CONSTRUCTS AND METHODS FOR IMPROVED ISOPRENE

BIOSYNTHESIS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application no. 61/579,909, filed on Dec. 23, 201 1, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] 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. The IspS enzyme catalyzes the remo v al of pyrophosphate from

dimethyiallyl pyrophosphate (DMAPP) to yield isoprene and inorganic pyrophosphate. Plant isoprene synthases have been cloned and characterized from a number of plant species, and the sequences are publically available. [0003] 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.

[0004] 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

(Chlamydomonas) and bacteria (E. coii) do not possess MVA pathway enzymes, and normally rely exclusively on the highly-regulated, rate-limiting MEP pathway for terpenoids.

[0005] This invention addresses the need for additional methods for producing isoprene using microbial systems. BRIEF SUMMARY OF THE INVENTION

[0006] 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

cyanobacteria and green microalgae, and utilized for isoprene production. 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.

[0007] The present invention relates to isoprene production in E. coii and Synchocyslis resulting from heterologous expression of MVA enzymes in the bacteria and the

cyanobacteria, respectively. In this invention, 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).

[0008] 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). In some instances, 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.

[0009] 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.

[0010] Provided herein are compositions and methods for improved cellular isoprene production. In some embodiments, provided is a recombinant expression cassette for producing isoprene in a cell, the recombinant expression cassette 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

decarboxylase), and MVK2 (Phospho-mevalonic acid kinase), wherein the nucleic acid coding sequence of the at least one member of the MVA pathway is preceded by a

Translation Initiation Region (TIR) comprising a Ribosomal Binding Site (RBS). In some embodiments, the recombinant expression cassette further comprises the nucleic acid coding sequence of Isoprene Synthase (IspS) preceded by a TIR comprising an RBS.

[0011] In some embodiments, 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. In some embodiments, where 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). In some embodiments, 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). In some embodiments, the nucleic acid coding sequences are arranged on the recombinant expression cassette in any order. In some embodiments, 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.

[0012] In some embodiments, 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. For example, in some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, the RBS is selected from any of the RBS sequences shown in SEQ ID NOs: 1 -17.

[0013] In some embodiments, 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. One of skill will recognize that each TIR can be the same or different from the other TIRs on the recombinant expression cassette in any combination. For example, the restriction site for each TIR can be different from the restriction site of every other TIR on the recombinant expression cassette. Similarly, 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. In some embodiments, 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.

[0014] In some embodiments, 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

embodiments, each TIR independently is selected from the group consisting of SEQ ID NOs: 19-25.

[0015] In some embodiments, 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. In some embodiments, the HMGS polypeptide has a sequence of SEQ ID NO:27. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of HMGS is SEQ ID NO:26. In some embodiments, the nucleic acid coding sequence of HMGS is codon-optimized for expression in a particular host cell.

[0016] In some embodiments, 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. In some embodiments, the HMGR polypeptide has a sequence of SEQ ID NO:29. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of HMGR is SEQ ID NO:28. In some embodiments, the nucleic acid coding sequence of HMGR is codon-optimized for expression in a particular host cell.

[0017] In some embodiments, 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. In some embodiments, the ATOB polypeptide has a sequence of SEQ ID NO:31. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of MVKI is SEQ ID NO:32. In some embodiments, the nucleic acid coding sequence of MVKI is codon-optimized for expression in a particular host cell. [0019] in some embodiments, 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. In some embodiments, the MVD polypeptide has a sequence of SEQ ID NO:35. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of MVD is SEQ ID NO:34. In some embodiments, the nucleic acid coding sequence of MVD is codon-optimized for expression in a particular host cell.

[0020] In some embodiments, 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. In some embodiments, the MVK2 polypeptide has a sequence of SEQ ID O:37. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of M VK2 is SEQ ID NO:36. In some embodiments, the nucleic acid coding sequence of M VK2 is codon-optimized for expression in a particular host cell.

[0021] In some embodiments, the nucleic acid coding sequence of MVK2 encodes a polypeptide having at feast 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. In some embodiments, the MVK2 polypeptide has a sequence of SEQ ID NO:37. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of MVK2 is SEQ ID NO:36. In some embodiments, the nucleic acid coding sequence of MVK2 is codon-optimized for expression in a particular host cell.

[0022] In some embodiments, 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. In some embodiments, the IPP isomerase polypeptide has a sequence of SEQ ID NO:39. In some embodiments, 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. In some embodiments, the nucleic acid coding sequence of IPP isomerase is SEQ ID NO:38. In some embodiments, the nucleic acid coding sequence of IPP isomerase is codon-optimized for expression in a particular host cell.

[0023] In some embodiments, 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. In some embodiments, the cell and control cell further express heterologous IspS. In some embodiments, the nucleic acid coding sequence of IspS is codon- optimized for expression in a host cell. For example, 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).

[0024] In some embodiments, 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. In some embodiments, the recombinant expression cassette comprises a single terminator. In some embodiments, the recombinant expression cassette comprises more than one promoter and/or more than one terminator. For example, 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.). In some embodiments, the recombinant expression cassette comprises a separate promoter for each nucleic acid coding sequence.

[0025] In some embodiments, 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. In some embodiments, the first flanking region is preceded by a promoter. In some embodiments, the second flanking region is followed by a terminator. In some embodiments, 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.

[0026] In some embodiments, the flanking regions of the recombinant expression cassette comprise a nucleic acid sequences of a specific gene in the host cell. In some embodiments, the flanking regions of the recombinant expression cassette comprise sequences of a gene expressed in bacteria, cyanobacteria and/or microalgae. For example, the flanking regions can be nucleic acid sequences of a gene, wherein the first and second flanking regions are not identical sequences. In some embodiments, the 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).

[0027] In some embodiments, the flanking regions are used for homologous recombination into the genome of a host ceil, such as bacteria, cyanobacteria, or green microalgae. In some embodiments, 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. For example, the flanking regions can be about 500 bp of sequence and located on either side of the transgene

(including promoter, nucleic acid coding sequence(s), and terminator), wherein the sequence of the flanking regions are of a gene (i.e., a portion of a gene) present in the host cell. Non- limiting examples of genes in the host cell include , a neutral (Neu) site (ORF slr0168 ), PsbA2 (ORF slrl31 1), GlgA (ORF sll()945), and GlgX (ORF slr0237).

[0028] In some embodiments, the 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.

[0029] In some embodiments, 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. In some embodiments, the cell is selected from bacteria, cyanobacteria, and green microalgae. In some embodiments, 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.

[0030] In some embodiments, 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.

[0031] Further provided are methods for producing isoprene in a ceil, e.g., a cell lacking the MVA pathway, e.g., bacteria, cyanobacteria, or green microalgae. In some embodiments, 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. In some embodiments, the method further comprises recombinantly expressing IspS.

[0032] In some embodiments, 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.

[0033] In some embodiments, HMGS, HMGR, ATOB, IPP isomerase, MVK1 , MVD, and/or MVK2 are recombmantly expressed from at least one recombinant expression cassette as described above. For example, 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. In some embodiments, the recombinant expression cassette further comprises the nucleic acid coding sequence of IspS preceded by a TIR comprising an RBS. In some embodiments, the recombinant expression cassette comprises the nucleic acid coding sequences of HMGS, HMGR, ATOB, IPP isomerase, MVK1, MVD, and MVK2. In some embodiments, the nucleic acid coding sequence of each member of the MVA pathway is preceded by a TIR comprising an RBS. In some embodiments, each nucleic acid coding sequence is included on a single transcript upon expression. In some embodiments, 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.

[0034] In some embodiments, at least one recombinant expression cassette is targeted to the host cell's genome at a specific site of integration via double homologous recombination. In some instances, 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. [0035] In some embodiments, operons of the MVA pathway are recombined into a specific genomic location. In some embodiments, at least one member of the MVA pathway targeted to a specific site of integration and at least one member of the MVA pathway is targeted to another specific site of integration. In some embodiments, at least one member of the upper MVA pathway (e.g., IIMGS, HMGR and ATOB) is targeted to a first genomic location and at least one member of the lower MVA pathway (e.g., IPP isonierase, MVK1, MVD, and

MVK.2) is recombined into second genomic location. For example, to express operons of the pathway in various genomic locations, a recombinant expression cassette can contain nucleic acid coding sequences for HMGS, HMGR and ATOB and flanking regions for the PsbA2 gene., and 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. Similarly, 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.

[0036] In some embodiments, the method further comprises introducing the recombinant expression cassette to the cell prior to the expressing step. In some embodiments, 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. In some embodiments, the method further comprises harvesting the isoprene emitted from the cell. [0037] One of skill will appreciate that, in practicing the method of producing isoprene in a cell, that the embodiments of the recombinant expression cassette and cell described above can be used. For example, 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. In addition, 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.

[0038] In some embodiments, 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. For example, 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. [0039] In some embodiments, 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. In some embodiments the carbon source is quantitatively converted to isoprene, e.g., with an efficiency of 18, 25, 30, 50, 60, 70, 80% or higher. In some embodiments, 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. In some embodiments, 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.

[004(5] Further included is 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. In some embodiments, 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. Also provided are methods for producing isoprene in a cell comprising recombinantly expressing at least one member of the MEP pathway in the ceil and culturing the cell in the presence of a carbon source, thereby producing isoprene. In some embodiments, 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. In some embodiments, the recombinant expression cassette comprises the nucleic acid coding sequences of IspG, Dxr, IspE, IspH, Dxs, IspD, IspF, and Ipi. In some embodiments, the method further comprises recombinantly expressing IspS, e.g., from the same recombinant expression cassette or from a separate recombinant expression cassette.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1. Biosynthesis of IPP and DMAPP. Both the MVA pathway (left) and the MEP pathway (right, labelled DXP) lead to the formation of DMAPP, the substrate of isoprene synthase (IspS), Prv^ Pyruvate; GAP= Glyeeraldeliyde-3-phosphate; ATP= Adenosine-5''- triphosphate; NAD(P)H= Nicotinamide adenine dinucleotide (phosphate).

[0043] Figure 2, 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).

[0044] 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.

[0045] Figure 4. 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.

[0046] Figure 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).

[0047] Figure 6. Relative in vivo isoprene production. (A) kispS induced at different cell densities. (B) Effect of organic carbon sources on isoprene production. LB- Luria Broth; Gly= glycerol; Fru= fructose; Glc= glucose; Prv= pyruvate; Xyl= xylose. All contain the same total amount of carbon. 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.

[0048] Figure 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. The lower M VA pathway- genes (MK (MVK I), PMK (MVK2), PMD (MVD), and Fni (TPP isomerise)) were isolated from S. pneumoniae with native TIRs (C), and with E. coli TIRs introduced (D). 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).

[0049] Figure . 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 (D) pET28 with the MVA pathway using cell-specific RBSs (HsIirAIKlDK2) i). Atten, attenuation.

[0050] 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).

[0051] Figure 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. [0052] 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).

[0053] 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.

Translation initiation regions (TTRs), containing a ribosomal binding site and illustrated as black boxes, were placed in front of each MVA pathway gene, apart from those genes at the start of each operon, which used the native Synechocystis TIR of the PsbA2 gene within the upstream PsbA2. flanking region. (A) The Pueraria moniana isoprene synthase gene (IspS) was codon-optirnized for expression in Synechocystis and integrated within a neutral site (Neu) of the Synechocystis genome using the Neu flanking regions for homologous recombination. (B) 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. (C) 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. pathway super-operon 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) allowed for the selection of transformant lines. [0054] Figure 13. GC analysis of gases in the headspace of transform ant cyanobacterial cultures. 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.

[0055] 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 -

AGlgX: :IspS-AN<?w. Note the greater yield of isoprene by strains transformed with constructs (3) SRAFKiDK ₂ -A ?i^2::IspS-AN«i, (4) SRA-A wM2::FK _i DK ₂ -A67g ::IspS-AA¾/ _> and (5) §RA^pshA2: FK.O& ₁ -AG!gX: :lspS-ANeu, as opposed to the (1) lspS-ApsbA2 control.

[0056] Figure 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. 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. The percentage of assimilated carbon that was diverted to isoprene, after 196 hours of photoautotrophic growth, is expressed as a ratio of the measured isoprene to half of the measured dty cell weight (about 50% of dry biomass is carbon). 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. Note the greater isoprene -to-carbon ratio achieved by strains transformed with constructs (3) $RA¥KiOK ₂ -ApsbA2: :IspS-ANeu, (4) and (5) SRA-ApsbA2: :¥K DK ₂ -AGlgX: :lspS-ANeu as opposed to the (1) lspS-ApsbA2 control.

[0058] Figure 17. 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.

[0059] Figure 18. 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.

DETAILED DESCRIPTION OF THE INVENTION L Introduction [0060] 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. Accordingly, 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. li. Definitions

[0061] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4 ^M ed. 2007); Sambrook et at , MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Coid Springs Harbor, NY 1989); Raven et al. PLANT BIOLOGY (7 ^TH ed. 2004). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure,

[0062] 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. As used herein, 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

(Phospho-mevalonic acid kinase). One of skill will appreciate that species homologs of each member of the MVA pathway share activities, and thus can be used interchangeably in various combinations with other members of the MVA pathway, e.g., in recombinant or heterologous systems, to produce isoprene. Most bacteria, cyanobacteria, and green microalgae lack the MVA pathway.

