[go: up one dir, main page]

WO2013096863A1 - Constructions et procédés pour la biosynthèse améliorée d'isoprène - Google Patents

Constructions et procédés pour la biosynthèse améliorée d'isoprène Download PDF

Info

Publication number
WO2013096863A1
WO2013096863A1 PCT/US2012/071416 US2012071416W WO2013096863A1 WO 2013096863 A1 WO2013096863 A1 WO 2013096863A1 US 2012071416 W US2012071416 W US 2012071416W WO 2013096863 A1 WO2013096863 A1 WO 2013096863A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression cassette
cell
isoprene
recombinant expression
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2012/071416
Other languages
English (en)
Inventor
Anastasios Melis
Andreas Zurbriggen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Publication of WO2013096863A1 publication Critical patent/WO2013096863A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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)
    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • 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)
    • C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)
    • 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)
    • C12Y203/01016—Acetyl-CoA C-acyltransferase (2.3.1.16)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
    • C12Y203/0301—Hydroxymethylglutaryl-CoA synthase (2.3.3.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01036—Mevalonate kinase (2.7.1.36)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04002—Phosphomevalonate kinase (2.7.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/03—Intramolecular oxidoreductases (5.3) transposing C=C bonds (5.3.3)
    • C12Y503/03002—Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)

