator sequences derived from the genus Zymomonas , and e. a multiple cloning site between the promoter and the terminator.
  • a shuttle vector according to claim 1, wherein the selectable marker comprises a. an ampicillin cassette, b. a kanamycin cassette, c. a chloramphenicol cassette, d. a tetracycline cassette, and e. a spectinomycin cassette.
  • a shuttle vector according to claim 2 wherein a. the chloramphenicol cassette is amplified via PCR, b. the chloramphenicol cassette comprises a promoter, the chloramphenicol selectable marker gene and a terminator, c. a Sail restriction site is contained on the forward primer (SEQ ID NO 2), and a Xbal restriction on the reverse primer (SEQ ID NO 3), d. the PCR product is cloned into the Sail and Xbal restriction sites of the pZM- DUET vector.
  • a shuttle vector according to claim 2 wherein a. the tetracycline cassette is amplified via PCR, b. the tetracycline cassette comprises a promoter, the tetracycline selectable marker gene and a terminator, c. a Xmal restriction site is contained on the forward primer (SEQ ID NO 4) and a Xbal restriction site on the reverse primer (SEQ ID NO 5), d. the PCR product is cloned into the Sail and Xbal restriction sites of the pZM- DUET vector.
  • one of the origins of replication is an Escherichia coli derived origin replication and is selected from pBR322, pUC, pi 5 A, pMBl, CoIEl or one of their derivatives.
  • ZM4 Zymomonas mobilis
  • a. the Zymomonas mobilis derived origin of replication is amplified via PCR
  • b. an EcoRI restriction site on the forward primer and a Notl restriction site on the reverse primer
  • the PCR product is cloned into the EcoRI and Notl restriction sites of the pZM-
  • Ppdc pyruvate decarboxylase
  • Pgap glyceraldehyde-3 -phosphate dehydrogenase
  • Peno phosphopyruvate hydratase
  • a shuttle vector according to claim 1 wherein the multiple cloning site is located between the promoter sequence and the terminator and contains one or more ribosome-binding sites.
  • SEQ ID. NO 1 DNA plasmid pZM-DUET starting vector (2589 bp) ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attcccttttttttttgcggcatt ttgccttcct gtttgctc acccagaaac gctggtgaaa gtaaagatg ctgaagatca gttgggtgca cgagtgggttgca cgagtgggttgca acatcgaact ggatctcaac agcggtaaga tccttgagag tttcg
  • SEQ ID. NO 2 DNA Primer AmpR KanaR CamR Sall FW (36 bp) gcgcgtcgac ttcaaatatg tatccgctca tgagac
  • SEQ ID. NO 9 Zymomonas mobilis DNA Ribosome-binding site 1 (20 bp) gagcgagaag gaggtaaagt
  • SEQ ID. NO 14 DNA Primer pZMDuet_AmpR_Xmal_RV (34 bp) gcgccccggg gtctcatgag cggatacata trig

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Abstract

L'invention concerne un système de vecteur navette efficace pour l'expression de protéines hétérologues et homologues dans une bactérie appartenant au genre Zymomonas. Les vecteurs comprennent au moins deux promoteurs de gènes constitutifs, chacun avec un site de clonage multiple en aval pour l'incorporation de gènes étrangers. Un site de liaison de ribosomes comprend la séquence de promoteur à proximité de l'extrémité 3', tandis que des sites de liaison de ribosomes supplémentaires sont situés dans le site de clonage multiple. Ceci permet la transcription d'ARNm polycistroniques conduisant à l'expression de multiples protéines. De plus, l'incorporation de différentes origines de réplication dérivées de Zymomonas et diverses cassettes de gènes marqueurs sélectionnables facilitent la transformation de plus d'un plasmide en Zymomonas.
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CN115747242A (zh) * 2022-08-25 2023-03-07 湖北大学 消除质粒、质粒组合、基因编辑试剂盒、制备方法及应用
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CN115717150A (zh) * 2022-08-25 2023-02-28 湖北大学 一种穿梭质粒、构建方法及应用
CN115747242A (zh) * 2022-08-25 2023-03-07 湖北大学 消除质粒、质粒组合、基因编辑试剂盒、制备方法及应用
CN115717150B (zh) * 2022-08-25 2023-08-29 湖北大学 一种穿梭质粒、构建方法及应用
CN115747242B (zh) * 2022-08-25 2023-10-24 湖北大学 消除质粒、质粒组合、基因编辑试剂盒、制备方法及应用
CN115806922A (zh) * 2022-09-19 2023-03-17 湖北大学 运动发酵单胞菌的基因工程菌株及其应用
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