[0063] 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 , As used herein, 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). One of skill will appreciate that species homologs of each member of the MEP pathway sha re activities, and thus can be u sed interchangeably in various combinations with other members of the MEP pathway, e.g., in recombinant or heterologous systems, to produce isoprene. [0064] 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.

[0065] As used herein, a TTR. (Translation Initiation Region) refers to the region in front of the start codon of a coding sequence, in a polycistro ic construct (e.g., a superoperon with multiple coding sequences expressed on a single transcript), this includes the regions between the stop codon of a preceding coding sequence and the start codon of the following coding sequence. 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). One of skill will appreciate that the elements of a TIR can be rearranged, e.g., so that the restriction site doubles as a spacer for positioning the start codon.

[0066] 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. For example, an RBS in a bacterial (e.g., E. coli) cell is selected to bind to bacterial (E. coif) ribosomes (e.g., the 16S rRNA), an RBS in a cyanobacterial ceil (e.g., Synechocystis) is selected to bind to cyanobacterial ribosomes (e.g., the 16S rRNA), and an RBS in a green microalgae cell is selected to bind to ribosomes (e.g., the 18S rRNA) in green microalgae, etc. One of skill will appreciate that the cell or expression system can be manipulated to include heterologous ribosomes that bind to a particular RBS.

[0067] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer 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.

[0068] The phrase "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. Such 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] The term "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. A "termination signal" or "terminator" refers to an element that terminates transcription.

[0070] The term "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. [0071] The term "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,

[0072] The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second

polynucleotide. For example, the 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.

[0073] The terms "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.

[0074] The term "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, γ- 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.

[007S] 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.

[0076] "Conservatively 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. Such 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. One of skill will recognize that 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) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0077] As to 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.

[0078] The terms "identical" or percent "identity," in the context of two or more 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

ncbi.nlm.nih.gov/BLAST/. Such 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.

[0079] 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). Generally, an expression cassette is inserted into an expression vector (e.g., a plasmid) to be introduced into a host cell.

[0080] 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. [0081] 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. For example, 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). Similarly, 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.

[0082] "Recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, 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 (e.g., a coding sequence) 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 αί, 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. For instance, human manipulated restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences. A 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). [0083] The terms "culture," "culturing," "grow," "growing," "maintain," "maintaining," "expand," "expanding," etc., 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.

[0084] The terms "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 Typically 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 .

[0085] 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. For example, 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). In the context of the present disclosure, 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. For example, 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. One of skill will recognize that controls can be designed for assessment of any number of parameters. [0086] One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. 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

[0087] 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. In some embodiments, the cell naturally produces at least some biosynthetic precursors, e.g., Acetyl- CoA. Tn some embodiments, e.g., those involving isoprene production via the MEP pathway, 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.

[0088] Any microorganism can be used in the present method so long as it remains viable after being transformed with a heterologous expression cassette. In some embodiments, the microorganism is bacterial. In some embodiments, the bacteria is a cyanobacteria. Examples of 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.

[0089] 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. Examples of algae that can be used are a microalga (e.g.,

Chlamydomonas reinhardtii, or other member of the genus Chlamydomonas; a member of the genus Dunaliella, or a member of the genus Chlorella). In some embodiments, 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. [0090] 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.

IV. Recombinant methods

[0091] 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.

[0092] The 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,

[0093] 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. For example, in 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. Further, commercial services are available that can supply synthetic genes of the desired sequence. In addition, 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. [0094] The nucleic acid coding sequences for desired biosynthetic enzymes can be incorporated into an expression cassette. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression cassette, and into an expression vector for introduction to a cell. 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.

[009S] 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. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. Examples include lactose promoters (Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator) and 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). Another example is the tac promoter. (See deBoer et al. ( 1983) Proc. N ^' atl. Acad. Set USA, 80:21 -25.)

[0Θ96] 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);

Kreuteweiser et al, Ecotoxicol Environ. Safety 28: 14-24 (1994)); heat shock inducible regulatory elements (Takahashi et al. Plant Physiol 99:383-390 (1992); Yabe el al, Plant Cell Physiol. 35: 1207-1219 (1994); Ueda t al, Mol. Gen. Genet. 250:533-539 (1996)); and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, lPTG-inducible expression (Wilde et al, EMBO J. 1 1 : 1251 - 1259 (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.

[0097] 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). Further, 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 ).

[0098] As will be appreciated by those of ordinary skill in the art, the invention is not limited with respect to the precise promoter or expression vector used. Although 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 λ 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. For example, 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. In addition, reference may be made to the relevani iexis and literature, which describe expression vectors and their suitability to any particular host cell.

[0099] 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

Cyanobacteria: New Biotechnological Applications" in Handbook of Microalgal Culture: Biotechnology and Applied Phycology, ed. Amos Richmond, Oxford, UK, 2004).

Homologous recombination can occur between, the expression vector and the homologous region in one or more genomic copies present in the host cell. Typically, 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.

[0100] One of skill in the art understands that other methods for constructing and integrating a recombinant expression vector into a host's genome can be used, such as, but not limited to, site-directed mutagenesis (see, e.g., Melnikov et aL, J. Bacterial, (2009), 191 :4 58), the FLP system (see, e.g., Martinez-Morales et aL, J. Bacterial (1999),

181 :7143), transgene integration into the chloroplast genome (see, e.g., Verma, D. and Daniell, PL, Plant Physiol (2007), 145: 1 129), and synthetic biology (see, e.g., Wang et aL, Frontiers in Microbiology, (2012), 3: 1). [0101] 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. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) 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. Those of ordinary skill in the art can transfect a host ceil with a desired sequence using these or other methods. [01Θ2] A variety of methods are available for identifying a transfected/ transformed cell. For example, 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. In addition, 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.

[0103] Once a cell has been transformed with the expression vector, 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. [0104] 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).

[ί)1θ5] 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 ). [0106] Examples of Type I IPP isomerases that can be used 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). Tn some embodiments, the Type I isomerase is from bacteria or a fungus, such as a yeast.

[0107] Examples of Type Π ΓΡΡ 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).

V. Cell culture techniques

[01 Θ8] 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). For photosynthetic microorganisms, 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.

[01Θ9] Photobioreactors can have different configurations. For example, although 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. For example, 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.

[011(5] 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. 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 _? , t o support the growth of the photosynthetic microorganisms. In embodiments in which the CO? is less than 100%, the remainder of the gaseous mixture is air. The 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.

[0111] 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.

[0112] In vertical tubular photobioreactors, CO _? -rich gases are typically slowly bubbled at the bottom of the liquid phase. In horizontal tubular photobioreactors, 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? 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. In such an embodiment, 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 _? ). Alternatively, in some embodiments, 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.

[0113] Examples of 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

WO/2011034567 and WO2012/145692. 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.

VI. Collection of soprene

[0114] Volatile isoprene hydrocarbons produced by genetically modified microorganisms can be harvested using known techniques. Isoprene hydrocarbons are not miscible in aqueous solution, and thus rise to the surface of the media and float or vaporize, Isoprene can be siphoned off from the surface and sequestered in suitable containers. Depending on the prevailing culture temperature, isoprene can exist in vapor form above the media in a culture container (e.g., if the culture is over the isoprene boiling temperature T=34°C). Isoprene vapor can be piped off the culture container and condensed into liquid fuel form upon cooling or low-level compression. [0115] In contained culture conditions, e.g., a culture container or photobioreactor, 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. 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. Concentrations of isoprene (or other volatile hydrocarbon) collected in this manner vary between 1%, when a solvent is used to trap the isoprene, and 100% isoprene when no solvent is used, but a condenser-temperature low enough is employed to enable retention of isoprene in the liquid phase. [0116] 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. In such embodiments, cultures are fed periodically with an organic carbon source or CO?., e.g., ever 2.4 to 48 hours. In addition, 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). [0117] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence database entries, internet sites, patents, and patent applications cited herein are incorporated by reference in their entireties for ail purposes.

VIL Examples

A. Materials and Methods for Bacteria

[0118] Cloning procedures, bacterial strains, plasmids, and media: The nucleotide sequence of the Pueraria montana (kudzu vine) isoprene synthase (klspS) gene (Sharkey et al. (2005) Plant Physiol. 137:700, GenBank accession No AY316691), without its predicted chloroplast transit peptide, was employed. Codon use optimization was applied to the klspS gene to make it more suitable for expression in E. coli, using the Gene Designer software (DNA2.0, USA).

10119] 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 .

Table 1: Strains and plasmids

Rosetta(DE3) F- ompT, hsdSB(rB-mB-), gal, dcm(DE3), plasmid Novagen encoding rare fRNAs, BL 21 (DE3) derivative

C43(DE3) derivative of C41 (DE3) transformed with the F- Avidis S.A

ATPase subuni! gene and cured

Plasmids

pGEX6Pl P _T7 . lacf, bla, oriR ColEl, glutathione-S-transferase Amersham Pharmacia

(GST) tag

pJFl i9EH P _IAC, lacf, bla, oriR ColEl , ¾ tag lab stock pET28 P _7, !acf, l an, oriR ColEl, H ₆ tag Novagen pGEX6Pl -klspS>45 klspS (45-608) with an N-terminal, cleavable GST this work

tag under control of P-r; (lacf, kan, oriR ColE )

pGEX6Pi-kIspS>60 klspS (60-608) with a N-terminaL cleavable GST this work

tag under control of P ? (iacF, kan, oriR ColE )

pGEX6Pl -kispS>63 klspS (63-608) with an N-terminal, cleavable GST this work

tag under control of P-π (lacP, kan, oriR ColEl)

pGEX6PI -kIspS>78 klspS (78-608) with an N-terminai, cleavable GST this work

tag under control of Ρ _τ -, (lacl\ kan, oriR CoiEl)

pJF- kIspS>45 kispS (45-608) untagged under control of P _rac (3acl ^q , this work

bla, oriR ColEl)

pJF- kIspS>46 klspS (46-608) untagged under control of P _tac (lacs' ¹ . this work

bla, oriR ColEl)

pJF- kIspS 51 klspS (57-608) untagged under control of P _iac (lacf, this work

bla, oriR,ColEl)

pJF- kIspS>57 klspS (57-608) untagged under control of P _tac ( iaclq. this work

bla, oriR ColEl)

pJF- kIspS>60 klspS (60-608) untagged under control of P _tac (lacf, this work

bla, oriR ColEl)

pJF- kIspS>61 klspS (61-608) untagged under control of P _tac (lacf, this work

bla, oriR ColEl)

pJF- kIspS>78 klspS (78-608) untagged under control of P _tac (lacf, this work

bla, oriR ColEl)

pJF- H6-kIspS klspS (45-608) with N-terminal ¾ tag under control this work

of P _iac (lacf, bla, oriR ColEl)

pJF- kIspS-I¼ klspS (45-608) with C -terminal H ₆ tag under control this work

ofP _f c _c (lacf, bla, oriR ColEl )

pET28- ¾-k!spS ki pS with an N-terminal cleavable Hf, tag under this work

control of P _T7 (lacf, kan, oriR ColEl )

pET28- kispS-H ₆ k!spS (45-608) with C-terminal H ₆ tag under control this work

of P _tac (lacf, bla, oriR ColEl)

pET2S- HmgS (hS) and HmgR (hR) of E.faecaiis, atoB (A) this work

(hShRA)x(K I D 21)y of E. coii, mvKl (Kl), mvD (D), mvK2 (K2) and f i

(I) of S. pneumoniae under control of P _T7 (lacf, kan, oriR ColEl); x, y represent different intergenic

regions

pET28- SGHiERDF DXS (S), IspG (G), IspH (H), Ipi (i), IspE (E), Dxr this work

(R), fspD (D), and IspF (F) of E. coli under control of P _T7 (lacf, kan, oriR ColEl)

[0120] Standard procedures were used for isol ation of plasniid DNA , restriction analysis, PGR amplification, ligation and transformation. 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.