Definitions

  • isoprene (2 -methyl- 1 ,3-butadiene) is a volatile CsHs terpenoid that is emitted from the leaves of many herbaceous and deciduous plant species.
  • Isoprene synthesis is catalyzed by an isoprene synthase (IspS) enzyme, which is encoded by a nuclear gene for a ehloroplast- iocaiized protein.
  • IspS enzyme catalyzes the remo v al of pyrophosphate from IspS enzyme.
  • DMAPP dimethyiallyl pyrophosphate
  • Plant isoprene synthases have been cloned and characterized from a number of plant species, and the sequences are publically available.
  • DMAPP and IPP are precursors of all cellular isoprenoids. Rates and yield of isoprenoid production by fermentative microbes can be improved by enhancing cellular metabolic flux toward DMAPP.
  • Biological systems employ two independent and distinct biosynthetic pathways by which to generate DMAPP and IPP.
  • the mevalonic acid (MVA) pathway functions in eukaryotes (animals, yeast, fungi, the cytoplasm of plant cells), archaea, and a limited number of eubacteria.
  • the methyleryihriiol phosphate (MEP) pathway functions in most bacteria, cyanobacteria, and algal and plant plastids.
  • the MEP pathway is also referred to as the DXP pathway (1 -deoxy-D-xyluiose-5 -phosphate).
  • the MVA biosynthetic pathway requires acetyl-CoA as the primary feedstock molecule, whereas the MEP pathway requires pyruvate and glyceraldehyde-3-phosphate (G3P) as initial substrates for IPP and DMAPP biosynthesis. These pathways are outlined in Figure 1. Most organisms also contain an IPP isomerase that catalyzes the intercoiiversion of IPP and DMAPP.
  • Microbial production of isoprene is advantageous over plants, as containment and sequestration of the volatile isoprene is relatively easy from microbial fermentors. Further, microbial systems are amenable to large-scale production, and are easily modified for comparison and combination. In addition, many microbial ceils lack the MVA pathway, and thus do not contain regulator '- mechanisms to control isoprene synthesis via the MVA pathway, such as feedback inhibition. This allows for unregulated isoprene synthesis at the expense of other cellular processes, e.g., growth and replication, up to the limit of carbon source available. For example, cyanobacteria (Synechocystis), green microalgae
  • This invention addresses the need for additional methods for producing isoprene using microbial systems.
  • the invention is based, in part, on the discovery that the MVA pathway can be efficiently expressed in bacteria, and in photosyntbetic microorganisms, such as
  • the MVA paihway has evolved to operate in non-photosynthetic organisms, where the oxygen partial pressure in the cell is lower than ambient. Typically, the pathway operates under anaerobic or anoxic conditions in a cell and is inhibited by oxygen.
  • the invention provides methods for expressing the MVA pathway in bacteria and an oxygen-evolving photo synthetic microorganism.
  • the present invention relates to isoprene production in E. coii and Synchocyslis resulting from heterologous expression of MVA enzymes in the bacteria and the
  • the MVA enzymes are typically expressed together on a superoperon, where each member of the pathway is preceded by a Translation Initiation Region (TIR) comprising a ribosomal binding site (RBS).
  • TIR Translation Initiation Region
  • RBS ribosomal binding site
  • the examples provide illustrative data showing that when E. eo/? ' -specific RBS sequences were used, isoprene production was increased over 150-fold, in some instances 800-fold, over E. coii expressing only Isoprene Syntase (IspS).
  • IspS Isoprene Syntase
  • the yield of isoprene improved from 0.2 mg Isp/ L culture with the native MEP pathway to -320 mg Isp/ L with the heterologously-expressed MV A pathway in E. coii, comprising a 1 ,600-fold increase in isoprene production.
  • the results show that over-expression of various members of the MEP pathway with E. coft- specific TIR sequences improved isoprene yield at least 10- to 12-fold over E. coii expressing only IspS.
  • the examples additionally provides illustrative data showing that when optimized RBS sequences and double homologous recombination in Synechocystis were used, isoprene production was improved by 10-fold or greater as compared to Synechocystis expressing only Isoprene Syntase (IspS). Moreover, in this example, the isoprene to biomass carbon partitioning ratio in Synechocystis improved from 0.1% isoprene-to-biomass (w:w), as previously achieved, to 1%, and as high as 10%.
  • the MVA pathway with TIRs specific for bacteria, cyanobacteria, or green microalgae increased isoprene yield 5-6 fold compared to expression with non-cell type specific TIRs.
  • compositions and methods for improved cellular isoprene production comprising a nucleic acid coding sequence of at least one member of the MVA pathway (e.g., MVA enzyme) selected from the group consisting of HMGS (Hydroxy-methyl-glutaryl synthase), HMGR (Hydroxy- methyl-glutaryl reductase), ATOB (Aceryl-CoA acetyla.se), Isopentenyl-pyrophosphate (IPP) isomerase, MVKl (Mevalonic acid kinase), MVD (Di-phospho-mevalonic acid
  • MVA pathway e.g., MVA enzyme
  • nucleic acid coding sequence of the at least one member of the MVA pathway is preceded by a
  • the recombinant expression cassette further comprises the nucleic acid coding sequence of Isoprene Synthase (IspS) preceded by a TIR comprising an RBS.
  • IspS Isoprene Synthase
  • the recombinant expression cassette includes nucleic acid coding sequence for at least 2, 3, 4, 5, 6, or 7 members of the MVA pathway, wherein each nucleic acid coding sequence is preceded by a TIR.
  • the recombinant expression cassette comprises nucleic acid coding sequence for 2 or more members of the MVA pathway, the 2 or more nucleic acid coding sequences are included on a single transcript upon expression (e.g., expression of the 2 or more nucleic acid coding sequences is driven by a single promtor and they are part of a single operon).
  • the recombinant expression cassette comprises the nucleic acid coding sequence ofHMGS, HMGR, ATOB, IPP isomerase, MVKl, MVD, and MVK2, wherein each coding sequence is preceded by a TIR, and wherein each nucleic acid coding sequence is included on a single transcript upon expression (e.g., expression of the nucleic acid coding sequences is driven by a single promoter and they are part of a single operon).
  • the nucleic acid coding sequences are arranged on the recombinant expression cassette in any order.
  • the nucleic acid coding sequences are arranged in the following order: HMGS-HMGR-ATOB-IPP isomerase-MVKl-MVD-MVK2. In other embodiments, the nucleic acid coding sequences are arranged in the following order: HMGS- HMGR-ATOB-IPP isomerase- VKl -MVK2- VD.
  • the RBS effectively binds ribosomes in the cell, e.g., resulting in a translation initiation rate of at least 1000, 2000, 5000, 10,000 or higher; binds at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher percentage of ribosomes as a native RBS in the cell; or binds with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher affinity as a native RBS in the cell.
  • the RBS comprises at least 3, 4, 5, 6, 7, or 8 contiguous nucleotides complementary to the 16S rRNA (for expression in a prokaryotic cell) or the 18S rRNA (for expression in a eiikaryotic cell).
  • the RBS comprises at least 3, 4, 5, 6, 7, or 8 contiguous nucleotides complementary to the I6S rRNA of bacteria (e.g., E. coli) or cyanobacteria (e.g., Synechocystis).
  • the RBS comprises at least 3, 4, 5, 6, 7, or 8 contiguous nucleotides complementary to the 18S rRNA of green microalgae (e.g. , Chlamydomonas).
  • the RBS is selected from any of the RBS sequences shown in SEQ ID NOs: 1 -17.
  • the at least one TIR further comprises a spacer of about 4-12, 5-10, 6-9, 6-8 nucleotides. In some embodiments, the at least one TIR further comprises a restriction site (e.g. , about 6- 10 or 6-8 nucleotides). In some embodiments, the at least one TIR has a configuration of: Restriction site-RBS-spacer or Spacer- RBS-Restriction site.
  • each TIR can be the same or different from the other TIRs on the recombinant expression cassette in any combination.
  • the restriction site for each TIR can be different from the restriction site of every other TIR on the recombinant expression cassette.
  • the spacer for each TIR can be different from the spacer of every other TIR on the recombinant expression cassette, and the RBS for each TIR can be different from the RBS of every other TIR on the recombinant expression cassette.
  • the RBS of each TIR is the same, or the RBSs of 2, 3, 4, 5, 6, or all of the TIRs are the same.
  • each TIR independently comprises a sequence is selected from the group consisting of SEQ ID NOs: 1-17. In some embodiments, each TIR mdependently is selected from the group consisting of SEQ ID NOs: 1-17. In some embodiments, each TIR independently comprises a sequence at least 90 or 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 19-25. In some embodiments, each TIR mdependently is at least 90 or 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 19-25. In some embodiments, each TIR independently comprises a sequence is selected from the group consisting of SEQ ID NOs: 19-25. In some
  • each TIR independently is selected from the group consisting of SEQ ID NOs: 19-25.
  • the nucleic acid coding sequence of HMGS encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:27, or a species homolog thereof, wherein the HMGS polypeptide retains HMGS activity.
  • the HMGS polypeptide has a sequence of SEQ ID NO:27.
  • the nucleic acid coding sequence of HMGS is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:26, or a species homolog thereof, wherein the encoded HMGS polypeptide retains HMGS activity.
  • the nucleic acid coding sequence of HMGS is SEQ ID NO:26.
  • the nucleic acid coding sequence of HMGS is codon-optimized for expression in a particular host cell.
  • the nucleic acid coding sequence of HMGR encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:29, or a species homolog thereof, wherein the HMGR polypeptide retains HMGR activity.
  • the HMGR polypeptide has a sequence of SEQ ID NO:29.
  • the nucleic acid coding sequence of HMGR is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID MO:28, or a species homolog thereof, wherein the encoded HMGR polypeptide retains HMGR activity.
  • the nucleic acid coding sequence of HMGR is SEQ ID NO:28.
  • the nucleic acid coding sequence of HMGR is codon-optimized for expression in a particular host cell.
  • the nucleic acid coding sequence of ATOB encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:3 I, or a species homolog thereof, wherein the ATOB polypeptide retains ATOB activity.
  • the ATOB polypeptide has a sequence of SEQ ID NO:31.
  • the nucleic acid coding sequence of ATOB is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:30, or a species homolog thereof, wherein the encoded ATOB polypeptide retains ATOB activity.
  • the itcleic acid coding sequence of ATOB is SEQ ID NO:30. In some embodiments, the nucleic acid coding sequence of ATOB is codon-optimized for expression in a particular host cell. [0018] In some embodiments, the nucleic acid coding sequence of MVK1 encodes a polypeptide having at least SO, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:33, or a species homolog thereof, wherein the MVK1 polypeptide retains MVK1 activity. In some embodiments, the MVK1 polypeptide as a sequence of SEQ ID NO:33.
  • the nucleic acid coding sequence of MVK1 is at feast 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID O:32, or a species homolog thereof, wherein the encoded MVK1 polypeptide retains MVK1 activity.
  • the nucleic acid coding sequence of MVKI is SEQ ID NO:32.
  • the nucleic acid coding sequence of MVKI is codon-optimized for expression in a particular host cell.
  • the nucleic acid coding sequence of MVD encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:35, or a species homolog thereof, wherein the MVD polypeptide retains MVD activity.
  • the MVD polypeptide has a sequence of SEQ ID NO:35.
  • the nucleic acid coding sequence of MVD is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:34, or a species homolog thereof, wherein the encoded MVD polypeptide retains MVD activity.
  • the nucleic acid coding sequence of MVD is SEQ ID NO:34.
  • the nucleic acid coding sequence of MVD is codon-optimized for expression in a particular host cell.
  • the nucleic acid coding sequence of MVK2 encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:37, or a species homolog thereof, wherein the MVK2 polypeptide retains MVK2 activity.
  • the MVK2 polypeptide has a sequence of SEQ ID O:37.
  • the nucleic acid coding sequence of MVK2 is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:36, or a species homolog thereof, wherein the encoded MVK2 polypeptide retains MVK2 activity.
  • the nucleic acid coding sequence of M VK2 is SEQ ID NO:36.
  • the nucleic acid coding sequence of M VK2 is codon-optimized for expression in a particular host cell.
  • the nucleic acid coding sequence of MVK2 encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID O:37, or a species homolog thereof, wherem the MVK2 polypeptide retains MVK2 activity.
  • the MVK2 polypeptide has a sequence of SEQ ID NO:37.
  • the nucleic acid coding sequence of MVK2 is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:36, or a species homolog thereof, wherem the encoded MVK2 polypeptide retains MVK2 activity.
  • the nucleic acid coding sequence of MVK2 is SEQ ID NO:36.
  • the nucleic acid coding sequence of MVK2 is codon-optimized for expression in a particular host cell.