Table 2: Oligonucleotide primers (SEQ ID NOs: 48-64)

pETi529-

PI 4 53 IspE down Ti 5 ' ^■ CACGCGTCGACTTAAAGCATGGCTCTGTG

Rl

5 ^* - pET1529-

PI 5 54

Dxs up TIR2 CTGAGGATCCAAGGAG^¾∑4CCATGAGTTTTGATATTGCCAAATA

C

pET1529-

P16 55

ispD_up_TIR2 5 ' - CCGGAATTCAAGG AGA TATA CATGGCAACC ACTCATTTG pETI529-Ipi-

PI 7 56

1 up TiR2 5'- CGACGAG rCA AGGAG^¾ TA CATGCA AACGGA AC ACG pET1529-

PI S 57

Dxi up TIR2 5'- CTGAGGATCC AAGGAG 4 TA TA CCATG A AGCAACTCACCATTC pET1529-

PI 9 58 5 ' - CGACGAGCTCAAGGAG4 TA TA CCATGCGGACACAGTGG

IspE up TIR2

pET1529-

P20 59 fci up TIR2 5'- CTGACCATGGCAACGACAAATCGTAAGGACG

pET1529-

P2] 60 i i down TIR 5 ' - CGCGGATCCTTACGCCTTTTTCATCTGATC

pET1529-

P22 61 mvkl up 11 R 5 ' - CGCGGATCC AAGGAG iir.4 TA CATGACAAA AA AAGTTGGTGTC

pET1529-

P23 62 mvkl down T 5 ' - CGACGAGCTCTCAC AGGCTCTCTATCCATG

IR2

pET1529-

D -

P24 63 mvdl up 11 R

CGACGAGCTCA AGGAG4 TA TA CCATGTATCATAGCCTTGGT AAC pET!529-

P25 64 mvdl down T 5 ' - C ACGCGT CG ACTT AAC AGCAATC ATCTTGA C

IR2:

Bo!d indicates restriction enzyme site. Underline indicates RBS. Italics indicates spacer.

[0121] Cells were grown at 37°C in Luria-Bertani (LB) media supplemented with appropriate antibiotics (100 pg/mf ampicilhn; 25 g/ml chloramphenicol; 50 μg/ml kanainycin). Chemicals were obtained from Fisher Scientific (USA) or Sigma- Aldrich (USA).

[0122] Protein expression: E. coii Rosetta ceils transformed with plasmtd ρΕΤ28-¾&*/¾!? 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. Cells were grown in 1 L of LB medium within a 2 L Erlenmever flask on a rotar shaker at 37°C to OD ₆ oo ⁼ 0.8. The flasks were cooled to 15°C, protein expression was induced with of 0.1 mM isopropyl β-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.

[0123] 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 β-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.

[0124] The supernatant was mixed with 2 ml ofNr ⁴¹ -NTA slurry (Qiagen, USA), equilibrated with buffer T ^' , and incubated for 1 h at 4°C. The sluny was poured into a column, the flow-through was discarded and the slurry washed at 4°C with buffer Ϋ and buffer I ²⁵ (50 mM Tris/HCl, pH 8.0, 400 mM NaCl, 10 mM β-mercaptoethanol, 0.1% Triton X-! OO, 25 mM imidazole). He-kTspS was eluted with buffer I ²⁰⁰ (50 mM Tris/HCl, pH 8.0, 400 mM NaCl, 10 mM β-mercaptoethanoi, 200 mM imidazole). Fractions were analyzed by SDS-PAGE and fractions containing ¾-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.

[0125] Conditions for klspS function: Purification of the klspS protein through a Ni ⁱ - NTA column, instead of a glut athione agarose one, caused loss of klspS enzymatic activity. This was traced to the inhibitory effect of ⁺ , which was inadvertently present in the purification extract. The effect of a tag on protein activity was also investigated by in vivo and in vitro isoprene synthesis measurements using E. coli extracts. Assays showed that the presence of a hexahistidine tag on the N- terminal end of the protein (He-klspS) caused a lowering of the enzymatic activity to a level of about 10% of that in the control (klspS without a tag). When the ¾ tag was present on the C-terminal end of the protein (klspS-He), solubility of ihe recombinant protein in ihe E. coli ceil was substantially lower, to the point where it was almost exclusively found in inclusion bodies.

[0126] 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. 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. Following the protease cleavage, five amino acids (GPLGS) 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).

[0127] in vitro IspS activity assay: In vitro isoprene production activity of the kTspS recombinant enzyme was assayed in 100 μΐ reaction mixtures containing 50 mM Bicine, pH 8.0, 30 mM NaCi, 50 mM Mgi¾, 5 mM KC1, 5% glycerol, 1 mM DTT and 9 ,ug recombinant klspS protein. The reaction was initiated upon the addition of various concentrations of DMAPP (Echelon, USA), For inhibitor, temperature, and H studies, 500 μί of reaction mixtures were employed containing 100 uM DMAPP as the substrate. The effect of the pH was measured using the following buffer systems: Na-Citrate, 3.0; Na- Acetate, 5.0; MOPS, 6.5-7.5; Bicine, 7.5-9.0; CAPS, 10.0-1 1.0, at a concentration of 10 mM each,

[0128] inhibitors, if any, and DMAPP were added simultaneously to the reaction mixtures. Reaction mixtures were incubated for 15 min at 42°C. For 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).

[0129] in vivo IspS activity assay: 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 main cultures were grown at 37°C to an ODeoo = 0.2, induced with 0.1 mM IPTG, and sealed. 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. [0130] To test whether the identity and properties of the plasmid affects isoprene production, three different expression vectors were compared, namely pJFl 19EH, pET28 and pACYC. Over-expression ofklspS from pET28 and pACYC showed similar in vivo isoprene production rates, whereas activity from pJF 1 19EH was somewhat lower. Furthermore, comparative performance analysis of different E. coli strains revealed that Rosetta cells perform 30-40% better than C43 or YYC202 cells.

[0131] Product analysis: Chemical identification of the in vitro and in vivo assay reaction product(s) was implemented by gas chromatography-mass spectrometry (GC-MS). All GC- MS analyses were conducted with an Agilent 6890GC/5973 MSD equipped with a DB-XLB column (0.25 mm i.d. x 0.25 μηι x 30 m, J &W Scientific). The DB-XLB column is suitable for the separation of a wide variety of molecules ranging from small organic molecules and hydrocarbons to pesticides, herbicides, PCBs and PAHs. The oven temperature was initially maintained at 40°C for 4 min, followed by a temperature increase of 5°C/min to 80°C. 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.

B. 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.

[0133] The isolated klspS enzyme was incubated with 100 μ,Μ DMAPP in the presence of 50 mM MgCb at 35°C for 15 min. Volatile compounds accumulating in the headspace of the sealed reaction mixture were tested by GC analysis, revealing the presence of isoprene, catalyzed from DMAPP by the recombinant klspS. The same reaction mixture (without the klspS protein) did not yield isoprene. The apparent K _m of the klspS enzyme was determined to be 2.5 mM. The maximal enzymatic activity rate was V _max = 4.1 μηιοΐ (mg protein)" min while the L _at was 4.4 s ^{" 1} (Fig. 2A). Therefore, the k _cs.t /K _m ratio was determined to be 1 ,760: 1. [0134] 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 \ In the latter study, the isoprene synthase activity was measured with an N-tenninal hexahistidine ¾-kIspS fusion protein that was purified using a Ni-NTA agarose column. As discussed in the Materials and Methods section, use of the N-terniinal hexahistidine tag (MRGSHHHHHHGS) caused a lowering of the enzymatic activity by 90% compared to the activity of the untagged protein. Moreover, when purified with a N -NTA resin, 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). The activity of the recombinant klspS protein in the presence of different divalent cations was normalized to that measured in the presence of Mg ² \ Relative activities of 36%, 12%, 4,9%, 4.7%, 1.9%, and 0.6% were measured in the presence of Mn ² ', Fe ^" , Co ² ', Cu ^' , Ni", and Zn ^{2 r} , respectively (Fig. 2B). Neither Ca" nor EDTA supported any measurable levels of klspS activity ⁷ . Application of risedronate, a potent inhibitor of the farnesyl-PP synthase, blocked isoprene production, suggesting that risedronate is also a potent inhibitor of klspS. In contrast to DMAPP, its isomeric IPP form did not serve as a klspS substrate under these experimental conditions, and could not be converted to isoprene by the in vitro enzymatic reaction assay (Fig. 2B). The results indicate that isoprene synthase activity is inhibited by the presence of an N-tenninal tag and/or the use of Ni ^{2 f} -NTA resin in the recombinant protein purification procedure.

[0135] Isoprene production in plants is induced upon heat stress. Thus, we characterized the temperature dependence of klspS activity . Optimum in vitro activity was observed at 42°C (Fig, 2C). At higher than optimum temperatures, the activity of the enzyme declined rapidly. At 29°C and 46°C, the activity of the klspS enzyme was about 50% of the maximum (Fig. 2C). The effect of the pH of the reaction medium was also measured using different buffer systems. Optimum enzymatic activity was measured at around pH 7.5, but the enzyme showed a broad spectrum of activities from H 6.5 to 9.5 (Fig. 2D).

C. Example 2: In vivo activity of IspS in E. coli [0136] We observed 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).

[0137] in addition, we carried out GC-MS analysis of the reactor headspace gas of the UspS transformant was compared to an isoprene standard (Fig. 4). Isoprene was positively identified upon comparison of the GC peaks with a retention time of 1.5- 1.6 mm in the transformant (Fig, 4A) and isoprene standard (Fig. 4C). MS analysis of the dominant peak at 1 ,5 min retention time confirmed the identification, revealing distinct mass spectral lines [lines 53, 67, and 68] that signify isoprene hydrocarbons (Fig, 4B and 4D). The isoprene- specific peak and MS lines were not present in headspace samples from the recipient strain or from the not-induced transformant.

[0138] Upon purification, 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.

[0139] Therefore, we investigated the in vivo activity of several klspS protein constructs with variable portions of the N -terminal domain removed. Constructs were made ranging from the full-length of the putative klspS (amino acids 45-608) to a highly truncated (267- 608) derivative. Protein klspS constructs (267-608), (78-608) and (63-608) were all expressed in the transformant E. coli but showed little or no catalytic activity (Fig. 5). This was attributed to removal of the highly conserved RR(X)gW terpene synthase motif (amino acid residues 61 through 71, Fig, 10) from these klspS constructs. 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).

D. 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. Comparing the induction of klspS protein synthesis and accumulation of isoprene vapour at different culture densities (ODeoo 0.06, 0.12, 0.25, 0.5, 0.8, and 1.80), isoprene production was highest when cultures were induced at ODsoo S 0,25 (Fig. 6A).

[0141] We also tested the effect of different organic carbon substrates in driving isoprene production, LB media were supplemented with an equivalent amount of organic carbon, given in the form of different compounds. Results showed that the chemical nature of the organic carbon supplement had an impact on cell productivity and isoprene yield. The yield of isoprene production declined in the following order of compounds: glycerol > fructose > glucose > LB > xylose (Fig. 6B). No isoprene could be detected when the growth medium was supplemented with pyruvate, which is the starting material for the MEP pathway. Use of glycerol as a substrate for microbial fermentation is advantageous due to the increasing availability of glycerol as a by-product of biodiesel production.

[0142] We next sought to improve the yield of isoprene production in E. coli by (i) overexpressing the E. coli endogenous MEP pathway genes, and (ii) overexpressing heterologous MVA pathway genes from Enterococcus faecalis and Streptococcus pneumoniae. The klspS transformant isoprene production system can be used to determine the relative efficiencies of the MEP and MVA pathways in terms of the generation of DMAPP, and also of the efficacy of the MVA pathway to bypass the MEP pathway in the generation of isoprenoids. There are no additional catalytic steps to be considered between TPP/DMAPP generation and the klspS activity. All MEP and MVA pathway constructs for these experiments were cloned as one operon into pET28, where transcription is driven from a T7 Promoter (Ρτ _? ).

E. Example 4: Isoprene production via ovcrcxpression of native MEP

pathway in E. coli [0143] Over-expression of MEP pathway genes in various superoperon combinations was implemented with one of two different TIRs placed in front of each gene. 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. In the final step, 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 ). Yield of isoprene production was measured from the beadspace of the cultures following an overnight incubation of induced cells. E. coli transformed with the kispS gene yielded about 1.25 mg isoprene L ^"1 culture (Fig. 7E, Table 3). Co- transformation with the pET plasmid reduced isoprene yield, down to 0.4 mg Isp L ^~! culture. This was attributed to an effect of the pET plasmid on E. coli cellular metabolism. Co-transformation with kispS and the GR-TTR2 construct (Fig. 7A) improved yield to 2.36 mg Isp L ^"1 culture, i.e., a 6-fold improvement in the yield of isoprene. This result helped identify the Dxr.DXP reductoisomerase and/or the IspG: HMBPP synthase as a rate limiting steps in the MEP pathway. Co-transformation with kispS and the HSDFi-TIRi construct yielded 1.76 mg Isp L ^"1 culture [a 4.4-fold

improvement], while co-transformation with the HSDFi-TIR2 construct yielded 2.64 mg Isp L ^"1 culture [a 6.6-fold improvement]. These enhancements in yield are similar to the improvement seen upon co-transformation with GR-TIR2 (Fig. 7E).

[0145] Co-transformation with kispS and the full GREHSDFi-TIRI MEP pathway superoperon yielded 3.04 mg Isp L ^{" 1} culture [a 7.6-fold improvement], while co- transformation witii klspS and the GREHSDFi-TIR2 MEP pathway superoperon (Fig. 7A) yielded 0.96 mg Isp L ^"1 culture [a 2.4-fold improvement. The results indicate that TIR1 is better suited for the expression of this particular superoperon than TIR2. Co-transformation with kispS and the SGHiERDF-TIR2 construct (Fig. 7B) yielded 5,04 mg Isp L ^{" 1} culture [a 12.6-fold improvement] (Fig, 7E and Table 3). The results indicate that the order of the members of the MEP pathway in the superoperon affects relative expression levels and, consequently, the yield of isoprene production.

Table 3: Yield of isoprene production by E. coli Rosetta cells transformed with kispS

(i). Effect of klspS co-transformations using an (ii) empty vector, (iii) MEP pathway, (iv) MVA pathway with native TTRs, (v) MVA pathway with cell-specific TIRs, and (vi) MVA pathway with cell-specific TIRs and specified culture conditions. Glycerol-to-isopreiie ratios were estimated assuming all isoprene is found in the overhead space. The isoprene-to- bio mass carbon-partitioning ratio was calculated from the ratio of Isp in the overhead space to dry cell weight after 18 h incubation time. Total carbon per dry cell weight (DCW) was estimated according to Luria (I960) The bacteria, vol. 1. Academic Press, New York, pp 1 - 34.

F. Example 5: Isoprene production via heterologous expression of MVA pathway in E. coli

[0146] 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 β-carotene production in E. coli (Yoon et at (2009) J. Biotechnol.

140:214). The results indicated that a combination of the upper half from E. ja ^' ecalis and the lower half from S, pneumoniae performed best. The prokaryotic MVA pathways in that study were cloned as operons using native RBSs. In addition, the Streptococcus pneumoniae genes overlap, and might not be optimal for expression in E. coii. [0147] We used an RBS calculator (Salis et al. (2009) Nat. Biotechnol, 27:946) to estimate the translation initiation rate [from < 1 to 100,000+ on a proportional scale] of the lower MVA pathway of S. pneumoniae in E, coli. According to the RBS calculator, 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 Furthermore, the TGT start codon of FNI in Streptococcus pneumoniae was changed to ATG. By way of comparison, we reached a 25- fold increase in isoprene yields with the lower portion of the MVA pathway of S. pneumoniae using the native RBSs (and the upper portion of E. faecalis using E. co //-specific RBSs), whereas a 150-fold increase in yield was reached with the E. co//- specific RBSs used for the whole MVA pathway, [0148] Most bacteria, including E. coli, utilize the MEP pathway for terpenoid

biosynthesis, and thus do not possess MVA pathway genes or regulatory mechanisms (Fig. 1). We sought to investigate whether heterologous expression of the entire MVA pathway in E. coli would increase isoprene production rates and yields. Genes for the upper MVA pathway (HMGS (Hs) and HMGR (Hr)) were obtained from Ente coccus faecalis. The HMGR of Entemcoccus faecalis combines thiolase activity and HMG-CoA reductase activity, i.e., the first and third reaction of the MVA pathway. We added AtoB off. coli (Fig. 7C and ID), an additional thiolase, to drive more aceiyl-CoA towards the MVA pathway.

[0149] 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

(HsHrA)2(KlDK2I)n (Fig. 7C).

[0150] in addition, we designed an operon in which these genes were cloned sequentially upon introducing TIR1 or TIR2 between individual genes, and then ligated to the upper MVA pathway construe! to produce (HsHrAIK 1 DK2) 1 and (HsHr AIK 1 DK2 )2 (Fig. 7D). [0151] isoprene production between the construct with overlapping genes

((HsHrA)l(KlDK2I)n and (HsHrA)2(KlDK2I)n) (Fig. 7E, bar#8) was compared wiih that of the artificial TIRl and T1R2 operons ((HsHrAIK 1 DK2) 1 and (HsHrAIK 1DK2)2) (Fig. 7E, bar#9). Constructs with overlapping genes yielded up to 10 mg Tsp L ^'1 culture. In constrast, constructs in which artificial TIRs were inserted between each coding sequence yielded up to 56,4 mg isoprene L ^{" !} culture (Fig. 7E), The results sho that isoprene production via the heterologous MVA pathway in E. coli, using constructs with cell-specific TTR sequences preceding each coding sequence in the artificial operon, results in far higher yields of isoprene. [0152] Examples of GC analyses of E. coli culture headspace gases were conducted with the various transformants (Fig. 8). Results are shown for a klspS plus pET co-transformant (Fig. 8A), a klspS plus SGHiE DF MEP pathway co-transformant (Fig. 8B), a klspS plus (HsHrA) 1 (K 1 DK2I)n MVA pathway co-transformant using the native RBSs (Fig. 8C), and a klspS plus (HsHr AIK 1 DK2) 1 MVA pathway co-transformant using E. eo/z-specific TTRls (Fig. 8D). This comparative analysis revealed dramatic differences in the isoprene:ethanoi peak ratios in the culture headspaces of the different transformants. 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. G. Example 6: isoprene production versus cell growth upon heterologous expression of MVA pathway in E. coli

[0153] Comparative kinetic studies of cell growth and isoprene production were conducted with the klspS plus (HsHrAIKlDK2)l MVA pathway co-transformants. Representative results are shown in Fig. 9, where IPTG induction was initiated at two different cell densities, ODgoo = 0.25 and Ο!¾οο = 0.7. In both cases, cell growth was arrested upon initiation of the isoprene production process, about 6 h after induction. During this stage of fermentation, organic substrate in the culture was quantitatively converted to isoprene (Fig. 9). This phenomenon was not observed for the MEP pathway transformants, where isoprene production and growth occurred in tandem. This observation indicates that E. coli cultures are kept in a transient stationary (live enzyme) phase. That is, the cells are actively producing isoprene through the unregulated MVA pathway at the expense of endogenous systems, e.g., cell growth.

[0154] The isoprene/QDeoo ratios achieved were substantially different under the two IPTG induction conditions (Fig. 9). When induced at 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. When induced at ODeocH). ^™ , MVA transformants produced 320 mg Isp L ^{" 1} culture and attained a final 0.44 g Isp/g DCW ratio. 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. [0155] The results show that when IPTG induction of isoprene production is initiated at a culture ODeoo ^;;; 0-7, instead of ODeoo - 0.25, the yield of isoprene is increased 800-fold (320 mg Isp L ^{" 1} culture) compared to the klspS transforniant. Moreover, a higher

isoprene/biomass ratio was reached under these conditions, suggesting differential partitioning of endogenous substrate between biomass and isoprene in different growth stages of the culture. In contrast, metabolic engineering of the MEP pathway increased isoprene levels by about 12-13 fold. These observaiions show ihat ihe MVA pathway, when expressed in E. coli, introduces a bypass in the flux of cellular substrate to IPP and DMAPP, overcoming the limitations imposed upon the innate regulation of the MEP pathway by the ceil. Thus, over-expression of the MEP pathway in bacteria may be limited owing to control mechanisms present in the native host, which is avoided with heterologous expression of the MVA pathway.

H, Materials and Methods for Cyanobacteria

[0156] Cloning procedures, bacterial strains and plasmids: The Escherichia coli strain DH50C was used for routine subcloning and plasmid propagation, grown in LB media with appropriate antibiotics at 37°C, according to standard protocols. Genomic DNA of

Streptococcus pneumoniae (ATCC no. 6314), Enterococcus faecalis (strain V583; ATCC no. 700802) and 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. The isoprene synthase (IspS) gene from Pueraria montana, (Sharkey ei. al (2005) Plant Physiol 137:700) Genbank accession no. AY316691) was optimized for codon-useage in Synechocystis, without the predicted chloroplast transit peptide Lindberg, et al. (2010) Metahol Engin 12:70). Nucleotide sequences of the primers used to amplify these genes are listed in Table 2, and show the positions of introduced restriction sites to aid cloning, as well as introduced TIR sequences in-front of genes. Two different TIR sequences were employed: TIR1 , composed of the ribosomal binding site AGGAGG and spacer TAATAT; and T1R2, composed of ribosomal binding site AAGGAG and spacer ATATACC (Zurbriggen et al. (2012) Bioenergy Res.)

[0157] Four regions of the Synechocystis genome were chosen as sites of integration for IspS and the MVA pathway operons via double homologous recombination: ( I) A neutral (Neu) site ORF slr()168, encoding a hypothetical protein; (2) PsbA2 (slrl31 1), Dl protein; (3) GlgA (sll0945), a glycogen synthase; and (4) GlgX (slr0237), a putative isoamylase-type debranching enzyme.

[0158] 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)

73 G3gA upstream G3gAus_EcoRI_F

CCGGAATTCGCCATGTCCCAAATTCTTGATCC

74 GlgA upstream GlgAus_NdeI_R GGAATTCCATATGACCGTCGTTATTCCACTAATT

GAG

75 GigA Gig Ads BamHI CGCGGATCCCAATTGATGGCCATGCGTTATGG

downstream F

76 GlgA GlgAds_Saci_R GAGAGAGAGAGCTCGAGCGATCAAGACCACCAT

downstream TAGG

Underlined portion demotes introduced restriction enzyme sites added to aid cloning.

[0159] Transforniants were screened by PGR for proper transgene incorporation and complete DNA cyanobactenai copy segregation for the introduced transgene. Primers (SEQ ID NOs: 77-84) for amplifying the operons are listed in Table 5.

Table 5: Oligonucleotide primers used for screening Synechocystis genome for IspS and the MVA pathway transgene incorporation (SEQ ID NOs: 77-

[0160] Cyanobacterial strains, growth conditions and transformation procedures: Synechocystis sp. (Williams JOK (1988) Methods Enzymol 167:766; Lindberg, et. al. (2010) Metahol Engin 12:70; Bentley et al (2012) Biotech Bioeng 109: 100) was used as the recipient strain in this invention, and is referred to as the wild type. Wild type and transformant strains were maintained on solid BG-1 1 media supplemented with 10 m ^' M TES- NaOH (pH 8.2), 0.3% sodium thiosulfate, and 5 niM glucose. Where appropriate, kanamycin (Km) and spectinomycin (8m) were used at a concentration of 25 μ§/πΐΙ_-, and

chloramphenicol (Cm) at 15 ig/niL. 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 μηιοΐ photons m ' s Growth conditions employed when measuring the production of isoprene from Synechocystis cultures are described in the following section.

[0161] 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 μηιοΐ 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. The sealed bioreactor headspace allowed for the trapping, accumulation and concentration of photosynthetically produced isoprene as a volatile product (WO2012/145692; Bentley et al. (2012) Biotech Bioeng 109: 100) [0162] Gas from the headspace of sealed bioreactors was sampled and analyzed by gas chromatography using a Shimazu 8A GC (Shimazu, Columbia, MD, USA) equipped with a flame ionization detector (FID) and a Porapak 80/100 column appropriate for detection of short-chain hydrocarbons. 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 μπι 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. [0163] 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.

[0164] Western Blot Analysis: Cyanobacterial cells from liquid culture were harvested by centrifugaiion. Cells were disrupted by French Press and then centrifuged to remove cellular debris. The supernatant was collected as the soluble protein fraction. Solubilized proteins were separated by 8DS-PAGE (Bio-Rad, USA), After electrophoresis the proteins were transferred to PVDF membrane for immunodetection using the rabbit immune serum containing specific polyclonal antibodies against each of the MVA pathway proteins (ProSci Inc, LISA). Cross-reactions were visualized by incubating the PVDF membrane in a solution containing 0.5 mg/ml 3,3-diaminobenzidine (DAB) (Sigma, USA) and 0.03 % H ₂ 0 ₂ in 50 mM acetate buffer, pH 5.0 at room temperature.

I. Example 7: Isoprene production via heterologous expression of MVA pathway in Synechoeystis

[0165] Heterologous expression of the isoprene synthase gene in cyanobacteria, e.g.

Synechoeystis sp., conferred upon these microorganisms the property of isoprene (CsHg) hydrocarbons production. However, 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. Fieterologous expression of the isoprene synthase gene in combination with the mevalonic acid (MVA) pathway in cyanobacteria, e.g. Synechoeystis sp., provided additional substrate flux to 1PP and DMAPP precursors to 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. An optimized heterologously expressed MVA pathway in Synechoeystis sp., comprised further improvement in the yield of isoprene production. Heterologous expression of the M VA pathway in Synechoeystis introduced a bypass in the flux of endogenous cellular substrate to IPP and DMAPP, overcoming the limitations imposed upon the regulation of the native MEP pathway by the cell. [0166] The MEP pathway (Fig. 11) is native to cyanobacteria, e.g. Synechoeystis sp. 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. [0167] 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. Translation initiation regions (TIRs), containing a ribosomai binding site, were placed in front of each MVA pathway gene, apart from those genes at the start of each operon, which used the native Synechocystis TTR of the PsbA2 gene within the upstream PsbA2 flanking region. The Pueraria montana isoprene synthase gene (IspS) was codon- optimized for expression in Synechocystis and integrated within a neutral site (TMeu) of the Synechocystis genome using the Neu flanking regions for homologous recombination. A chloramphenic l-resistaiice selectable marker (Cm ¹ ) of transformant lines. 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. 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.