  • the nucleic acid coding sequence of IPP isomerase encodes a polypeptide having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:39, or a species hornolog thereof, wherein (he IPP isomerase polypeptide retains IPP isomerase activity.
  • the IPP isomerase polypeptide has a sequence of SEQ ID NO:39.
  • the nucleic acid coding sequence of IPP isomerase is at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO:38, or a species homoiog thereof, wherein the encoded IPP isomerase polypeptide retains IPP isomerase activity.
  • the nucleic acid coding sequence of IPP isomerase is SEQ ID NO:38.
  • the nucleic acid coding sequence of IPP isomerase is codon-optimized for expression in a particular host cell.
  • the recombinant expression cassette comprises a sequence having at least 80, 85, 90, 95, 96, 97, 98, 99, or higher percent identity to SEQ ID NO: 18, wherein, upon expression in a ceil, the cell produces more isoprene than a control ceil lacking the recombinant expression cassette.
  • the cell and control cell further express heterologous IspS.
  • the nucleic acid coding sequence of IspS is codon- optimized for expression in a host cell.
  • the IspS gene from Pueraria Montana (Sharkey et al., Plant Physiol, 137:700; GenBank accession no. AY316691) can be optimized for codon-usage in Svnechocystis, without the predicted chioroplast transit peptide (see, e.g., Lindberg et al., Metahol. Engin. 12:70).
  • the recombinant expression cassette comprises a single promoter driving expression of the at least one nucleic acid coding sequence, e.g., so that the nucleic acid coding sequences are included in a single operon.
  • the recombinant expression cassette comprises a single terminator.
  • the recombinant expression cassette comprises more than one promoter and/or more than one terminator.
  • the recombinant expression cassette can have a promoter driving expression of a first set of nucleic acid coding sequences, and a second promoter driving expression of a second set of nucleic acid coding sequences (and a third and fourth, etc.).
  • the recombinant expression cassette comprises a separate promoter for each nucleic acid coding sequence.
  • the recombinant expression cassette comprises flanking regions, wherein a first flanking region is adjacent to, e.g., upstream of, the promoter and a second flanking region is adjacent to, e.g., downstream of, the terminator.
  • the first flanking region is preceded by a promoter.
  • the second flanking region is followed by a terminator.
  • the flanking regions are located on either side of, but not immediately adjacent to, the nucleic acid coding sequence(s) of the recombinant expression cassette.
  • flanking regions of the recombinant expression cassette comprise a nucleic acid sequences of a specific gene in the host cell.
  • the flanking regions of the recombinant expression cassette comprise sequences of a gene expressed in bacteria, cyanobacteria and/or microalgae.
  • the flanking regions can be nucleic acid sequences of a gene, wherein the first and second flanking regions are not identical sequences.
  • flanking regions contain sequences of a gene expressed in cyanobacteria, such as, but not limited to, a neutral ( eu) site (ORF sir0168), PsbA2 (ORF s1rl31 1), GlgA (ORF sllQ945), and GlgX (ORF slr0237).
  • a neutral ( eu) site ORF sir0168
  • PsbA2 ORF s1rl31 1
  • GlgA ORF sllQ945
  • GlgX ORF slr0237
  • flanking regions are used for homologous recombination into the genome of a host ceil, such as bacteria, cyanobacteria, or green microalgae.
  • the flanking regions comprise a sequence having at least 50, 100, 200, 300, 400, 500, 600, or more base pairs of a gene in the host cell.
  • the flanking regions can be about 500 bp of sequence and located on either side of the transgene
  • genes in the host cell include , a neutral (Neu) site (ORF slr0168 ), PsbA2 (ORF slrl31 1), GlgA (ORF sll()945), and GlgX (ORF slr0237).
  • a neutral (Neu) site ORF slr0168
  • PsbA2 ORF slrl31 1
  • GlgA ORF sll()945
  • GlgX ORF slr0237
  • flanking regions comprise a nucleic acid sequences that can undergo double homologous recombination into the host cell's genome at a site of integration.
  • Methods of homologous recombination in cyanobacteria are described in detail in, for example, Flores et al., "Gene Transfer to Cyanobacteria in the Laboratory and in Nature” in The Cyanobacteria: Molecular Biology, Genomics, and Evolution, Caister Academic Press, Seville, Spain, 2008.
  • the cell lacks the MVA pathway, and thus lacks the ability to regulate isoprene production resulting from the MVA pathway. Accordingly, further provided is a cell comprising the recombinant expression cassette as described above, wherein the cell lacks the MVA pathway.
  • the cell is selected from bacteria, cyanobacteria, and green microalgae.
  • the cell comprises heterologous nucleic acid coding sequences for HMGS, HMGR, ATOB, IPP isomerase, MVK1, IvWD, and MVK2, wherein each coding sequence is preceded by a TIR comprising an RBS, as described above.
  • the nucleic acid coding sequences are included on more than one recombinant expression cassette, e.g., 2, 3, or 4 recombinant expression cassettes. In some embodiments, the nucleic acid coding sequences are included on a single recombinant expression cassette in the cell. In some embodiments, the cell further comprises a nucleic acid coding sequence for IspS, wherein the IspS coding sequence is on the same or a different recombinant expression cassette as the nucleic acid coding sequences for HMGS, HMGR, ATOB, IPP isomerase, MVK 1 , MVD, and/or MVK 2.
  • the method comprises recombinantly expressing at least one member of the MVA pathway (i.e., HMGS, HMGR, ATOB, IPP isomerase, MVK1, MVD, and MVK2) in the cell, and culturing the ceil in the presence of a carbon source, thereby producing isoprene in the cell.
  • the method further comprises recombinantly expressing IspS.
  • the carbon source is selected from glycerol, fructose, xylose, glucose, and LB. In some embodiments, the carbon source is C0 2 . In some embodiments, the culturing is carried out at 20-45°C, e.g., room temperature, 37°C, 30-42 °C, 3G-40°C, or 32-38°C.
  • HMGS, HMGR, ATOB, IPP isomerase, MVK1 , MVD, and/or MVK2 are recombmantly expressed from at least one recombinant expression cassette as described above.
  • the recombinant expression cassette can comprise a nucleic acid coding sequence of at least one member of the MVA pathway selected from the group consisting of HMGS, HMGR, ATOB, IPP isomerase, MVK ! , MVD, and MVK2, wherein the nucleic acid coding sequence of the at least one member of the MVA pathway is preceded by a TIR comprising an RBS.
  • the recombinant expression cassette further comprises the nucleic acid coding sequence of IspS preceded by a TIR comprising an RBS.
  • the recombinant expression cassette comprises the nucleic acid coding sequences of HMGS, HMGR, ATOB, IPP isomerase, MVK1, MVD, and MVK2.
  • the nucleic acid coding sequence of each member of the MVA pathway is preceded by a TIR comprising an RBS.
  • each nucleic acid coding sequence is included on a single transcript upon expression.
  • the nucleic acid coding sequences are in the following order: HMGS-HMOR-ATOB-IPP isomerase-MVKl-MVD-MVK2. In other embodiments, the nucleic acid coding sequences are in the following order: HMGS-HMGR-ATOB-IPP isomerase-MVKl-MVK2.-MVD.
  • At least one recombinant expression cassette is targeted to the host cell's genome at a specific site of integration via double homologous recombination.
  • the site of integration includes any region of the host cell genome able to express a recombinant expression cassette.
  • Sites of integration include but are not limited to, a neutral (Neu) site (GenBank Accession no. BAA10047), PsbA2 gene (GenBank Accession no. X13547), GlgA gene, and GlgX gene.
  • operons of the MVA pathway are recombined into a specific genomic location.
  • at least one member of the upper MVA pathway e.g., IIMGS, HMGR and ATOB
  • at least one member of the lower MVA pathway e.g., IPP isonierase, MVK1, MVD, and
  • MVK.2 is recombined into second genomic location.
  • a recombinant expression cassette can contain nucleic acid coding sequences for HMGS, HMGR and ATOB and flanking regions for the PsbA2 gene.
  • other recombinant expression cassettes can contain nucleic acid coding sequences for IPP isomerase, MVK I, MVD, and MVK2 and flanking regions for the GlgA gene or the GlgX gene.
  • the Isps operon can be targeted to the neu site using a recombinant expression cassette with neu flanking regions on either side of the nucleic acid coding sequence of IspS.
  • the method further comprises introducing the recombinant expression cassette to the cell prior to the expressing step.
  • the method further comprises introducing a recombinant expression cassette comprising IspS, e.g., simultaneously or consecutively, with the recombinant expression cassette comprising the nucleic acid coding sequence of at least one member of the MVA pathway.
  • the method further comprises harvesting the isoprene emitted from the cell.
  • members of the MVA pathway can be included on more than one recombinant expression cassette, as described above, and that the TIRs and RBSs can be selected as described above.
  • the recombinant expression cassette can have one or more promoters driving expression of the members of the MVA pathway, and/or one or more terminators of transcription.
  • the method results in an increase in the amount of isoprene produced by the cell compared to a control ceil not recombinant! ⁇ ' expressing the at least one member of the M VA pathway.
  • the method results in at least a 2-, 5-, 6-, 8-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 200-, 500-, 600-, 1000-, 1200-, 1600-, 2000-fold or higher fold increase in the amount of isoprene produced by the ceil compared to a control cell not recombinantly expressing the at least one member of the MVA pathway.
  • the method results in a cessation of cell growth, i.e., the cells no longer replicate, or grow/ replicate at a greatly reduced rate. In some embodiments, the method results in cell growth 20, 10, 5, 1% or lower compared to a control cell not recombinantly expressing the at least one member of the MVA pathway.
  • the carbon source is quantitatively converted to isoprene, e.g., with an efficiency of 18, 25, 30, 50, 60, 70, 80% or higher.
  • the mass ratio of isoprene to dry cell weight (Isp/DCW) is at least 0.25, 0.5, 0.7, 0.8, 0.9, 1 .0 or higher.
  • the isoprene -to-biomass (w:w) ratio is at least 0.3%, 0.5%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 1 1.0%, 12.0%, 13.0%, 14.0%, 15.0% or more.
  • a recombinant expression cassette for producing isoprene in a cell comprising the nucleic acid coding sequences for at least one member of the MEP pathway selected from the group consisting of IspG, Dxr, IspE, IspH, Dxs, IspD, IspF, and Ipi, wherein each nucleic acid coding sequence is preceded by a TIR comprising an RBS.
  • the components of the recombinant expression vector can be selected as described above, e.g. , the TIR can also include a restriction site and/ or spacer, and the RBS can be selected to specifically bind the ribosomes in the ceil.
  • the recombinant expression cassette comprises the nucleic acid coding sequence of IspG, Dxr, IspE, IspH, Dxs, IspD, IspF, and Ipi. In some embodiments, the recombinant expression cassette further comprises IspS. [0041] Similarly, further provided is a ceil comprising a recombinant expression cassette comprising at least one member of the MEP pathway as described above. In some embodiments, the cell further comprises a recombinant expression cassette comprising the nucleic acid coding sequence of IspS.
  • the nucleic acid coding sequence of at lea st one member of the MEP pathway is on at least one recombinant expression cassette wherein each nucleic acid coding sequence is preceded by a T1R comprising an RBS.
  • the recombinant expression cassette comprises the nucleic acid coding sequences of IspG, Dxr, IspE, IspH, Dxs, IspD, IspF, and Ipi.
  • the method further comprises recombinantly expressing IspS, e.g., from the same recombinant expression cassette or from a separate recombinant expression cassette.
  • IspS isoprene synthase
  • Prv ⁇ Pyruvate the substrate of isoprene synthase
  • GAP Glyeeraldeliyde-3-phosphate
  • ATP Adenosine-5''- triphosphate
  • NAD(P)H Nicotinamide adenine dinucleotide (phosphate).
  • FIG. 1 Enzymatic characterization of recombinant klspS.
  • A Lineweaver-Burk diagram of klspS activity as a function of DMAPP concentration. The K m value was 2.5 mM and t eat was 4.4 s " '.
  • B Effect of divalent cations on klspS activity. Concentrations of divalent cations were 10 mM.
  • C Temperature profile.
  • D pH profile. The buffers were Citrate (pH 3.0), Acetate (pH 5.0), MOPS (pH 6.5-7.5), Bicine (pH 7.5-9.0), and CAPS (pH 10.0-1 1.0), all at 10 mM. Each value is shown as mean ⁇ SE (n > 3).
  • Figure 3 GC profiles of headspace gases from control (not- induced) and IP ' TG- induced Rosetta transformant. Both strains contained the klspS transgene on a pJF plasmid. The non-induced sample shows a single eihanol peak with a retention time of 2.9 min, and the induced sample shows the eihanol peak and the isoprene peak with retention time of 2.9 and 3.5 min.
  • FIG. 1 GC-MS analysis.
  • A) and C) show the GC chromatogram of the overhead space of the klspS transformant and the isoprene standard, respectively.
  • B) and D illustrate the mass spectrum of the dominant peak at 1.5 min retention time of the klspS transformant and the isoprene standard, respectively.