[01 8] Examples of GC analyses of Synechocystis culture headspace gases were conducted with the various transformants (Fig. 13). An aliquot of 100% C0 ₂ was bubbled slowly though a 700 mL liquid culture to fill the 467 mL headspace with CO2. The headspace of the bioreactor was then sealed to allow for the accumulation of isoprene. GC analysis of headspace gases from transformant cultures was measured following 24 h incubation of the cultures in the light. 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.

[0169] Isoprene accumulation in the headspace of the gaseous aqueous two-phase bioreactor was measured by GC analysis after 196 hours of photoautotrophic growth.

Aliquots of 100% CO _? % ^r ere administered to the culture every 24 h to support

photoautotrophic growth and isoprene is presented as a cumulative yield over 196 hr.

Compared to the IspS-ApsbA2 control, the 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.

[0170] Biomass accumulation in cyanobaeterial strains carrying the different MVA super - operons (Fig. IS) was measured by dry cell weight (DCW) after 196 hours of

photoautotrophic growth, which was supported by aliquots of 100% CO? administered to the liquid culture every 24 h. The strains (e.g., lspS-ApsbA2; IspS-ANeu; SRAFKiDK?- ApsbA2vlspS-ANeu; SRA-ApsbA2 :FKiOK ₂ -AGlgA :lspS-ANeu; and SRA-

ApsbA2: :FK _\ O ₂ -AGlgX: 'IspS-ANeu) showed similar levels of biomass accumulation. The site of integration of the superoperon including the upper MVA pathway components and the lower MVA pathway components did not dramatically affect the dry cell weight of the biomass. [0171] Carbon-partitioning towards isoprene in cyanobaeterial strains carrying the MVA super-operons was measured (Fig. 16). The percentage of assimilated carbon that was diverted to isoprene, after 196 hours of photoautotrophic growth, is expressed as a ratio of the measured isoprene to half of the measured dry cell weight (about 50% of dry biomass is carbon). 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.

[0172] The kinetics of isoprene production and biomass accumulation in cyanobaeterial strains carrying the different MVA super-operons was measured. Aliquots of 100% C0 ₂ were administered to the culture every 24 h to support photoaittotrophic growth, after which the headspace of the bioreactor was sealed to allow for the accumulation of isoprene.

Cumulative isoprene production was measured over 196 h of photoautotrophic growth (Fig. 17A), and the corresponding biomass accumulation was measured as dry cell weight (Fig. 17B). The transformant strains expressing super-operons (e.g., SRA-ApsbA2: :FKiDK ₂ - AGlgA :l$pS-ANeu and SRA-ApsbA2 :FKiDK ₂ -AG% : -IspS-ANeu.) produced more isoprene compared to control transformants (e.g., SRAFKj ΌΚ2- ApshA2, and IspS-ANeu).

[0173] Western blot analysis of proteins from cyanobacterial strains showed the expression of the proteins encoded by the introduced genes of the MVA super-operons. The wild-type recipient Synechocystis strain (left lane) and a iransformant strain carrying the recombinant MV pathway super-operon (right lane) were probed with specific polyclonal antibodies for the MVA pathway enzymes including HmgS, HmgR, AtoB, Fni, MK, PM.D and PMK. The corresponding Coomasste stained gel (B) is shown as a protein loading control.

[0174] 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. Application of the mevalonic acid (MVA) pathway bypasses the regulated rate-limiting steps of the methylerythritoi (MEP) pathway and can improve the isoprene to biomass carbon partitioning ratio in cyanobacteria (e.g., Synechocystis) to 1 %, and even 10% isoprene-to-biomass (w:w), which is dramatically increased from 0.1%, as previously achieved (Bentley and Melts, (2012) Biotech. Bioeng. 109: 100: WO2012/145692).

VIST. Illustrative Sequences and Supplementary Materials TTR1 (Restriction site-RBS l-Spacer):

SEQ ID NO 1 (N) ₆ .. ₈ AGGAGG(N) ₆ ^' ₉

TIR2 (Restriction site-RBS2-Spacer):

SEQ ID NO 2 (N) ₆ - ₈ AAGGAG(N) ₆ _9

TTR3 (Restriction site-RBS l -Spacer):

SEQ ID NO 3 (N) ₆ . ₈ GGAGG(N) ₆ . ₉

TIR4 (Restriction site-RBS2-Spacer):

SEQ ID NO 4 (N) ₆ _ _S AGGAG(N) ₆ .9

TTR5 (Restriction site-RBS2~ Spacer):

SEQ ID NO 5 (N) ₆ .. ₈ AAGGA(N) ₆ .9 '

TIR6 (Restriction site-RBS2-Spacer):

SEQ ID NO 6 (N) ₆ . ₈ TAAGGAG(N) ₆ _9

TTR7 (Restriction site-RBS2-Spacer):

SEQ ID NO 7 (N) ₆ .8AAAGGAG(N) ₆ . ₉ TTR8 (Restriction site-RBS2-Spacer):

SEQ ID NO 8 (N) ₆ .8CAAGGAG(N) ₆ .9

TIR9 (Restriction site-RBS2-Spacer):

SEQ ID NO 9 (N) ₆ _8GAAGGAGfN) ₆ _9 TIR 10 (Restriction site-RBS I -Spacer) :

SEQ ID NO 10 (N) ₆ ..8AAGGAGG(N)6-9

TIR1 1 (Restriction site-RBS 1 -Spacer):

SEQ ID NO 1 1 (N) ₆ .8CAGGAGG(N) ₆ _9

TIR 12 (Restriction site-RBS 1 -Spacer):

SEQ ID NO 12 (N) _6- 8GAGGAGG(N) ₆ -9

TIR 13 (Restriction site-RBS 1 -Spacer):

SEQ ID NO 13 (N) ₆ -8TAGGAGG(N) ₆ -9

TIR 14 (Restriction site-RBS I -Spacer) :

SEQ ID NO 14 (N) ₆ ..8AAAGGAGG(N) ₆ .. ₉ TIR15 (Restriction site-RBS 1 -Spacer) :

SEQ ID NO 15 (N) ₆ _8CAAGGAGG(N) ₆ _ ₉

TIR 16 (Restriction site-RBS 1 -Spacer):

SEQ ID NO 16 (N) ₆ .8GAAGGAGG(N) ₆ .9

TIR17 (Restriction site-RBS 1 -Spacer):

SEQ ID NO 17 (N) ₆ _8TAAGGAGG(N) ₆ -9

MV . superoperon organization:

TIR-HMGS-TTR-HMGR-TIR-ATOB-TIR-FNI-TTR-MVK1-TIR-MVO-TIR-MVK2

HMGS = Hydroxy-methyl-glutaryl synthase

HMGR = Hydroxy-methyl-glutaryl reductase

ATOB AcetylcoA acetylase

FNI ^;;; IPP isomerase

MVK1 - Mevalonic acid kinase

MVD = Di-phospho-mevalonic acid decarboxylase

MVK2 = Phospho-raevalonic acid kinase

SEQ ID NO: 18 (TIR sequences SEQ ID NOs: 19-25 are underlined, and appear in order)

AAGAAGGAGATATACCATGGCAACAATTGGGATTGATAAAATTAGTTTTTTTGTGCC CCCTT

ATTATATTG^

GGTATTGGGCAAGACCAAATGGCGGTGAACCCAATCAGCCAAGATATTGTGACATTT GCAGC CAATGCCGCAGAAGCGATCTTGACCAAAGAAGATAAAGAGGCCATTGATATGGTGATTGT CG GGACTGAGTCCAGTATCGATGAGTCAAAAGCGGCCGCAGTTGTCTTACATCGTTTAATGG GG ATTCAACCTTTCGCTCGCTCTTTCGAAATCAAGGAAGCTTGTTACGGAGCAACAGCAGGC TT ACAGTTAGCTAAGAATCACGTAGCCTTACATCCAGATAAAAAAGTCTTGGTCGTAGCGGC AG ATATTGCAAAATATGGCTTAAATTCTGGCGGTGAGCCTACACAAGGAGCTGGGGCGGTTG CA ATG TAGTTGCTAGTGAA.CCGCGCATTTTGGCTTTAAAA.GAGGAT ATGTGATGCTGACGCA AGATATCTATGACTTTTGGCGTCCAACAGGCCACCCGTATCCTATGGTCGATGGTCCTTT GT CAAACGAAACCTACATCCAATCTTTTGCCCAAGTCTGGGATGAACATAAAAAACGAACCG GT CTTGATTTTGCAGATTATGATGCTTTAGCGTTCCATATTCCTTACACAAAAATGGGCAAA AA AGCCTTATTAGCAAAAATCTCCGACCAAACTGAAGCAGAACAGGAACGAATTTTAGCCCG TT ATGAAGAAAGTATCGTCTATAGTCGTCGCGTAGGAAACTTGTATACGGGTTCACTTTATC TG GGACTCATTTCCCTTTTAGAAAATGCAACGACTTTAACCGCAGGCAATCAAATTGGTTTA TT CAGTTATGGTTCTGGTGCTGTCGCTGAATTTTTCACTGGTGAATTAGTAGCTGGTTATCA AA ATCATTTACAAAAAGAAACTCATTTAGCACTGCTGGATAATCGGACAGAACTTTCTATCG CT GAATATGAAGCCATGTTTGCAGAAACTTTAGACACAGACATTGATCAAACGTTAGAAGAT GA ATTAAAATATAGTATTTCTGCTATTAATAATACCGTTCGTTCTTATCGAAACTAAGGATC CA GGAGGTAATATATGAAAACAGTAGTTATTATTGATGCATTACGAACACCAATTGGAAAAT AT AAAGGCAGCTTAAGTCAAGTAAGTGCCGTAGACTTAGGAACACATGTTACAACACAACTT TT AAAAAGACATTCCACTATTTCTGAAGAAATTGATCAAGTAATCTTTGGAAATGTTTTACA AG CTGGAAATGGCCAAAATCCCGCACGACAAATAGCAATAAACAGCGGTTTGTCTCATGAAA TT CCCGCAATGACGGTTAATGAGGTCTGCGGATCAGGAATGAAGGCCGTTATTTTGGCGAAA CA ATTGATTCAATTAGGAGAAGCGGAAGTTTTAATTGCTGGCGGGATTGAGAATATGTCCCA AG CACCTAAATTACAACGTTTTAATTACGAAACAGAAAGCTACGATGCGCCTTTTTCTAGTA TG ATGTATGATGGATTAACGGATGCCTTTAGTGGTCAGGCAATGGGCTTAACTGCTGAAAAT GT GGCCGAAAAGTATCATGTAACTAGAGAAGAGCAAGATCAATTTTCTGTACATTCACAATT AA AAGCAGCTCAAGCACAAGCAGAAGGGATATTCGCTGACGAAATAGCCCCATTAGAAGTAT CA GGAACGCTTGTGGAGAAAGATGAAGGGATTCGCCCTAATTCGAGCGTTGAGAAGCTAGGA AC GCTTAAAACAGTTTTTAAAGAAGACGGTACTGTAACAGCAGGGAATGCATCAACCATTAA TG ATGGGGCTTCTGCTTTGATTATTGCTTCACAAGAATATGCCGAAGCACACGGTCTTCCTT AT TTAGCTATTATTCGAGACAGTGTGGAAGTCGGTATTGATCCAGCCTATATGGGAATTTCG CC GATTAAAGCCATTCAAAAACTGTTAGCGCGCAATCAACTTACTACGGAAGAAATTGATCT GT ATGAAATCAACGAAGCATTTGCAGCAACTTCAATCGTGGTCCAAAGAGAACTGGCTTTAC CA GAGGAAAAGGTCAACATTTATGGTGGCGGTATTTCATTAGGTCATGCGATTGGTGCCACA GG TGCTCGTTTATTAACGAGTTTAAGTTATCAATTAAATCAAAAAGAAAAGAAATATGGAGT GG CTTCTTTATGTATCGGCGGTGGCTTAGGACTCGCTATGCTACTAGAGAGACCTCAGCAAA AA AAAAACAGCCGATTTTATCAAATGAGTCCTGAGGAACGCCTGGCTTCTCTTCTTAATGAA GG CCAGATTTCTGCTGATACAAAAAAAGAATTTGAAAATACGGCTTTATCTTCGCAGATTGC CA ATCATATGATTGAAAATCAAATCAGTGAAACAGAAGTGCCGATGGGCGTTGGCTTACATT TA ACAGTGGACGAAACTGATTATTTGGTACCAATGGCGACAGAAGAGCCCTCAGTTATTGCG GC TTTGAGTAATGGTGCAAAAATAGCACAAGGATTTAAAACAGTGAATCAACAACGCTTAAT GC GTGGACAAATCGTTTTTTACGATGTTGCAGATCCCGAGTCATTGATTGATAAACTACAAG TA AGAGAAGCGGAAGTTTTTCAACAAGCAGAGTTAAGTTATCCATCTATCGTTAAACGGGGC GG CGGCTTAAGAGATTTGCAATATCGTACTTTTGATGAATCATTTGTATCTGTCGACTTTTT AG TAGATGTTAAGGATGCAATGGGGGCAAATATCGTTAACGCTATGTTGGAAGGTGTGGCCG AG TTGTTCCGTGAATGGTTTGCGGAGCAAAAGATTTTATTCAGTATTTTAAGTAATTATGCC AC GGAGTCGGTTGTTACGATGAAAACGGCTATTCCAGTTTCACGTTTAAGTAAGGGGAGCAA TG GCCGGGAAATTGCTGAAAAAATTGTTTTAGCTTCACGCTATGCTTCATTAGATCCTTATC GG GCAGTCACGCATAACAAAGGAATCATGAATGGCATTGAAGCTGTAGTTTTAGCTACAGGA AA TGATACACGCGCTGTTAGCGCTTCTTGTCATGCTTTTGCGGTGAAGGAAGGTCGCTACCA AG GCTTGACTAGTTGGACGCTGGATGGCGAACAACTAATTGGTGAAATTTCAGTTCCGCTTG CT TTAGCCACGGTTGGCGGTGCCACAAAAGTCTTACCTAAATCTCAAGCAGCTGCTGATTTG TT AGCAGTGACGGATGCAAAAGAACTAAGTCGAGTAGTAGCGGCTGTTGGTTTGGCACAAAA TT TAGCGGCGTTACGGGCCTTAGTCTCTGAAGGAATTCAAAAAGGACACATGGCTCTACAAG CA CGTTCTTTAGCGATGACGGTCGGAGCTACTGGTAAAGAAGTTGAGGCAGTCGCTCAACAA TT AAAACGTCAAAAAACGATGAACCAAGACCGAGCCATGGCTATTTTAAATGATTTAAGAAA AC AATAAGAGCTCAGGAGGTAATATATGAAAAATTGTGTCATCGTCAGTGCGGTACGTACTG CT ATCGGTAGTTTT AOT