  • FIG. 5 Relative in vivo isoprene production using different klspS constructs. klspS amino acids 45-608 was predicted by ChloroP to be the mature protein. Successive N- terminal truncations were evaluated on isoprene production yields. Construct klspS 267-608 represents the globular C-tenninal domain bearing the active site pocket. Each value is shown as mean ⁇ SE (n > 3).
  • FIG. 1 Relative in vivo isoprene production.
  • A kispS induced at different cell densities.
  • B Effect of organic carbon sources on isoprene production.
  • Values are relative isoprene amounts per gram dry cell weight (DCW). Each value is shown as mean ⁇ SE (n > 3). The normalized 100% value corresponds to 2.25 mg isoprene per gram DCW.
  • FIG. 7 Isoprene production by Rosetta cells harbouring different MEP and MVA pathway expression cassettes. Construction of isoprenoid pathway plasmids: First, genes including T1R1/TIR2 were cloned sequentially to produce two parts which were fused in a final step. The MEP pathway was constructed in 2 expression cassettes (plasmid series A and B). GREHSDFi (A) was cloned using both TTR1 and TTR2, whereas for SGHiERDF (B), only TIR2 was used. The upper MVA pathway (HmgS, HmgR, and AtoB) was constructed from HmgS and HmgR from E. faecalis and AtoB from E. coli.
  • HmgS, HmgR, and AtoB The upper MVA pathway was constructed from HmgS and HmgR from E. faecalis and AtoB from E. coli.
  • MK MVK I
  • PMK MVK2
  • PMD PMD
  • Fni TPP isomerise
  • C native TIRs
  • D E. coli TIRs
  • Arrows indicate genes, black and grey boxes represent artificial TIRs and promoter/terminator, respectively.
  • E Isoprene production by Rosetta from plasmid pJF-klspS and pET containing various isoprenoid pathway constructs. Each value is shown as mean ⁇ SE (n > 3).
  • FIG. 1 Isoprene production measurements. Gas samples of Rosetta cells transformed with constructs in Figure 7 are shown. Ail strains have klspS on a pJF plasmid and the second plasmid (pET28) contains the various constructs.
  • A Control, empty pET28 plasmid;
  • B pET28 with the full MEP pathway (SGHiEFDF);
  • C pET28 with the MVA pathway using native RBSs (HsHrA I(K IDK2I)n), and
  • Figure 9 Isoprene production by Rosetta expressing klspS and the MVA pathway with cell-specific RBSs (Fig. 7D). Cells were induced at ODeoo ;;; 0-25 (empty symbols) and 0,7 (filled symbols), respectively. Squares show isoprene amounts (left axis), whereas circles show cell densities at QDcoo (right axis).
  • FIG. 10 CLUSTAL W multiple sequence alignments of known proteins.
  • the putative chloroplast transit peptide CpTP is shown by the underlined amino acid sequences. Cys amino acids are highlighted, including conservative Ser substitutions. Tandem arginine (R) residues, occurring nine amino acids upstream of an absolutely conserved tryptophan residue, which are motifs conserved in ierpene synthases, are highlighted. Aspartate (D) residues of the absolutely conserved DDXXD terpene synthase motif are highlighted.
  • Figure 11 The mevalonic acid (MVA) and methyleryihritoi phosphate (MEP) biosynthetic pathways. MVA pathway enzymes: AtoB, acetyl-CoA acetyl transferase;
  • HmgS Hmg-CoA synthase
  • HmgR Hmg-CoA reductase
  • MK mevalonate kinase
  • PMK mevalonate 5-phosphate kinase
  • PMD mevalonate 5-diphoshate decarboxylase
  • Fni IPP isomerase.
  • MEP pathway intermediate metaboiiies DXP, deoxyxviuiose 5-phosphate; MEP, methylerythritol 4-phosphate; CDP-ME, diphosphocytidylyi methyl erythritol; CDP-MEP, CDP-ME 2 -phosphate; ME-cPP, methylerythritol 2,4-cyclodiphosphate; HMBPP, hydroxymethylbutenyl diphosphate.
  • MEP pathway enzymes Dxs, DXP synthase (slll945); Dxr, DXP reductoisom erase (sll0019); IspD, CDP-ME synthase (slr095i); IspE, CDP-ME kinase (sll071 1); IspF, ME-cPP synthase (sir 1542); IspG, HMBPP synthase (sfr2I36); IspH, HMBPP reductase (slr0348); Ipi, IPP isomerase (sill 556).
  • Figure 12 Constructs designed for the expression of the isoprene synthase and the MVA biosynthetic pathway in cyanobacteria. AH operons were under the transcriptional control of the native Synechocystis PsbA2 promoter (P) and terminator (T) sequences.
  • TTRs Translation initiation regions
  • IspS Pueraria moniana isoprene synthase gene
  • Neu neutral site
  • the upper MVA pathway operon (SRA) included HmgS, HmgR and AtoB, and was cloned into the PsbA2 site of the Synechocystis genome using the PsbA2 flanking sequences for homologous recombination, and replacing the native PsbA2 gene.
  • the lower MVA pathway operon (FK] DK 2 ) included Fni, MK, PMD and PMK, and was cloned into the GigX or GigA sites of the Synechocystis genome using the GigX and GigA flanking sequences, respectively, for homologous recombination.
  • D The complete M VA.
  • Transformant strains are: (1) SRAFKiOK 2 -ApsbA2; (2) IspS-AN «i; (3) SRA- ApsbA2: ⁇ K X DK 2 - AG!gA :I - spS-ANeu; (4) SRA-ApsbA2:: ⁇ K DK 2 -AGlgX:-JspS-ANeu.
  • Isoprene peaks labeled with asterisks, having a retention time of 3.5 niin, were identified by comparison with an isoprene vapor standard (5), which also has a retention time of 3.5 min.
  • Figure 14 Isoprene production in cyanobacterial strains carrying the MVA super- operons. Isoprene accumulation in the headspace of the gaseous/aqueous two-phase bioreactor was measured by GC analysis after 196 hours of photoautotrophic growth.
  • Transformant strains are: ( 1) isp$-ApsbA2; (2) IspS-ANeu; (3) SRA ⁇ KiDK 2 - ApsbA2 :lspS- ANeu; (4) SBA-ApsbA2:: ⁇ KiDK 2 -AGlgA::lspS-ANeu; (5) SKA- ApsbA2: :FKiDK 2 -
  • FIG. 15 Biomass accumulation in cyanobacterial strains carrying the different MVA super-operons. Biomass accumulation was measured by dry cell weight (DCW) after 196 hours of photoautotrophic growth, which was supported by aliquots of 100% C0 2 administered to the liquid culture every 24 h.
  • DCW dry cell weight
  • Transformant strains are: ( I) IspS-ApsbA2; (2) lsp$-ANeu; (3) $RAFK 1 OK 2 -ApsbA2:-lsp$-ANeu; (4) SRA-ApsbA2:: ⁇ K ] OK 2 -AGlgA::lspS- ANeu; (5) SRA-ApsbA 2: : ⁇ KiDK 2 -AGlgX: :lspS-ANeu. [0057] Figure 16. Carbon-partitioning towards isoprene in cyanobacterial strains carrying the MVA super-operons.
  • Transformant strains are: ( 1) lsp$-ApsbA2; (2) IspS-ANeu; (3) SRA ⁇ K ⁇ OK 2 -ApsbA2 JspS-ANeu; (4) : (5) SBA-ApsbA2:: ⁇ KiDK 2 -AGlgX::lspS-ANeu.
  • FIG. Kinetics of isoprene production and biomass accumulation in cyanobacterial strains carrying the different MVA super-operons. Cumulative isoprene producti on was measured over 196 h of photoautotrophic growth (A), and the corresponding biomass accumulation was measured as dry cell weight (B). Transformant strains are: white circle: SRAFKiDK 2 -ApsbA2; black circle: IspS-ANeu; white square: SRA-AAVM2::FKIDK 2 - AGigAvlspS-ANeu; black square: SRA ⁇ psbA2::FKiDK 2 -AGlgX :J$pS-ANeu.
  • FIG. Western blot analysis of proteins from cyanobacteri l strains showing expression of the proteins encoded by the introduced genes of the MVA super-operons.
  • A The wild-type recipient strain (left lane) and a transiormant strain carrying the recombinant MV pathway super-operon (right lane) were probed with specific polyclonal antibodies for the MVA pathway enzymes: HmgS, HmgR, AtoB, Fni, MK, PMD, PMK.
  • B The corresponding Coomassie stained gel is a control for equal protein loading, and the molecular weight markers are labeled in the far right lane.
  • the molecular weight for each protein of the MVA pathway is as follows: HmgS, 42 kDa; HmgR, 87 kDa; AtoB, 40 kDa; Fni, 38 kDa; MK, 31 kDa; PMD, 39 kDa; PMK, 37 kDa.
  • Microbes can be used for relatively inexpensive biosynthesis of desired molecules.
  • Isoprene is a small volatile molecule that can be emitted and harvested from the overhead space of a microbial culture, as opposed to more complicated extraction from intracellular spaces in plants.
  • the present disclosure shows that production of isoprene in microbial cells, which normally utilize the MEP pathway, can be vastly increased upon heterologous expression of the MVA pathway, which is normally utilized in eukaryotes.
  • Microbial cells that lack the MVA pathway can dedicate resources to production of isoprene without regulation, as the cellular machinery for regulation does not exist.
  • the cells cease other functions, such as cell growth and division, and dedicate resources to isoprene production with high efficiency.
  • Previous methods for cellular isoprene production typically rely on at least one endogenous enzymatic reaction that is subject to cellular control, thus reducing isoprene production.
  • the mevalonic acid (MVA) pathway is used by eukaryotic cells to synthesize terpenoids, e.g., isoprene.
  • the name derives from one of the precursors, mevalonic acid (or mevalonate).
  • the enzymes and precursors involved in the MVA pathway are shown in Figure 1.
  • a "member of the MVA pathway” refers to an enzyme, e.g., HMGS ( Hydrox -methyl-glutaryl synthase), PIMGR (Hydroxy-methyl-glutaryl reductase), ATOB (Acetyl-CoA acetylase), Isopentenyl- pyrophosphate (IPP) isomerase, MVK1 (Mevalonic acid kinase), MVD (Di-phospho-mevalonic acid decarboxylase), and MVK2
  • the methyl-erythritol 4-phospliate (MEP) pathway (also called the non-mevaloiiate or DXP pathway) is used for terpenoid production in prokar otes and the chloroplasts of plants.
  • the enzymes and precursors involved in the MEP pathway are also shown in Figure 1 ,
  • a "member of the MEP pathway” refers to an enzyme, e.g., DOXP synthase (Dxs), DOXP reductase (Dxr), IspD, IspE, IspF, HMB-PP synthase (IspG), and FiMB-PP reductase (IspFI).
  • isoprene has the chemical formula C-stig and can serve as starting material for a number of synthetic reactions resulting, e.g., in rubber, adhesives, and plastic.
  • a TTR. Translation Initiation Region
  • a polycistro ic construct e.g., a superoperon with multiple coding sequences expressed on a single transcript
  • the polycistronic construct can comprise any number of coding sequences,
  • a typical TTR includes a restriction site (used for cloning), followed by an RBS (Ribosome Binding Site, used for recruiting ribosomes (e.g., the 16S or 18S rRNA) to the transcript, and a spacer (which positions the start codon for translation).
  • RBS Rabosome Binding Site, used for recruiting ribosomes (e.g., the 16S or 18S rRNA) to the transcript, and a spacer (which positions the start codon for translation).
  • RBS Rabosome Binding Site
  • the term "effectively binds to ribosomes" or "effectively recruits ribosomes,” in reference to an RBS, indicates that the RBS binds to ribosomes in the relevant cell or expression system in a manner sufficient to initiate translation.
  • an RBS in a bacterial (e.g., E. coli) cell is selected to bind to bacterial (E.
  • ribosomes e.g., the 16S rRNA
  • an RBS in a cyanobacterial ceil e.g., Synechocystis
  • an RBS in a green microalgae cell is selected to bind to ribosomes (e.g., the 18S rRNA) in green microalgae, etc.
  • the cell or expression system can be manipulated to include heterologous ribosomes that bind to a particular RBS.
  • 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.
  • 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 iranscribed into RNA, and the RN A 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.
  • a coding sequence can include degenerate codons (relative to the native sequence) or sequences that provide codon preference in a specific host ceil [0069]
  • promoter refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RN A polymerase and other proteins to initiate transcription.
  • termination signal or “terminator” refers to an element that terminates transcription.
  • 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
  • complementary refers to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second
  • 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 m metics 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, ⁇ - 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.
  • the terms "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.
  • 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 e v ery 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 "consen'ativelv 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.
  • 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., the NCBI web site at
  • 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.
  • An "expression cassette” refers to a nucleic acid construct, which when introduced into a host cell results in transcription and/or translation of a RNA or polypeptide, respectively.
  • 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 terms "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 ei ah, 1989, Molecular Cloning: A Laboratory Manual, 18.1- 18.88.
  • 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 operabiy 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.
  • Recombinant refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism.
  • a recombinant polynucleotide or a copy or complement of a recombinant polynucleotide is one that has been manipulated using well known methods.
  • a recombinant expression cassette comprising a promoter operabiy 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 ei ⁇ , 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).
  • culture 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 C0 2 .
  • 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).
  • a cell comprising a recombinan t expression cassette comprising the members of the MVA pathway or MEP pathway could be compared to a negative control cell lacking the recombinant expression cassette.