TAAAGCCGCCATTGAACGTGCAAAAATCGATTCACAACACGTTGATGAAGTGATTAT GGGTA ACGTGTTACAAGCCGGGCTGGGGCAAAATCCGGCGCGTCAGGCACTGTTAAAAAGCGGGC TG GCAGAAACGGTGTGCGGATTCACGGTCAATAAAGTATGTGGTTCGGGTCTTAAAAGTGTG GC GCTTGCCGCCCAGGCCATTCAGGCAGGTCAGGCG CAGAGCATTGTGGCGGGGGGTATGGAAA ATATGAGTTTAGCCCCCTACTTACTCGATGCAAAAGCACGCTCTGGTTATCGTCTTGGAG AC GGACAGGTTTATGACGTAATCCTGCGCGATGGCCTGATGTGCGCCACCCATGGTTATCAT AT GGGGATTACCGCCGAAAACGTGGCTAAAGAGTACGGAATTACCCGTGAAATGCAGGATGA AC TGGCGCTACATTCACAGCGTAAAGCGGCAGCCGCAATTGAGTCCGGTGCTTTTACAGCCG AA ATCGTCCCGGTAAATGTTGTCACTCGAAAGAAAACCTTCGTCTTCAGTCAAGACGAATTC CC GAAAGCGAATTCAACGGCTGAAGCGTTAGGTGCATTGCGCCCGGCCTTCGATAAAGCAGG AA CAGTCACCGCTGGGAACGCGTCTGGTATTAACGACGGTGCTGCCGCTCTGGTGATTATGG AA GAATCTGCGGCGCTGGCAGCAGGCCTTACCCCCCTGGCTCGCATTAAAAGTTATGCCAGC GG TGGCGTGCCCCCCGCATTGATGGGTATGGGGCCAGTACCTGCCACGCAAAAAGCGTTACA AC TGGCGGGGCTGCAACTGGCGGATATTGATCTCATTGAGGCTAATGAAGCATTTGCTGCAC AG TTCCTTGCCGTTGGGAAAAACCTGGGCTTTGATTCTGAGAAAGTGAATGTCAACGGCGGG GC CAT CG CG C T CGG G CAT C C T AT CG G TG C C AG TG G T G C T CG T AT T C TG G T C AC AC T AT T A C ATG CCATGCAGG CACGCGATAAAACG CTGGGGCTGGCAACACTGTGCATTGGCGGCGGTCAGGGA AT TG C G ATG G TG AT TG AAC GG T T G AAT T AAAG C T TG C G G C C G C A C T CG AG AG AAG GAG AT AT ACCATGGCAACGACAAATCGTAAGGACGAGCATATCCTCTATGCCCTTGAGCAGAAAAGT TC C TAT AAT AG C T T TG AT G AG G T GG AG C TG AT T CAT TCTTCCTTGCCTCTT T A C AAT C TG G ATG AAATCGATCTTTCGACAGAGTTTGCTGGTCGAAAGTGGGACTTTCCTTTTTATATCAATG CC ATGACTGGTGGAAGTAATAAGGGAAGAGAAATCAATCAAAAGCTGGCTCAGGTGGCGGAA AC CTGTGGTATTTTATTTGTAACGGGTTCTTATAGCGCAGCCCTCAAAAATCCAACGGATGA TT CTTTTTCTGTCAAGTCTAGTCATCCCAATCTCCTCCTTGGAACCAATATTGGATTGGACA AG CCTGTCGAGTTAGGACTTCAGACTGTAGAAGAGATGAATCCTGTTCTATTGCAAGTGCAT GT CAATGTCATGCAGGAATTACTCATGCCCGAGGGAGAAAGGAAGTTTAGAAGCTGGCAATC GC ATCTAGCAGATTATAGCAAGCAAATTCCCGTTCCTATTGTCCTCAAGGAAGTGGGCTTTG GA ATGGATGCCAAGACAATCGAAAGAGCCTATGAATTCGGTGTTCGTACAGTGGACCTATCG GG TCGTGGTGGCACCAGCTTTGCCTATATCGAAAACCGTCGTAGTGGCCAGCGTGATTACCT CA ATCAATGGGGTCAGTCTACCATGCAGGCCCTTCTCAATGCCCAAGAATGGAAAGATAAGG TC GAACTCTTGGTTAGTGGAGGGGTTCGGAATCCGCTGGATATGATTAAGTGCTTGGTTTTT GG TGCTAAGGCTGTGGGATTGTCACGAACCGTTCTGGAATTGGTTGAAACCTACACAGTTGA AG AAGTGATTGGCATTGTCCAAGGCTGGAAAGCAGATCTACGCTTGATTATGTGTTCCCTTA AC TGTGCCACCATAGCAGATCTACAAAAAGTAGACTATCTTCTTTATGGAAAATTAAAAGAA GC AAATGATCAGATGAAAAAGGCGTAAGGATCCAGGAGGTAATATATGACAAAAAAAGTTGG TG TCGGTCAGGCACATAGTAAGATAATTTTAATAGGGGAACATGCGGTCGTTTACGGTTATC CT GCCATTTCCCTGCCTCTTTTGGAAGTGGAGGTGACCTGTAAGGTAGTTCCTGCAGAGAGT CC TTGGCGCCTTTATGAGGAGGATACCTTGTCCATGGCGGTTTATGCCTCACTGGAGTATTT GA AT AT C AC AG AAG C C TG CAT T C G T TG T G AG AT T G A C T C G G C T AT C C C TG AG AAA CG GGG G ATG GGTTCGTCAGCGGCTATCAGCATAGCGGCCATTCGTG CGGTATTTGAC'TACTATCAGGCTGA TCTGCCTCATGATGTACTAGAAATCTTGGTCAATCGAGCTGAAATGATTGCCCATATGAA TC CTAGTGGTTTGGATGCTAAGACCTGTCTCAGTGACCAACCTATTCGCTTTATCAAGAACG TA GGATTTACAGAACTTGAGATGGATTTATCCGCCTATTTGGTGATTGCCGATACGGGTGTT TA TGGTCATACTCGTGAAGCCATCCAAGTGGTTCAAAATAAGGGCAAGGATGCCCTACCGTT TT TGCATGCCTTGGGAGAATTAACCCAGCAGGCAGAAATTGCGATTTCACAAAAAGATGCTG AA GGGCTGGGACAAATCCTCAGTCAAGCACATTTACATTTAAAAGAAATTGGTGTCAGTAGC CT TGAGGCAGACTCTTTGGTTGAAACAGCTCTTAGTCATGGTGCTCTGGGTGCCAAGATGAG CG GTGGTGGGCTAGGAGGTTGTATCATAGCCTTGGTAACCAATTTGACACACGCACAAGAAC TA GCAGAAAGATTAGAAGAGAAAGGAGCTGTTCAGACATGGATAGAGAGCCTGTGAGAGCTC AG GAGGTAATATATGTATCATAGCCTTGGTAACCAATTTGACACACGCACAAGAACTAGCAG AA AGATTAG AGAGAAAGGAGCTGTTCAGACATGGATAGAGAGCCTGTGACAGTACGTTCCTAC GCAAATATTGCTATTATCAAATATTGGGGAAAGAAAAAAGAAAAAGAGATGGTGCCTGCT AC TAGCAGTATTTCTCTAACTTTGGAAAATATGTATACAGAGACGACCTTGTCGCCTTTACC AG CCAATGTAACAGCTGACGAATTTTACATCAATGGTCAGCTACAAAATGAGGTCGAGCATG CC AAGATGAGTAAGATTATTGACCGTTATCGTCCAGCTGGTGAGGGCTTTGTCCGTATCGAT AC TCAAAACAATATGCCTACGGCAGCGGGCCTGTCCTCAAGTTCTAGTGGTTTGTCCGCCCT GG TCAAGGCTTGTAATGCTTATTTCAAGCTTGGATTGGATAGAAGTCAGTTAGCGCAGGAAG CC AAGTTTGCCTCAGGCTCTTCTTCTCGGAGTTTTTATGGACCACTAGGAGCCTGGGATAAG GA TAGTGGAGAAATTTACCCTGTAGAGACAGACTTGAAACTAGCTATGATTATGTTGGTGCT AG AGGACAAGAAAAAACCAATCTCTAGCCGTGACGGGATGAAACTTTGTGTGGAAACCTCGA CG ACTTTCGACGACTGGGTTCGTCAGTCTGAGAAGGACTATCAGGATATGCTGATTTATCTC AA GGAAAATGATTTTGCCAAGATTGGAGAATTAACGGAGAAAAATGCCCTGGCTATGCATGC TA CGACAAAGACTGCTAGTCCAGCCTTTTCTTATCTGACGGATGCCTCTTATGAGGCTATGG AC TTTGTTCGTCAGCTTCGTGAGAAAGGAGAGGCCTGCTACTTTACCATGGATGCTGGTCCC AA TGTTAAGGTCTTCTGTCAGGAGAAAGACTTGGAGCATTTGTCAGAAATTTTCGGTCAGCG TT ATCGCTTGATTGTGTCAAAAACAAAGGATTTGAGTCAAGATGATTGCTGTTAAGTCGACA GG AGGTAATATATGATTGCTGTTAAAACTTGCGGAAAACTCTATTGGGC'AGGTGAATATGC TAT TTTAGAGCCAGGGCAGTTAGCTTTGATAAAGGATATTCCCATCTATATGAGGGCTGAGAT TG CTTTTTCTGACAGCTACCGTATCTATTCAGATATGTTTGATTTCGCAGTGGACTTAAGGC CC AATCCTGACTACAGCTTGATTCAAGAAACGATTGCTTTGATGGGAGACTTCCTCGCTGTT CG CGGTCAGAATTTAAGACCTTTTTCTCTAGCAATCTATGGCAAAATGGAACGAGAAGGGAA AA AGTTTGGTCTAGGTTCTAGTGGCAGCGTCGTTGTCTTGGTTGTCAAGGCTTTACTGGCTC TC TATAATCTTTCGGTTGATCAGAATCTCTTGTTCAAGCTGACTAGTGCTGTCTTGCTTAAG CG AGGAGACAATGGTTCCATGGGCGACCTTGCCTGTATTGCGGCAGAGGATTTGGTTCTCTA CC AGTCATTTGATCGCCAGAAGGTGGCTGCTTGGTTAGAAGAAGAAAACTTGGCGACAGTTC TG GAGCGTGATTGGGGATTTTCAATTTCACAAGTGAAACCAACTTTAGAATGTGATTTCTTA GT GGGATGGACCAAGGAAGTGGCTGTATCGAGTCACATGGTCCAGCAAATCAAGCAAAATAT CA ATCAAAATTTTTTAACTTCCTCAAAAGAAACGGTGGTTTCTTTGGTCGAAGCCTTGGAAC AG GGGAAATCAGAAAAGATTATCGAGCAAGTAGAAGTAGCCAGCAAGCTTTTAGAAGGCTTG AG TACAGATATTTACACGCCTTTGCTTAGACAGTTGAAAGAAGCCAGTCAAGATTTGCAGGC CG TTGCCAAGAGTAGTGGTGCTGGTGGTGGTGACTGTGGCATCGCCCTGAGTTTTGATGCGC AA TCAACCAAAACCTTAAAAAATCGTTGGGCCGATCTGGGGATTGAGCTCTTATATCAAGAA AG GATAGGACATGACGACAAATCGTAA HMGS from E.fiiecalis:

SEQ ID NO: 26 nucleic acid sequence

ATGGCAACAATTGGGATTGATAAAATTAGTTTTTTTGTGCCCCCTTATTATATTGATATG AC GGCACTGGCTGAAGCCAGAAATGTAGACCCTGGAAAATTTCATATTGGTATTGGGCAAGA CC AAATGGCGGTGAACCCAATCAGCCAAGATATTGTGACATTTGCAGCCAATGCCGCAGAAG CG ATCTTGACCAAAGAAGATAAAGAGGCCATTGATATGGTGATTGTCGGGACTGAGTCCAGT AT CGATGAGTCAAAAGCGGCCGCAGTTGTCTTACATCGTTTAATGGGGATTCAACCTTTCGC TC GCTCTTTCGAAATCAAGGAAGCTTGTTACGGAGCAACAGCAGGCTTACAGTTAGCTAAGA AT CACGTAGCCTTACATCCAGATAAAAAAGTCTTGGTCGTAGCGGCAGATATTGCAAAATAT GG CTTAAATTCTGGCGGTGAGCCTACACAAGGAGCTGGGGCGGTTGCAATGTTAGTTGCTAG TG AACCGCGCATTTTGGCTTTAAAAGAGGATAATGTGATGCTGACGCAAGATATCTATGACT TT TGGCGTCCAACAGGCCACCCGTATCCTATGGTCGATGGTCCTTTGTCAAACGAAACCTAC AT CCAATCTTTTGCCCAAGTCTGGGATGAACATAAAAAACGAACCGGTCTTGATTTTGCAGA TT ATGATGCTTTAGCGTTCCATATTCCTTACACAAAAATGGGCAAAAAAGCCTTATTAGCAA AA ATCTCCGACCAAACTGAAGCAGAACAGGAACGAATTTTAGCCCGTTATGAAGAAAGTATC GT CTATAGTCGTCGCGTAGGAAACTTGTATACGGGTTCACTTTATCTGGGACTCATTTCCCT TT TAGAAAATGCAACGACTTTAACCGCAGGCAATCAAATTGGTTTATTCAGTTATGGTTCTG GT GCTGTCGCTGAATTTTTCACTGGTGAATTAGTAGCTGGTTATCAAAATCATTTACAAAAA GA AACTCATTTAGCACTGCTGGATAATCGGACAGAACTTTCTATCGCTGAATATGAAGCCAT GT TTGCAGAAACTTTAGACACAGACATTGATCAAACGTTAGAAGATGAATTAAAATATAGTA TT TCTGCTATTAATAATACCGTTCGTTCTTATCGAAACTAA SEQ ID NO: 27 amino acid sequence

MATIGIDKI SFFVPPYYIDMTALAEARI DPGKFHIGIGQDQMAVNPI SQDIVTFAANAAEA ILTKED EAIDMVIVGTESS I DESKAAAWLHRLMGI QPFARSFEI KEACYGATAGLQLAKN HVALHPD KVLWAADIAKYGL SGGEPTQGAGAVA lLVASEPRILALKEDISrV LTQDIYDF RPTGHPYPMVDGPLS ETYI QSFAQV DEHKKRTGLDFADYDALAFHI PYTKMGKKALLAK I SDQTEAEQERI LARYEES IVYSRRVGNLYTGSLYLGLI SLLE ATTLTAGNQIGLFSYGSG AVAEFFTGELVAGYQNHLQKETHLALLDNRTELS IAEYEAMFAETLDTDIDQTLEDELKYS I SAI TVRS YR HMGR bom R faecalis:

SEQ ID NO:28 nucleic acid sequence

ATGAAAACAGTAGTTATTATTGATGCATTACGAACACCAATTGGAAAATATAAAGGCAGC TT AAGTCAAGTAAGTGCCGTAGACTTAGGAACACATGTTACAACACAACTTTTAAAAAGACA TT CCACTATTTCTGAAGAAATTGATCAAGTAATCTTTGGAAATGTTTTACAAGCTGGAAATG GC CAAAATCCCGCACGACAAATAGCAATAAACAGCGGTTTGTCTCATGAAATTCCCGCAATG AC GGTTAATGAGGTCTGCGGATCAGGAATGAAGGCCGTTATTTTGGCGAAACAATTGATTCA AT TAGGAGAAGCGGAAGTTTTAATTGCTGGCGGGATTGAGAATATGTCCCAAGCACCTAAAT TA CAACGTTTTAATTACGAAACAGAAAGCTACGATGCGCCTTTTTCTAGTATGATGTATGAT GG ATTAACGGATGCCTTTAGTGGTCAGGCAATGGGCTTAACTGCTGAAAATGTGGCCGAAAA GT ATCATGTAACTAGAGAAGAGCAAGATCAATTTTCTGTACATTCACAATTAAAAGCAGCTC AA GCACAAGCAGAAGGGATATTCGCTGACGAAATAGCCCCATTAGAAGTATCAGGAACGCTT GT GGAGAAAGATGAAGGGATTCGCCCTAATTCGAGCGTTGAGAAGCTAGGAACGCTTAAAAC AG TTTTTAAAGAAGACGGTACTGTAACAGCAGGGAATGCATCAACCATTAATGATGGGGCTT CT GCTTTGATTATTGCTTCACAAGAATATGCCGAAGCACACGGTCTTCCTTATTTAGCTATT AT TCGAGACAGTGTGGAAGTCGGTATTGATCCAGCCTATATGGGAATTTCGCCGATTAAAGC CA TTCAAAAACTGTTAGCGCGCAATCAACTTACTACGGAAGAAATTGATCTGTATGAAATCA AC GAAGCATTTGCAGCAACTTCAATCGTGGTCCAAAGAGAACTGGCTTTACCAGAGGAAAAG GT CAACATTTATGGTGGCGGTATTTCATTAGGTCATGCGATTGGTGCCACAGGTGCTCGTTT AT TAACGAGTTTAAGTTATCAATTAAATCAAAAAGAAAAGAAATATGGAGTGGCTTCTTTAT GT ATCGGCGGTGGCTTAGGACTCGCTATGCTACTAGAGAGACCTCAGCAAAAAAAAAACAGC CG ATTTTATCAAATGAGTCCTGAGGAACGCCTGGCTTCTCTTCTTAATGAAGGCCAGATTTC TG CTGATACAAAAAAAGAATTTGAAAATACGGCTTTATCTTCGCAGATTGCCAATCATATGA TT GAAAATCAAATCAGTGAAACAGAAGTGCCGATGGGCGTTGGCTTACATTTAACAGTGGAC GA AACTGATTATTTGGTACCAATGGCGACAGAAGAGCCCTCAGTTATTGCGGCTTTGAGTAA TG GTGCAAAAATAGCACAAGGATTTAAAACAGTGAATCAACAACGCTTAATGCGTGGACAAA TC GTTTTTTACGATGTTGCAGATCCCGAGTCATTGATTGATAAACTACAAGTAAGAGAAGCG GA AGTTTTTCAACAAGCAGAGTTAAGTTATCCATCTATCGTTAAACGGGGCGGCGGCTTAAG AG ATTTGCAATATCGTACTTTTGATGAATCATTTGTATCTGTCGACTTTTTAGTAGATGTTA AG GATGCAATGGGGGCAAATATCGTTAACGCTATGTTGGAAGGTGTGGCCGAGTTGTTCCGT GA ATGGTTTGCGGAGCAAAAGATTTTATTCAGTATTTTAAGTAATTATGCCACGGAGTCGGT TG TTACGATGAAAACGGCTATTCCAGTTTCACGTTTAAGTAAGGGGAGCAATGGCCGGGAAA TT GCTGAAAAAATTGTTTTAGCTTCACGCTATGCTTCATTAGATCCTTATCGGGCAGTCACG CA TAACAAAGGAATCATGAATGGCATTGAAGCTGTAGTTTTAGCTACAGGAAATGATACACG CG CTGTTAGCGCTTCTTGTCATGCTTTTGCGGTGAAGGAAGGTCGCTACCAAGGCTTGACTA GT TGGACGCTGGATGGCGAACAACTAATTGGTGAAATTTCAGTTCCGCTTGCTTTAGCCACG GT TGGCGGTGCCACAAAAGTCTTACCTAAATCTCAAGCAGCTGCTGATTTGTTAGCAGTGAC GG ATGCAAAAGAACTAAGTCGAGTAGTAGCGGCTGTTGGTTTGGCACAAAATTTAGCGGCGT TA CGGGCCTTAGTCTCTGAAGGAATTCAAAAAGGACACATGGCTCTACAAGCACGTTCTTTA GC GATGACGGT CGG AG CT CTGGTAAAG AAGTTG AGGCAG CG CTC AACAATT AAAACGT CAAA AAACGATGAACCAAGACCGAGCCATGGCTATTTTAAATGATTTAAGAAAACAATAA SEQ ID NO: 29 amino acid sequence

MKTWIIDALRTPIGKYKGSLSQVSAVDLGTHVTTQLLKRHSTISEEIDQVIFG VLQAGNG QNPARQIAINSGLSHEIPAMTWEVCGSGMKJ ILAKQLIQLGEAEVLIAGGIEKMSQAPKL QRFNYETESYDAP SSMMYDGLTDAFSGQAMGLTAENVAE YHVTREEQDQFSVHSQLKi¾J^Q AQAEGIFADEIAPLEVSGTLVEKDEGIRPNSSVEKLGTLKTVFKEDGTVTAG ASTINDGAS AL1 IASQEYAEAHGLPYLAI IRDSVEVGIDPAYMGISPIKA QKLLARNQLTTEEIDLYEIN EAFAATSIWQRELALPEEKVNIYGGGISLGHAIGATGARLLTSLSYQLNQKEKKYGVASL C IGGGLGLAMLLERPQQKKNSRFYQ SPEERLASLLNEGQISADTK EFENTALSSQIANHMI ENQISETEVPMGVGLHLTVDETDYLVPMATEEPSVIAALSNGAKIAQGFKTVNQQRLMRG QI VFYDVADPESLIDKLQVREAEVFQQAELSYPSIVKRGGGLRDLQYRTFDESFVSVDFLVD VK DA GANIWANILEGVAELFRE FAEQKILFSILSNYATESVVTMKTAIPVSRLSKGSNGREI AEKIVLASRYASLDPYRAVTHNKGIMNGIEAWLATGNDTRAVSASCHAFAVKEGRYQGLT S WTLDGEQLIGEISVPLALATVGGATKVLPKSQAAADLLAYTDAKELSRWAAVGLAQNLAA L RALYSEGIQKGHMALQARSLAMTVGATGKEVEAVAQQLKRQKTMNQDRAi^AILNDLRKQ

ATOBfrom.ff.cofc

SEQ ID NO: 30 nucleic acid sequence

ATGAAAAATTGTGTCATCGTCAGTGCGGTACGTACTGCTATCGGTAGTTTTAACGGTTCA CT CGCTTCCACCAGCGCCATCGACCTGGGGGCGACAGTAATTAAAGCCGCCATTGAACGTGC AA AAATCGATTCACAACACGTTGATGAAGTGATTATGGGTAACGTGTTACAAGCCGGGCTGG GG CAAAATCCGGCGCGTCAGGCACTGTTAAAAAGCGGGCTGGCAGAAACGGTGTGCGGATTC AC GGTCAATAAAGTATGTGGTTCGGGTCTTAAAAGTGTGGCGCTTGCCGCCCAGGCCATTCA GG CAGGTCAGGCGCAGAGCATTGTGGCGGGGGGTATGGAAAATATGAGTTTAGCCCCCTACT TA CTCGATGCAAAAGCACGCTCTGGTTATCGTCTTGGAGACGGACAGGTTTATGACGTAATC CT GCGCGATGGCCTGATGTGCGCCACCCATGGTTATCATATGGGGATTACCGCCGAAAACGT GG CTAAAGAGTACGGAATTACCCGTGAAATGCAGGATGAACTGGCGCTACATTCACAGCGTA AA GCGGCAGCCGCAATTGAGTCCGGTGCTTTTACAGCCGAAATCGTCCCGGTAAATGTTGTC AC TCGAAAGAAAACCTTCGTCTTCAGTCAAGACGAATTCCCGAAAGCGAATTCAACGGCTGA AG CGTTAGGTGCATTGCGCCCGGCCTTCGATAAAGCAGGAACAGTCACCGCTGGGAACGCGT CT GGTATTAACGACGGTGCTGCCGCTCTGGTGATTATGGAAGAATCTGCGGCGCTGGCAGCA GG CCTTACCCCCCTGGCTCGCATTAAAAGTTATGCCAGCGGTGGCGTGCCCCCCGCATTGAT GG GTATGGGGCCAGTACCTGCCACGCAAAAAGCGTTACAACTGGCGGGGCTGCAACTGGCGG AT ATTGATCTCATTGAGGCTAATGAAGCATTTGCTGCACAGTTCCTTGCCGTTGGGAAAAAC CT GGGCTTTGATTCTGAGAAAGTGAATGTCAACGGCGGGGCCATCGCGCTCGGGCATCCTAT CG GTGCCAGTGGTGCTCGTATTCTGGTCACACTATTACATGCCATGCAGGCACGCGATAAAA CG CTGGGGCTGGCAACACTGTGCATTGGCGGCGGTCAGGGAATTGCGATGGTGATTGAACGG TT GAATTAA SEQ ID NO: 31 amino acid sequence