  • 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 isoprene production.
  • 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. [0086]
  • 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. For example, if v alues for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. ill. Cells
  • the cells used to produce isoprene as described herein are genetically modified. That is, heterologous nucleic acid is introduced into the cells.
  • the genetically modified 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.
  • genes encoding desired enzymes can be heterologous to the cell, or native to the cell but operaiively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the cell.
  • any microorganism can be used in the present method so long as it remains viable after being transformed with a heterologous expression cassette.
  • the microorganism is bacterial.
  • the bacteria is a cyanobacteria.
  • bacterial host cells include, without limitation, those species assigned to the Escherichia, Enterobacter, Arcobacter, Azotobacter, Campylobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Streptomyces, Streptococcus, Staphylococcus, Synechococcus, Synechocysiis, and Paracocciis taxonomical classes.
  • Photosynthetic microorganisms that can be used to produce isoprene include cyanobacteria and green microalgae. Photosyrithetic microorganisms can be grown in fresh or salt water, e.g., in a photobioreactor. Examples of cyanobacteria include Nostoc,
  • Anahaena Spirulina, Synechococcus, Synechocysiis, Athrospira, Gleocapsa, Oscillatoria, and Pseudoanabaena.
  • algae examples include a microalga (e.g.,
  • the algae is a green algae, for example algae from the genus Tetraselmia, the genus Micr actinium, the genus Desmodesmm, the genus Scenedesmus, the genus Nannochloropsis or the ge us Botryococcus.
  • a non-limiting list of specific photosynthetic microorganisms for isoprene production includes Chiamydomonas reinhardiii, Scenedesmus obliq us, Chlorella vulgaris, Botryococcus hraunii, Botryococcus sudeticus, Dunaliella salina, and Haematococcus pluvialis.
  • Cyanobacteria that can be genetically modified include thermophilic
  • cyanobacteria such as Thermosynechococcus elongatas; and cyanobacteria of the genera Synechococcus, Synechocysiis and Anahaena, including the species Synechocyslis sp. PCC 6803 and Anahaena 7120.
  • Microorganisms used for isoprene production e.g., microorganisms lacking the MVA pathway, e.g., bacteria, cyanobacteria or green microalgae, are engineered to express heterologous enzymes that generate isoprene.
  • nucleic acid constructs described herein can be operabiy 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 enzym.es 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.
  • nucleic acid coding sequences for desired biosynthetic enzymes can be incorporated into an expression cassette.
  • a typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions (e.g., promoter), along with a ribosome binding site (RBS), e.g., a nucleotide sequence that is 3-9 nucleotides in lengih thai binds ribosomes in the cell, and which is locaied 3-1 1 nucleotides upstream of the initiation codon in E. coii. See Shine et al. (1975) Nature 254:34 and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1 , p. 349, 1979, Plenum Publishing, N.Y.
  • Regulatory regions include, for example, those regions that contain a promoter and an operator.
  • a promoter is operably linked to the desired nucleic acid sequence (e.g., operon or superoperon), to drive transcription of the nucleic acid sequence via an R A polymerase enzyme.
  • An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein- binding domain of the operator binds to the operator, thereby inhibiting transcription.
  • lactose promoters Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator
  • tryptophan promoters when complexed with tryptophan, TrpR repressor pro tein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator.
  • tac promoter See deBoer et al. ( 1983) Proc. N ' atl. Acad. Set USA, 80:21 -25.
  • Promoters can be either constitutive or inducible, e.g., under certain environmental conditions. Examples of environmental conditions include chemicals, anaerobic conditions, elevated temperature, or the presence of light.
  • Useful inducible regulatory elements include copper-inducible regulatory elements (Mett el al, Proc. Natl. Acad. Set USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988)); tetracycline and chlor-tetracycline-inducible regulatory elements (Gate et al., Plant J. 2:397-404 (1992); Roder et al, Mol Gen. Genet. 243:32-38 (1994); Gate, Meik Cell Biol. 50:41 1 -424 (1995)); ecdysone inducible regulatory elements (Christopherson et al, Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992);
  • An inducible regulatory element also can be, for example, a nitrate-inducibie promoter, e.g., derived from the spinach nitrite reductase gene (Back et al. Plant Mol Biol. 17:9 (1991)), or a light- inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol. Gen. Genet. 226:449 (1991 ); Lam and Chua, Science 248:471 (1990)), or a light.
  • a nitrate-inducibie promoter e.g., derived from the spinach nitrite reductase gene (Back et al. Plant Mol Biol. 17:9 (1991)
  • a light- inducible promoter such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol. Gen. Genet. 226:4
  • a promoter sequence that is responsive to light can be used, e.g., to drive expression in Chlamydomonas exposed to light (e.g., Hahn, Curr Genet 34:459-66, 1999; Loppes, Plant Mol Biol 45:215-27, 2001 ; Villand, Biochem J 321:51-1), 1997.
  • Other liglii-indueible promoter systems may also be used, such as the phytoclirome/PlF3 system (Shimizu-Sato, Nat Bioiechnol 20): 1041 -4, 2002).
  • a promoter can be used that is also responsive to heat can be employed to drive expression in algae such as Chlamydomonas (Muller, Gene 1 11 : 165-73, 1992; von Gromoff, Mol Cell Biol 9:391 1-8, 1989).
  • Additional promoters e.g., for expression in algae such as green microalgae, include the RbcS2. and PsaD promoters ⁇ see, e.g., Stevens et al., Mol. Gen. Genet. 251 : 23-30, 1996; Fischer & Rochaix, Mol Genet Genomics 265:888-94, 2001 ).
  • the invention is not limited with respect to the precise promoter or expression vector used.
  • any suitable expression vector may be used to incorporate the desired sequences
  • readily available expression vectors include, without limitation: plasmids, such as pET, pGex, pJF119EH, pSClOl, pBR322, pBBRlMCS-3, pUR, EX, pMRlOO, pCR4, pBAD24, pUC19; and bacteriophages, such as Ml 3 phage and ⁇ phage.
  • Certain expression vectors may only be suitable for particular host cells which can be readily determined by one of ordinary skill in the art.
  • the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector.
  • the expression vectors of the invention can contain flanking regions that are substantially identical in sequence to a sequence in the host's genome.
  • the flanking regions of the recombinant expression vector direct efficient homologous double recombination between the vector and regions of identical sequence in the host's genome (see, e.g., Thomason et al., Curr. Protoc. Mol Biol., Chapter 1 : Unit 1.16, 2007; Vermaas, Wim, J. Appl. PhycoL, (1996), 8:263; Vermaas, Wim. "Targeted Genetic Modification of
  • 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 expression vectors of the invention must be introduced or transferred into the host cell. Such methods for transferring the expression vectors into host cells are well known to those of ordinary skill in the art. For example, E, coli can be transformed with an expression vector using calcium chloride precipitate. Other salts, e.g., calcium phosphate, can also be used following a similar procedure.
  • electroporation i.e., the application of current to increase the permeability of cells to nucleic acid sequences
  • electroporation can be used to transfect the host microorganism.
  • a lso, microinjection of the nucleic acid sequencers provides the ability to transfect host microorganisms.
  • Other means such as lipid complexes, liposomes, and dendrimers, may also be employed.
  • lipid complexes, liposomes, and dendrimers may also be employed.
  • Those of ordinary skill in the art can transfect a host ceil with a desired sequence using these or other methods. [01 ⁇ 2]
  • a variety of methods are available for identifying a transfected/ transformed cell.
  • a culture of potentially transfected cells may be separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of the desired nucleic acid sequence.
  • cells can be selected based on antimicrobial resistance that has been conferred by genes intentionally contained within the expression vector, such as a kan, cam, chl, spcm, amp, gpt, neo, or hyg gene.
  • the cell can be transformed with one or more expression vector. If more than one expression vector is introduced, each vector can include a different selective criteria.
  • the cell is cultured and typically allowed to grow. For microbial hosts, 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. Tn this way, cellular production of acetyl-CoA, a starting material for IPP and DMAPP synthesis is ensured. When added, the intermediate is present in an excess amount in the culture medium.
  • An IPP isomerase, or species homolog or functional variant thereof, that is capable of catalyzing the conversion of IPP to DMAPP can be introduced to a cell for improved isoprene production.
  • the coding sequence for IPP isomerase can be included on the same or different expression cassette, and expressed on the same or different transcript, as the members of the MVA (or MEP) pathway.
  • the homologous enzyme retains amino acids residues that are recognized as conserved for the enzyme and that are necessary for IPP isomerase activity.
  • the homologous enzyme may have non-conserved amino acid residues replaced or found to be of a different amino acid, or amino acid(s) inserted or deleted, but which does not affect or has insignificant effect on the enzymatic activity of the homologous enzyme.
  • the homologous enzyme may be found in nature or be an engineered mutant thereof. The structures of various IPP isomerases have been determined.
  • the enzymes are well characterized with respect to the catalytic site and residues important for activity (see, e.g., Zhen et al, J. Mol. Biol. 366: 1447- 1458, 2007; Zhang el at, J. Mol. Biol 366: 1437- 1446, 2.007: Street et al, Biochemistry 33 (14): 4212-4217, 1994; Wouters, et al, J. Biol. Chem. 278 (14): 1 1903-11908, 2003; Bonanno, et al., Proc. Natl. Acad Sci USA 98: 12896- 12901 , 2001).
  • IPP isomerases are also referred to as isopentenyl-diphosphate delta-isomerases, isopentenylpyrophosphate delta-isomerases, isopentenylpyrophosphate isomerases, and methylbutenylpyrophosphate isomerases. Any enzyme with IPP isomerase activity can be used as described herein. An enzyme with IPP isomerase activity can be either Type 1 or Type II.
  • Type I are commonly found in Eukaryota and Eubacteria, such as (but not limited to) Escherichia coli, Saccharomyces cerevisiae, Homo sapiens, Salmonella enierica, Arabidopsis thaliana, Bacillus subtilis, Rhodobacter capsulatus, Citrobacter rodentium, Klebsiella pneumoniae, Enter obacter asburiae, Pichia pastoris.
  • Type 1 IPP isomerases utilize a divalent metal (typically Mn 2, , Mg i1 , or Ca 2 h ). in a protonation-deprotonation reaction.
  • Type II IPP isomerases are commonly found in Archaea and some bacteria, such as (but not limited to) Synechocystis sp., Methanothermobacier thermautotrophicus, Sulfolobus shibatae, Sireptomyces sp., Staphylococcus aureus.
  • Type 11 enzymes employ reduced flavin and metal cofactors (e.g., Mtf", Mg 2 ', or Ca 2 ).
  • Type I IPP isomerases examples include, but are not limited to, the sequences identified by the following accession numbers: Escherichia coli ( P_417365), Saccharomyces cerevisiae (NP 015208), Homo sapiens (NP 004499), Mus musculus (NP 663335), Salmonella enierica (NP_806649), Arabidopsis thaliana ( 97148), Bacillus subtilis (NP_390168), Caenorhabditis elegans (NP_498766), Sireptomyces coelicolor (NP_630823).
  • the Type I isomerase is from bacteria or a fungus, such as a yeast.
  • Type ⁇ ⁇ isomerases that can be used in the invention, include, but are not limited to, the sequences identified by the following accession numbers:
  • Synechocystis sp. (NP_441701 ), Methanothermobacter thermautotrophicus (NP_2751 1), Sulfolobus solfataricus (NP_341634), and Staphylococcus aureus (NP_375459).
  • 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 carbon source is C €>2, and such cells are conveniently cultured in a photobioreactor, i.e., a bioreactor that provides for a light source to the reactor.
  • a photobioreactor can be described as an enclosed, illuminated culture vessel designed for controlled biornass production of phototrophic liquid ceil suspension cultures. Isoprene and other volatile hydrocarbons can be conveniently harvested from photobioreactors.
  • Photobioreactors can have different configurations.
  • a tubular horizontal photobioreactor is offered as an illustrative example, tubular vertical
  • photobioreactors and photobioreactors having a variety of geometries, inclinations and shapes, including but not limited to tubular square, tubular oval or ellipsoid, tubular rectangular, flat panel, bubble column, air-lift, stirred tank, lake model, and immobilized substrate photobioreactors, can be used.
  • Tubular photobioreactors are examples of a design suitable for microalgae or cyanobacteria cultivation, designed to support cell growth and isoprene production.
  • the tube structure can be made from a variety of materials ranging from polyethylene to plexiglass and glass. Tube diameter and length can vary, e.g., depending on the size of the bioreactor.
  • the diameter may vary from about 5-30 cm and the length can vary from a few to tens or hundreds of meters in length. Those skilled in the art understand that far greater capacity reactor volumes can be attained with 5-30 cm diameter reactors upon increasing the overall length of the tubes.
  • the operator pumps aqueous media, e.g., water suitably fertilized and properly inoculated with the microalgae, to fill the space assigned to the aqueous portion of the reactor.
  • aqueous media e.g., water suitably fertilized and properly inoculated with the microalgae
  • the remainder of the reactor space is filled with C0 2 , for example, 100% C0 2 , or a C0 2 mix where the mix is at feast 10% C0 2 , at feast 20% C0 2 , at least 30% C0 2 , at least 40% CO ? , preferably greater than 50% CO?, e.g., at least 60% CO ? , at least 70% CO ? , at least 80% CO ? , or at least 90% or at least 95%, or greater, CO ?
  • gaseous/aqueous partition ratio can vary considerably, e.g., can be in the range of about 1 :9 to about 9: 1. In some embodiments, the ratio is in the range of from about 4:6 to about 6:4 or about 50:50.
  • Exemplary bioreactor assemblies include a port/valve for the introduction of CO ? and a separate port/ valve through which the O?/ isoprene-containing gas is removed.
  • CO? replacement and isoprene removal from the gaseous headspace of the photo-bioreactor takes place periodically, for example, every 3 hours, every 6 hours, every 1 8 hours, every 24 hours, or every 48 hours, or at longer periods of time, depending on the photosynthetic productivity of the cells. For example, when sunlight is the light source, on sunny summer days CO ? replacement and isoprene removal from the gaseous headspace of the photo-bioreactor is typically performed every 24 hours. On overcast and low-sunlight-intensity days, CO ? replacement and isoprene remo val occurs at longer periods of time.
  • CO ? -rich gases are typically slowly bubbled at the bottom of the liquid phase.
  • CO ? -rich gases are applied either by bubbling the liquid phase, or applied as a stream of gas over the surface of the liquid phase. In either case, care is taken to ensure that fresh CO ? gas is applied from a valve located at one end of the "control box" in the photobioreactor assembly, whereas gaseous products of photosynthesis and isoprene remo val takes place at the other end of the control box from a separate valve. This approach ensures that a stream of CO?
  • the gas flows from one end of the control box through the entire length of the bioreactor tube before arriving at and exiting from the other end of the control box.
  • the turbulence generated by the flowing stream of CO ? in the gaseous phase of the reactor pushes out and removes the products of photosynthesis (isoprene and O ? ).
  • the gaseous products of photosynthesis can be removed in a similar manner by using a stream of air to purge the products of photosynthesis, which then is followed by filling the gaseous head phase of the reactor with CO ? for further photosynthesis and production.
  • photobioreactors include cylindrical or tubular bioreactors, see, e.g. , U.S. Pat. Nos. 5,958,761 and 6,083,740; U.S. Appln. Pub. No. US2007/0048859; and International Appln. Pub. Nos. WO 2007/01 1343 and WO2007/098150.
  • High density photobioreactors are described in, for example, Lee, et al. (1994) Biotech. Bioeng. 44: 1 161.
  • Another example of a photobioreactor includes a sealed fed-batch bioreactor that operates spontaneously using a diffusion- based method for CC 'isoprene exchange in a
  • gaseous/aqueous two-phase photobioreactor see, e.g., Bentley, FK and Melis, A (2012) Biotech Bioeng 109: 100- 109.
  • Other suitable bioreactors are described, e.g., in
  • Photobioreactor parameters that can be optimized, automated and regulated for production of photosynihetic organisms are further described in (Puiz (2001) Appl Microbiol Biolechnol 57:287-293).
  • Examples of light sources that are used to provide the energy required to sustain photosynthesis include, for example, fluorescent bulbs, incandescent light, LEDs, and natural sunlight.
  • isoprene can be harvested by passing the gaseous phase that contains the volatile hydrocarbon and O? through a cooled condenser so that it can be collected, in some embodiments, the condensed isoprene is passed through a solvent, typically an organic solvent.
  • a solvent typically an organic solvent.
  • Alcohols such as methanol (boiling point 64.7 °C), ethanol (boiling point 78.0 °C), butanol (boiling point 1 17.7 °C), and pure hydrocarbons such as hexane (boiling point 69.0 °C), heptane (boiling point 98.4 °C), octane (boiling point 125.5 °C), and dodecane (boiling point 216.2 °C) readily form stable "blends" with isoprene, or other volatile hydrocarbon obtained in accordance with the methods of the invention, thereby facilitating its retention and stabilization in a liquid solution.
  • Bioreactors can be set up to be continually harvested (as is with the majority of the larger volume cuitivation systems), or harvested one batch at a time (for example, as with polyethylene bag cultivation).
  • a batch bioreactor is set up with, for example, nutrients in aqueous solution, and the organism is cultured until the batch is harvested.
  • a continuous bioreactor can be harvested, for example, either continually, daily, or at fixed time intervals.
  • cultures are fed periodically with an organic carbon source or CO?., e.g., ever 2.4 to 48 hours.
  • cultures can be periodically diluted with fresh growth medium, e.g., every 24 to 48 hours, or when cultures reach a certain density (e.g., about 0.7 g dw L "1 or greater).
  • Genomic DNA of Streptococcus pneumoniae ( AT ' CC No. 6314) and Enterococcus faecalis (strain V583; ATCC No. 700802) were used as template for PGR amplification.
  • the properties of plasmids and E. coli strains used are listed in Table 1 .
  • Plasmid DNA was prepared with a plasmid purification kit (Qiagen, USA). Restriction enzymes were purchased from Ne England Biolabs (USA) and used according to the recommendations of the v endor. Oligonucleotides were purchased from Bioneer (USA). Nucleotide sequences and related primer details are given in Table 2.
  • Bo!d indicates restriction enzyme site. Underline indicates RBS. Italics indicates spacer.
  • Protein expression E. coii Rosetta ceils transformed with plasmtd ⁇ 28-3 ⁇ 4&*/3 ⁇ 4!? and pGex6P 1 -klspS vtcre used to overproduce the klspS protein with a thrombin cleavable N- terminal hex alii stidine tag or a PreSciss on protease cleavable glutathione-S-transferase (GST) tag, respectively.
  • the flasks were cooled to 15°C, protein expression was induced with of 0.1 mM isopropyl ⁇ -D-thiogalacto-pyranoside (IPTG), and growth was allowed to continue overnight at 15 C C. Cells were harvested by centrifugatioii at 4,500 g for K) min at 4°C.
  • IPTG isopropyl ⁇ -D-thiogalacto-pyranoside
  • Hg-klspS piirification Cells were pelleted and resuspended in 10 ml of buffer I J (50 mM Tris/HCl, pH 8.0, 400 mM aCi, 0.3% Triton X- 100, 10 mM ⁇ -mercaptoethanol, 5 mM imidazole), and lysed in a French pressure cell (1 ,000 psi). To remove cell debris, the ceil lysate was eentrifuged at 13,000 g for 10 min at 4°C.
  • buffer I J 50 mM Tris/HCl, pH 8.0, 400 mM aCi, 0.3% Triton X- 100, 10 mM ⁇ -mercaptoethanol, 5 mM imidazole
  • He-kTspS was eluted with buffer I 200 (50 mM Tris/HCl, pH 8.0, 400 mM NaCl, 10 mM ⁇ -mercaptoethanoi, 200 mM imidazole). Fractions were analyzed by SDS-PAGE and fractions containing 3 ⁇ 4-kIspS were pooled. The purified protein was concentrated with Amicon Ultra 15 50 kD cut-off devices (Millipore, USA) to a final volume of 1 ml (2.-3 mg/ml), and the concentrate eentrifuged at 15,000 g for 5 min.
  • buffer I 200 50 mM Tris/HCl, pH 8.0, 400 mM NaCl, 10 mM ⁇ -mercaptoethanoi, 200 mM imidazole.
  • GST-klspS purification Cells were resuspended in 10 ml of buffer A ( 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , pH 7.4, 140 mM NaCl, 2.7 mM KCi, 10 mM MgCl 2 , 5% glycerol, 1 mM DTT) and lysed in a French pressure cell (1 ,000 psi). To remove cell debris, the cell lysate was eentrifuged at 13,000 g for 10 min at 4°C.
  • buffer A 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , pH 7.4, 140 mM NaCl, 2.7 mM KCi, 10 mM MgCl 2 , 5% glycerol, 1 mM DTT
  • the glutathione agarose resin (Thermo Fisher Scientific, U SA) was equilibrated with buffer A and the fusion protein was incubated with the resin for 1 h at 4°C.
  • the sluny was washed extensively with buffer B (50 mM Bicine, pH 8.0, 30 mM NaCl, 50 mM MgC12, 5 mM KCI, 5% glycerol and i mM DTT).
  • the fusion protein was cleaved on the column upon addition of 10 units PreScisston Protease (GE Healthcare, USA), and upon incubation of the reaction mixture for 16 h ai 4°C.
  • GPLGS five amino acids from the fusion construct remained on the N-terminal side of the klspS, slightly extending the N-terminal length of the recombinant protein.
  • the klspS protein was collected from the flow-through of the column and concentrated, as described above. A ny uncleaved OST-IspS protein that remained on the column was subsequently eluted with buffer B 3 ° (buffer B containing 50 mM glutathione).
  • inhibitors if any, and DMAPP were added simultaneously to the reaction mixtures. Reaction mixtures were incubated for 15 min at 42°C.
  • isoprene quantification 1.2 ml of the headspace gas of sealed flasks was analyzed by gas chromatography using a Shimadzu 8A GC (Shimadzu, USA) equipped with a flame ionization detector and a Porapak N 80/100 column, designed to detect short-chain hydrocarbons. The amounts of isoprene produced were estimated by comparison of the GC peak heights with those obtained with an isoprene standard (Acros Organics, USA).
  • E. coli strains were cultivated either in 3-(N- Morpholino)-propane-sulfonic acid (MOPS) minimal medium or in M9 minimal medium containing 0.4% glycerol and supplemented with appropriate antibiotics.
  • Pre-culrures were mitiated from a single colony and used to inoculate 100 ml of the main culture media in 500 ml Erlemneyer flasks at an ODeoo - 0.02.
  • the cultures were thereafter incubated at 37°C and the headspace contents were monitored by gas chromatography.
  • the yield of isoprene production was calculated by dividing the molar quantity of isoprene in the headspace with the molar quantity of glycerol added to the medium. On this basis, yields of isoprene would be a conservative estimate and could be greater: (i) if cells did not consume the entire amount of glycerol in the medium; and (ii) because isoprene remained partially dissolved in the liquid medium of the sealed culture. To estimate the latter, Henry's law was applied to sho w that about 16% of the isoprene produced would remain in the liquid medium and, therefore, it was not detected by the GC analysis.
  • the carrier gas (helium) flow rate was set to 1.2 ml/min.
  • the product identity was confirmed by comparing its retention time and mass spectrum to those for an isoprene standard sample (Sigma- Aldrich, USA). Size selection conditions were chosen to detect 48-100 D products and to eliminate smaller ⁇ e.g. ethanol) and potentially larger than 100 D molecules from the analysis.
  • Example 1 in vitro activity of IspS [0132] To probe the enzymatic activity of kudzu isoprene synthase (klspS), the mature form of klspS (amino acid residues 45-608), missing the putative transit pept ide, w as cloned into pGex6 I and expressed in Rosetta cells. The GST-klspS fusion protein was purified using a glutathione agarose resin, and the cleaved klspS fraction was eluted from the column. The klspS protein fraction collected was pure and uniform as judged by SDS-PAGE analysis, migrating to the expected molecular mass of 65 kD.
  • klspS kudzu isoprene synthase
  • the k cs.t /K m ratio was determined to be 1 ,760: 1.
  • the V max in this study was 52-fold greater than that measured by Sharkey et al. (2005), who reported a V ma x :;;: 0.079 umol (mg protein) "1 mm ⁇
  • the isoprene synthase activity was measured with an N-tenninal hexahistidine 3 ⁇ 4-kIspS fusion protein that was purified using a Ni-NTA agarose column.
  • N-terniinal hexahistidine tag MRGSHHHHHHGS
  • klspS lost a substantial portion of its enzymatic activity, caused by the strong inhibitory effect exerted on the enzyme by divalent Ni 2 cations. Accordingly, a more systematic analysis of the inhibitory effect of divalent cations on the catalytic activity of the isoprene synthase was undertaken (Fig. 2B).
  • Example 2 In vivo activity of IspS in E. coli [0136]
  • klspS amino acids 45-608 activity in E. coli in vivo by GC analysis of the gases in the culture headspace.
  • Both the not- induced and induced E. coli cultures accumulated ethanol in the reactor headspace, as evidenced by the pronounced peak with a retention time of 2.9 mm (Fig. 3A and 3B).
  • the klspS transformant strain produced isoprene, but only after the culture was induced by IPTG, as evidenced by the GC peak with a retention time of 3.5 min (Fig. 3B).
  • the IspS was found to be N-terminally blocked and could not be Edman sequenced. The mature protein start amino acid could therefore not be precisely determined. Based on the conserved amino acid sequences (Fig, 10) and predicted tertiary configuration of the protein (Koksal et al. (2010) J. Mol. Biol. 17:402), we sought to determine whether a truncated N-ferminus of the IspS protein would eliminate regulatory aspects of the catalytic turnover and possibly increase rates of isoprene synthesis.
  • This arginine motif has been suggested to play a role in diphosphate walking and cyclization and is present in all terpene synthases.
  • the klspS construct containing amino acid residues 60-608 showed 70% activity compared to the presumed wild-type mature kTspS protein (amino acids 45-608).
  • the amino acid 46-608 construct showed the same enzymatic activity as that of the control (Fig. 5).
  • Example 3 Isoprene production in E. coli [0140] E. coli transformant cultures were induced at different temperatures (37°C for 6 h, 20°C for 24 h and 4°C for 72. h) and with different IPTG concentrations (1 mM, 0.1 mM, and 0.01 mM). Best isoprene production activity results and yield of isoprene were obtained at 37°C for 6 h incubation and upon induction with 0.1 mM IPTG.
  • TIRl was composed of the ribosomal binding site (RBS) AGGAGG and spacer TAATAT, while TTR2 had RBS AAGGAG and spacer ATATACC. These were selected because RBS of TIRl showed perfect complementation to the 3' end-region of the E. coli 16S rRNA.
  • TIR2 is the sequence used by the pET vector series. The coding sequences for members of the MEP pathway (MEP enzymes) were cloned as two separate halves by sequentially adding genes to the artificial operon.
  • the two halves were ligated to form a single artificial superoperon.
  • This strategy permitted generation of the entire MEP biosynthetic pathway, but also allowed construction of plasmids carrying only selected genes (portions) of the MEP hiosynthetic pathway (Fig. 7A and 7B). The latter were designed to help enhance carbon flux through specific steps of the MEP pathway, thereby identifying and alleviating rate- limiting bottlenecks, [0144] E. coli were simultaneously co-transformed with various MEP pathway constructs and with a plasmid containing the kispS coding sequence, driven by a tac promoter (P tac ).
  • Table 3 Yield of isoprene production by E. coli Rosetta cells transformed with kispS
  • the MVA pathway can be divided in two parts: the upper half generates mevalonate from acetyl-CoA, and the lower half generates IPP and DMAPP from mevalonate.
  • the upper and lower portions of MVA pathways from Streptococcus pneumoniae, Enierococcus j ' aecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevi.si.ae have been compared for ⁇ -carotene production in E. coli (Yoon et at (2009) J. Biotechnol.
  • the translation initiation rate for MVKl is 6361.7, ⁇ 1 for MVD1, 2526.4 for MVK2, and 34.0 for FN1.
  • MVKl has a high value since it is the first gene in the operon and was cloned behind an E. coli TIR, whereas all other genes would be translated at a slower rate.
  • Introducing artificial T1R1 for all genes resulted in a translation initiation rate of 421 18.2 for MVKl , 38492.8 for MVDI, 22430.4 for MVK2, and 3238.9 for FNL
  • the TGT start codon of FNI in Streptococcus pneumoniae was changed to ATG.
  • the upper MVA pathway was independently expressed from TIR1 and TIR2.
  • the lower MVA pathway (mevaionate kinase (MVKl :K1), a phospho-mevalonate kinase (PMK:K2), a diphosphate-mevaloiiate decarboxylase (MVD:D) and an IPP isomerase (FNLI)) was obtained from Streptococcus pneumoniae. In the native S. pneumoniae genome, these genes overlap. To assess the effect of the overlap on E. coli expression, we first cloned the lower MVA pathway using the native TIRs (presence of overlaps) and ligated it downstream of the upper MVA pathway to produce (HsHrA)l( ' KIDK2I)n and
  • the IspiEtOH peak ratio increased from a low of 1:8 in the klspS plus pET co-transformant to a high of about 100: 1 in the klspS plus (HsHr AIK1 DK2) 1 co-transformant strain.
  • the isoprene/QDeoo ratios achieved were substantially different under the two IPTG induction conditions (Fig. 9).
  • QDcoo 0.25
  • MVA transformants produced 56 mg Isp/L, and attained a final isoprene to biomass carbon partitioning ratio of 0.25 g Isp/g DCW.
  • TM MVA transformants produced 320 mg Isp L " 1 culture and attained a final 0.44 g Isp/g DCW ratio.
  • the cells In the exponential growth phase (up to 6 h after induction), the cells produced 0.230 g lsp/g DCW, whereas in the stationary phase (from 6 h to 22 h after induction), the cells exclusively produced isoprene, resulting in a higher Isp/DCW ratio.
  • the carbon conversion efficiency from glycerol to isoprene was estimated to be 18% and the isoprene-to-biomass carbon partitioning ratio was 0.78. The results indicate that induction of the M VA pathway at higher cell density results in higher isoprene production.
  • Streptococcus pneumoniae ATCC no. 6314
  • Enterococcus faecalis strain V583; ATCC no. 700802
  • Escherichia coli were used as templates for PGR amplification of the MVA pathway genes.
  • Primers used to amplify the operons include SEQ ID NOs: 40-64 listed in Table 2.
  • the upper MVA pathway genes, HmgS and HnigR were cloned from E. faecalis, and AtoB from E. coli, while the lower MVA pathway genes, Fni, MK, PMD and PMK, were cloned from S. pneumoniae.
  • TIR1 composed of the ribosomal binding site AGGAGG and spacer TAATAT
  • T1R2 composed of ribosomal binding site AAGGAG and spacer ATATACC
  • Nucleotide sequences (SEQ ID NOs: 65-76) designed to amplify these regions and listed in Table 4 were cloned into the pBluescript SK+ plasmid (Stratagene, USA). These regions are referred to as ihe flank region and equate to approximately 500 bp of sequence flanking either side of the trangene construct (Fig, 12), and used for homologous recombination in Synechocysiis.
  • the IspS and Cm 1 selectable marker was inserted midway within the Neu site sequence, insertionaily disrupting the slr0168 ORF, to create plasmid pIspS-Cm'-ANeu, The upper MVA operon (SRA) and the MVA super-operon
  • SRAFKiDK 2 along with a Km 1 selectable marker, were inserted between the up- and down-stream flanking regions of PsbA2 to create plasmids pSRA-Km r -APsbA2 and pSRAFK] DK 2 -Km r -APsb A2, respectively.
  • the lower MVA operon (FKjDK 2 ) and the Sm r selectable marker were inserted between the up- and down-stream flanking regions of either GlgX or GlgA to create plasmids pFK 1 DK2-Sm r -A01gX and pFKjDK 2 -Sm r -AGlgA, respectively.
  • Promoter (P) and terminator (T) sequences consisting of approximately 200 bp upstream (promoter) and 200 bp downstream (ierminator) of the native Synechocysiis PsbA2 ORF, were cloned immediately before and after the po!yeistromc transgene constructs (Fig. 12), except for constructs with PsbA2 flanking regions that already contained these sequences.
  • Table 4 Oligonucleotide primers used to amplify regions of the Synechocysiis genome for sites of integration for ispS and the MVA pathway operons via double homologous recombination (SEQ ID I Os: 65-76)
  • Liquid cultures were grown i BG-11 containing 25 m ' M sodium phosphate buffer, pH 7.5. Liquid cultures for inoculum purposes and for SDS-PAGE analyses were maintained at 25°C under a slow stream of constant aeration and illumination at 20 ⁇ photons m ' s Growth conditions employed when measuring the production of isoprene from Synechocystis cultures are described in the following section.
  • Isoprene production and biomass accumulation assays Synechocystis cultures for isoprene production assays were grown photoautotrophically in 1 L gaseous/aqueous two- phase phot obi oreactors, which are described in detail in Bentley and Melis (2012). Photo- bioioreactors were seeded with a 700 ml culture of Synechocystis cells at an OD'/so nm of between 0.2 - 0.3 in BG 1 1 medium containing 25 mM sodium phosphate buffer, pH 7.5.
  • Inorganic carbon was delivered to the culture in the form of 500 mL aliquot s of 100% CC gas, which was slowly bubbled though the bottom of the liquid culture to fill the bioreactor headspace. Once atmospheric gases were replaced with 100% CO?, the headspace of the bioreactor was sealed and the culture was incubated under continuous illumination of 150 ⁇ photons m " 2 s "1 at 35°C, Slow continuous mechanical mixing was employed to keep cells in suspension and to promote balanced cell illumination and gaseous C0 2 diffusion into the liquid culture to support biomass accumulation. Uptake and assimilation of headspace C0 2 by cells was concomitantly exchanged for 0 2 during photoautotrophic growth.
  • Quantitation of isoprene production was performed on the basis of an isoprene vapour calibration curve constructed by the GC analysis of a series dilution of a vaporized pure isoprene standard (Acros Organics, Fair Lawn, NJ, USA). Isoprene in the gaseous headspace was further identified by gas chromatography-mass spectrometr (GC- MS) analysis through the comparison of retention time and mass spectrum with the vaporized pure isoprene standard. GC-MS analyses were performed with an Agilent 6890GC/5973 MSD equipped with a DB-XLB column (0.25 mm i.d. x 0.25 ⁇ x 30 m, J &W Scientific).
  • Oven temperature was initially maintained at 4()°C for 4 min, followed by a temperature increase of 5°C/min to 80°C, and a carrier gas (helium) flow rate of 1.2 ml per minute.
  • Cyanobacterial biomass accumulation was measured gravimetrically as dry cell weight, where 5 mL samples of culture were filtered through 0.22 iim Millipore filters and the immobilized cells dried at 90°C for 6 h prior to weighing the dry ceil weight.
  • Synechoeystis sp. conferred upon these microorganisms the property of isoprene (CsHg) hydrocarbons production.
  • yields were low because of limited endogenous substrate flux through the highly regulated native 2 ⁇ C-meihyl-D-er ⁇ r thritol-4-phosphate (MEP) pathway in the cell.
  • the genes of the M VA pathway were derived from Enlerococcus faecalis, Escherichia coii and Streptococcus pneumoniae and heterologolously expressed in Synechoeystis sp. PCC 6803.
  • the MVA pathway required 3 molecules of Acetyl-CoA as immediate substrates, whereas the MEP pathway involves the condensation of pyruvate and giyceraidehyde-3-phosphate (GAP). Both pathways yield isopentenyl diphosphate (IPP) and dirnethylallyl diphosphate (DMAPP) as end products.
  • IPP isopentenyl diphosphate
  • DMAPP dirnethylallyl diphosphate
  • Constructs were designed for the expression of the isoprene synthase and the MVA hiosynthetic pathway in cyanobacteria (Fig. 12). Ail operons were placed under the transcriptional control of the native Synechocystis PsbA2 promoter (P) and terminator (T) sequences. Primers (SEQ ID NOs: 65-76) used to PGR amplify regions of the Synechocystis genome serving as sites of integration for the operons via homologous recombination are listed in Table 4.
  • TIRs Translation initiation regions
  • IspS isoprene synthase gene
  • the upper MVA pathway operon (SRA) included HrngS and HmgR from E. faecalis and AtoB from E. coli, and were cloned inf o the PsbA2 site of the Synechocystis genome using the PsbA2 flanking sequences for homologous recombination, and replacing the native PsbA2 gene, A kanamycin-resistance selectable marker (Km 1 ) was added for selection of transformant lines.
  • SRA The upper MVA pathway operon
  • the lower MVA pathway operon (FK)DK 2 ) included Fni, MK, PMD and PMK from 5 pneumoniae, and was cloned into the GlgX or GlgA sites of the Synechocystis genome using the GlgX and GlgA flanking sequences, respectively, for homologous recombination.
  • a spectinomycin-resistance selectable marker (Sm 1 ) was added for selection of transformant lines.
  • the complete MVA pathway superoperon was derived by combining the two halves of the pathway in a single construct, which had PsbA2 flanking regions to aid homologous recombination at the Synechocystis PsbA2 site, and a kanamycin-resistance selectable marker (KmR) was added for selection of transforman t lines.
  • KmR kanamycin-resistance selectable marker
  • results are shown for the following transformant strains: ( 1) SRAFKiOK 2 -ApsbA2; (2) IspS-ANeu; (3) SRA.-Ap$bA2 F l OK 2 -AGlgA lspS-ANeu; and (4) SRA-ApsbA2::FKiDK 2 -AGIgX JspS-ANeu.
  • This comparative analysis revealed dramatic differences in the isoprene peaks in the culture headspaees of the different transformants.
  • Isoprene accumulation in the headspace of the gaseous aqueous two-phase bioreactor was measured by GC analysis after 196 hours of photoautotrophic growth.
  • photoautotrophic growth and isoprene is presented as a cumulative yield over 196 hr.
  • strains producing the greater yield of isoprene were the SRAFKiDK 2 -ApsbA2::lspS-ANeu (3), SRA-ApsbA2::FKiDK 2 -AG!gA::lspS-ANeu (4), and SRA-ApsbA2: :FK OK 2 -AGlgX: :hpS-ANeu (5) transformants.
  • the strains e.g., lspS-ApsbA2; IspS-ANeu; SRAFKiDK?- ApsbA2vlspS-ANeu; SRA-ApsbA2 :FKiOK 2 -AGlgA :lspS-ANeu; and SRA-
  • the super-operon transformant strains (e.g., SRAFKj OK 2 -ApsbA2: • IspS-ANeu, S A-ApsbA2::FKiOK 2 -AGlgA::JspS-ANeu, mid SBA-ApsbA2::FKiDK 2 -AGlgX::lspS-ANeu) had the highest isoprene-to-carbon compared to the lspS-ApsbA2 control.
  • This example illustrates that heterologous expression of the isoprene synthase gene in combination with the mevalonic acid (MVA) pathway in cyanobacteria, e.g. Synechocystis sp., pro vides additional substrate flux of IPP and DMAPP precursors io isoprene, resulting in photosynthetic isoprene yield improvement by 10-fold or greater, as compared to that measured in cyanobacteria transformed with the isoprene synthase gene only.
  • MVA mevalonic acid
  • MVA mevalonic acid
  • MEP methylerythritoi
  • TIR1 1 (Restriction site-RBS 1 -Spacer):
  • TIR 12 (Restriction site-RBS 1 -Spacer):
  • TIR17 (Restriction site-RBS 1 -Spacer):
  • HMGS Hydroxy-methyl-glutaryl synthase
  • HMGR Hydroxy-methyl-glutaryl reductase
  • MVK2 Phospho-raevalonic acid kinase
  • FNI IPP isomerase

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des constructions d'expression recombinants pour la production microbienne améliorée d'isoprène et des procédés de production et d'utilisation de ces constructions pour la production d'isoprène.
PCT/US2012/071416 2011-12-23 2012-12-21 Constructions et procédés pour la biosynthèse améliorée d'isoprène Ceased WO2013096863A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161579909P 2011-12-23 2011-12-23
US61/579,909 2011-12-23

Publications (1)

Publication Number Publication Date
WO2013096863A1 true WO2013096863A1 (fr) 2013-06-27

Family

ID=48669570

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/071416 Ceased WO2013096863A1 (fr) 2011-12-23 2012-12-21 Constructions et procédés pour la biosynthèse améliorée d'isoprène

Country Status (1)

Country Link
WO (1) WO2013096863A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170145441A1 (en) * 2015-08-17 2017-05-25 INVISTA North America S.à.r.l. Methods, hosts, and reagents related thereto for production of unsaturated pentahydrocarbons, derivatives and intermediates thereof
US10662415B2 (en) 2017-12-07 2020-05-26 Zymergen Inc. Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation
US10696991B2 (en) 2017-12-21 2020-06-30 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone
US11162115B2 (en) 2017-06-30 2021-11-02 Inv Nylon Chemicals Americas, Llc Methods, synthetic hosts and reagents for the biosynthesis of hydrocarbons
US11505809B2 (en) 2017-09-28 2022-11-22 Inv Nylon Chemicals Americas Llc Organisms and biosynthetic processes for hydrocarbon synthesis
US11634733B2 (en) 2017-06-30 2023-04-25 Inv Nylon Chemicals Americas, Llc Methods, materials, synthetic hosts and reagents for the biosynthesis of hydrocarbons and derivatives thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090203102A1 (en) * 2007-12-13 2009-08-13 Cervin Marguerite A Compositions and methods for producing isoprene
WO2010031077A1 (fr) * 2008-09-15 2010-03-18 Danisco Us Inc. Production d’isoprène augmentée en utilisant la mévalonate kinase et l’isoprène synthase
US20100086978A1 (en) * 2008-09-15 2010-04-08 Beck Zachary Q Increased isoprene production using the archaeal lower mevalonate pathway
US20100196977A1 (en) * 2008-12-30 2010-08-05 Chotani Gopal K Methods of producing isoprene and a co-product
US20110014672A1 (en) * 2009-06-17 2011-01-20 Chotani Gopal K Isoprene production using the dxp and mva pathway
US20110159557A1 (en) * 2009-12-23 2011-06-30 Danisco Us Inc. Compositions and methods of pgl for the increased production of isoprene

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090203102A1 (en) * 2007-12-13 2009-08-13 Cervin Marguerite A Compositions and methods for producing isoprene
WO2010031077A1 (fr) * 2008-09-15 2010-03-18 Danisco Us Inc. Production d’isoprène augmentée en utilisant la mévalonate kinase et l’isoprène synthase
US20100086978A1 (en) * 2008-09-15 2010-04-08 Beck Zachary Q Increased isoprene production using the archaeal lower mevalonate pathway
US20100196977A1 (en) * 2008-12-30 2010-08-05 Chotani Gopal K Methods of producing isoprene and a co-product
US20110014672A1 (en) * 2009-06-17 2011-01-20 Chotani Gopal K Isoprene production using the dxp and mva pathway
US20110159557A1 (en) * 2009-12-23 2011-06-30 Danisco Us Inc. Compositions and methods of pgl for the increased production of isoprene

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170145441A1 (en) * 2015-08-17 2017-05-25 INVISTA North America S.à.r.l. Methods, hosts, and reagents related thereto for production of unsaturated pentahydrocarbons, derivatives and intermediates thereof
US11162115B2 (en) 2017-06-30 2021-11-02 Inv Nylon Chemicals Americas, Llc Methods, synthetic hosts and reagents for the biosynthesis of hydrocarbons
US11634733B2 (en) 2017-06-30 2023-04-25 Inv Nylon Chemicals Americas, Llc Methods, materials, synthetic hosts and reagents for the biosynthesis of hydrocarbons and derivatives thereof
US11505809B2 (en) 2017-09-28 2022-11-22 Inv Nylon Chemicals Americas Llc Organisms and biosynthetic processes for hydrocarbon synthesis
US10662415B2 (en) 2017-12-07 2020-05-26 Zymergen Inc. Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation
US10696991B2 (en) 2017-12-21 2020-06-30 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone
US11193150B2 (en) 2017-12-21 2021-12-07 Zymergen Inc. Nepetalactol oxidoreductases, nepetalactol synthases, and microbes capable of producing nepetalactone

Similar Documents

Publication Publication Date Title
Lindberg et al. Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism
Zurbriggen et al. Isoprene production via the mevalonic acid pathway in Escherichia coli (Bacteria)
Xue et al. Enhancing isoprene production by genetic modification of the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis
CN103243065B (zh) 一种生产法尼烯的菌株及其应用
US9382554B2 (en) Methods for isoprene and pinene production in cyanobacteria
CN103215315B (zh) 利用蓝藻生产异戊二烯
WO2013096863A1 (fr) Constructions et procédés pour la biosynthèse améliorée d'isoprène
US12391950B2 (en) Fusion constructs as protein over-expression vectors
JP2010539902A (ja) イソプレノイドの生産
BRPI0513837B1 (pt) Célula hospedeira saccharomyces cerevisiae geneticamente modificada; composição compreendendo a mesma e método para aumentar a produção de isoprenoides
US20200208178A1 (en) Production of beta-phellandrene using genetically engineered photosynthetic microorganisms
CN104789512B (zh) 异戊二烯的生产菌以及生产异戊二烯的方法
US10889835B2 (en) Production of monoterpene blends by unicellular photosynthetic microorganisms
EP3225691A1 (fr) Procédé de production d'un composé d'isoprénoïde
US20150225743A1 (en) Biocatalyst for conversion of methane and methanol to isoprene
US20180094281A1 (en) Production of fragrant compounds
KR102286815B1 (ko) 알파-휴물렌 생산용 형질전환 메탄자화균 및 이의 용도
WO2020180736A2 (fr) Production de cannabinoïdes à l'aide de microorganismes photosynthétiques génétiquement modifiés
KR102346076B1 (ko) α-bisabolene 생산용 형질전환 메탄자화균 및 이의 용도
CN116179460B (zh) 一种提高大肠杆菌鲁棒性的重组菌及其构建方法
Melis Andreas Zurbriggen, Henning Kirst &
KR20250113943A (ko) 메탄으로부터 이소프렌 생산능을 갖는 형질전환체
CN120700011A (zh) 合成单萜的基因工程菌及其构建方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12860708

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12860708

Country of ref document: EP

Kind code of ref document: A1