MKNCVIVSAVRTAIGSFNGSLASTSAIDLGATVIKAAIERAKIDSQHVDEVI GNVLQAGLG QNPARQALLKSGLAETVCOFT KVCGSGLKSVALAAQAIQAGQAQSIVAGGHENMSLAPYL LDAKARSGYRLGDGQVYDVILRDGLMCATHGYHMGITAENVAKEYGITRE QDELALHSQRK AAAAIESGAFTAEIVP \A, ^r TRKKTFVFSQDEFPKANSTAEALGALRPAFDKAGTVTAGNAS GI DGAAALVIΜΞΕSAALAAGLTPLARIKSYASGGVPPALMGMGPYPATQKALQLAGLQL AD IDLIEANEAFAAQFLAVGKNLGFDSEKVWVNGGAIALGHPIGASGARILVTLLHAMQARD KT LGLATLCIGGGQGIAMVIERLN

MVK ΐ from S. pneumoniae R6

SEQ ID NO:32 nucleic acid sequence

ATGACAAAAAAAGTTGGTGTCGGTCAGGCACATAGTAAGATAATTTTAATAGGGGAACAT GC GGTCGTTTACGGTTATCCTGCCATTTCCCTGCCTCTTTTGGAAGTGGAGGTGACCTGTAA GG TAGTTCCTGCAGAGAGTCCTTGGCGCCTTTATGAGGAGGATACCTTGTCCATGGCGGTTT AT GCCTCACTGGAGTATTTGAATATCACAGAAGCCTGCATTCGTTGTGAGATTGACTCGGCT AT CCCTGAGAAACGGGGGATGGGTTCGTCAGCGGCTATCAGCATAGCGGCCATTCGTGCGGT AT TTGACTACTATCAGGCTGATCTGCCTCATGATGTACT GAAATCTTGGTCAATCGAGCTGAA ATGATTGCCCATATGAATCCTAGTGGTTTGGATGCTAAGACCTGTCTCAGTGACCAACCT AT TCGCTTTATCAAGAACGTAGGATTTACAGAACTTGAGATGGATTTATCCGCCTATTTGGT GA TTGCCGATACGGGTGTTTATGGTCATACTCGTGAAGCCATCCAAGTGGTTCAAAATAAGG GC AAGGATGCCCTACCGTTTTTGCATGCCTTGGGAGAATTAACCCAGCAGGCAGAAATTGCG AT TTCACAAAAAGATGCTGAAGGGCTGGGACAAATCCTCAGTCAAGCACATTTACATTTAAA AG AAATTGGTGTCAGTAGCCTTGAGGCAGACTCTTTGGTTGAAACAGCTCTTAGTCATGGTG CT CTGGGTGCCAAGATGAGCGGTGGTGGGCTAGGAGGTTGTATCATAGCCTTGGTAACCAAT TT GACACACGCACAAGAACTAGCAGAAAGATTAGAAGAGAAAGGAGCTGTTCAGACATGGAT AG AGAGCCTGTGA SEQ ID NO: 33 amino acid sequence

MTKKVGVGQAHSKI ILIGEHA\A/YGYPAI SLPLLEVEVTCKVVPAESPVv ⁷ RLYEEDTLS AVY ASLE YLNI TEAC I RCE I DS AI PEKRGMGSS AAI S I AAI RAVFDY YQADLPHDVLE I LVNRAE MI HMNPSGLDAKTCLSDQPI RFI K VGFTELEMDLSAYLVIADTGVYGHTREAI QWQNKG KDALPFLHALGELTQQAEIAI SQKDAEGLGQI LSQAHLHLKEIGVSSLEADSLYETALSHGA LGAKMSGGGLGGCI I ALYTMLTH AQE LAERLE E KGAV QT I Ξ S L

MVP from S. pneumoniae R6:

SEQ ID NO:34 nucleic acid sequence

ATGTATCATAGCCTTGGTAACCAATTTGACACACGCACAAGAACTAGCAGAAAGATTAGA AG AGAAAGGAGCTGTTCAGACATGGATAGAGAGCCTGTGACAGTACGTTCCTACGCAAATAT TG CTATTATCAAATATTGGGGAAAGAAAAAAGAAAAAGAGATGGTGCCTGCTACTAGCAGTA TT TCTCTAACTTTGGAAAATATGTATACAGAGACGACCTTGTCGCCTTTACCAGCCAATGTA AC AGCTGACGAATTTTACATCAATGGTCAGCTACAAAATGAGGTCGAGCATGCCAAGATGAG TA AGATTATTGACCGTTATCGTCCAGCTGGTGAGGGCTTTGTCCGTATCGATACTCAAAACA AT ATGCCTACGGCAGCGGGCCTGTCCTCAAGTTCTAGTGGTTTGTCCGCCCTGGTCAAGGCT TG TAATGCTTATTTCAAGCTTGGATTGGATAGAAGTCAGTTAGCGCAGGAAGCCAAGTTTGC CT CAGGCTCTTCTTCTCGGAGTTTTTATGGACCACTAGGAGCCTGGGATAAGGATAGTGGAG AA ATTTACCCTGTAGAGACAGACTTGAAACTAGCTATGATTATGTTGGTGCTAGAGGACAAG AA AAAACCAATCTCTAGCCGTGACGGGATGAAACTTTGTGTGGAAACCTCGACGACTTTCGA CG ACTGGGTTCGTCAGTCTGAGAAGGACTATCAGGATATGCTGATTTATC'TCAAGGAAAAT GAT TTTGCCAAGATTGGAGAATTAACGGAGAAAAATGCCCTGGCTATGCATGCTACGAC AAGAC TGCTAGTCCAGCCTTTTCTTATCTGACGGATGCCTCTTATGAGGCTATGGACTTTGTTCG TC AGCTTCGTGAGAAAGGAGAGGCCTGCTACTTTACCATGGATGCTGGTCCCAATGTTAAGG TC TTCTGTCAGGAGAAAGACTTGGAGCATTTGTCAGAAATTTTCGGTCAGCGTTATCGCTTG AT TGTGTCAAAAACAAAGGATTTGAGTCAAGATGATTGCTGTTAA

SEQ I D NO : 35 amino acid sequence

YHSLGNQFDTRTRTS RKI RRERS CS DMDRE PVTVRS Y ANI AI I KYWGKKKEKEMVPATS S I SLTLENMYTETTLS PLPAIWTADEFYINGQLQNEVEHAKMSKI I DRYRPAGEGFVRIDTQNN MPTAAGLSSSSSGLSALVKACNAYFKLGLDRSQLAQEAKFASGSSSRSFYGPLGAWDKDS GE

lYPVETDLKL ilMLVLEDKKKPI SSRDGMKLCVETSTTFDDWVRQSE DYQDMLIYLKEND FAKIGELTEKNALAMHATT TAS PAFSYLTDASYEA31DFVRQLREKGEACY FTMDAGPNVKV FCQEKDLEHLSE I FGQRYRLIVSKTKDLSQDDCC MVK2 from S. pneumoniae R6:

SEQ ID NO: 36 nucleic acid sequence

ATGATTGCTGTTAAAACTTGCGGAAAACTCTATTGGGCAGGTGAATATGCTATTTTAGAG CC AGGGCAGTTAGCTTTGATAAAGGATATTCCCATCTATATGAGGG CTGAGATTGCTTTTTCTG ACAGCTACCGTATCTATTCAGATATGTTTGATTTCGCAGTGGACTTAAGGCCCAATCCTG AC TACAGC'TTGATTC'AAGAAAC'GATTGCTTTGATGGGAGACTTCCTCGCTGTTCGCGGT CAGAA TTTAAGACCTTTTTCTCTAGCAATCTATGGCAAAATGGAACGAGAAGGGAAAAAGTTTGG TC TAGGTTCTAGTGGCAGCGTCGTTGTCTTGGTTGTCAAGGCTTTACTGGCTCTCTATAATC TT TCGGTTGATCAGAATCTCTTGTTCAAGCTGACTAGTGCTGTCTTGCTTAAGCGAGGAGAC AA TGGTTCCATGGGCGACCTTGCCTGTATTGCGGCAGAGGATTTGGTTCTCTACCAGTCATT TG ATCGCCAGAAGGTGGCTGCTTGGTTAGAAGAAGAAAACTTGGCGACAGTTCTGGAGCGTG AT TGGGGATTTTCAATTTCACAAGTGAAACCAACTTTAGAATGTGATTTCTTAGTGGGATGG AC CAAGGAAGTGGCTGTATCGAGTCACATGGTCCAGCAAATCAAGCAAAATATCAATCAAAA TT TTTTAACTTCCTCAAAAGAAACGGTGGTTTCTTTGGTCGAAGCCTTGGAACAGGGGAAAT CA GAAAAGATTATCGAGCAAGTAGAAGTAGCCAGCAAGCTTTTAGAAGGCTTGAGTACAGAT AT TTACACGCCTTTGCTTAGACAGTTGAAAGAAGCCAGTCAAGATTTGCAGGCCGTTGCCAA GA GTAGTGGTGCTGGTGGTGGTGACTGTGGCATCGCCCTGAGTTTTGATGCGCAATCAACCA AA ACCTTAAAAAATCGTTGGGCCGATCTGGGGATTGAGCTCTTATATCAAGAAAGGATAGGA CA TGACGACAAATCGTAA

SEQ I D NO : 3 7 amino ac id sequence

MIAYKTCGKLY AGEYAI LEPGQLAL I KDI PI YMRAE IAFSDSYRI YSDMFDFAVDLRPNPD YSLI QET I LMGDFLAVRGQ LRPFSLAI YGKMEREG KKFGLGS SGSVVVLVVKALLALYNL SVDQNLLFKLTSAVLLKRGDNGSMGDIJACIAAEDLVLYQSFDRQKVAA LEEENLATVLERD G FS I SQVKPTLECDFLVG TKEVAVSSHMVQQI KQNINQNFLTSS KETWSLVEALEQGKS BKI I EQVBVASKLLBGLSTDI YTPLLRQLKBASQDLQAVAKSSGAGGGDCGIALSFDAQSTK TL KNRWADLG I E LLYQER I GHDD KS

FNI (IPP isomerase) from S. pneumoniae R6:

SEQ ID NO:38 nucleic acid sequence

ATGACGACAAATCGTAAGGACGAGCATATCCTCTATGCCCTTGAGCAGAAAAGTTCCTAT AA TAGCTTTGATGAGGTGGAGCTGATTCATTCTTCCTTGCCTCTTTACAATCTGGATGAAAT CG ATCTTTCGACAGAGTTTGCTGGTCGAAAGTGGGACTTTCCTTTTTATATCAATGCCATGA CT GGTGGAAGTAATAAGGGAAGAGAAATCAATCAAAAGCTGGCTCAGGTGGCGGAAAC'CTG TGG TATTTTATTTGTAACGGGTTCTTATAGCGCAGCCCTCAAAAATCCAACGGATGATTCTTT TT CTGTCAAGTCTAGTCATCCCAATCTCCTCCTTGGAACCAATATTGGATTGGACAAGCCTG TC GAGTTAGGACTTCAGACTGTAGAAGAGATGAATCCTGTTCTATTGCAAGTG CATGTCAATGT CATGCAGGAATTACTCATGCCCGAGGGAGAAAGGAAGTTTAGAAGCTGGCAATCGCATCT AG CAGATTATAGCAAGCAAATTCCCGTTCCTATTGTCCTCAAGGAAGTGGGCTTTGGAATGG AT GCCAAGACAATCGAAAGAGCCTATGAATTCGGTGTTCGTACAGTGGACCTATCGGGTCGT GG TGGCACCAGCTTTGCCTATATCGAAAACCGTCGTAGTGGCCAGCGTGATTACCTCAATCA AT GGGGTCAGTCTACCATGCAGGCCCTTCTCAATGCCCAAGAATGGAAAGATAAGGTCGAAC TC TTGGTTAGTGGAGGGGTTCGGAATCCGCTGGATATGATTAAGTGCTTGGTTTTTGGTGCT AA GGCTGTGGGATTGTCACGAACCGTTCTGGAATTGGTTGAAACCTACACAGTTGAAGAAGT GA TTGGCATTGTCCAAGGCTGGAAAGCAGATCTACGCTTGATTATGTGTTCCCTTAACTGTG CC ACCATAGCAGATCTACAAAAAGTAGACTATCTTCTTTATGGAAAATTAAAAGAAGCAAAT GA T C G ATG AAAAGG CG T A

SEQ I D NO : 3 9 amino ac id seuqence

MATTNRKDEHI LYALEQ S SYNS FDEVELI HS SLPLYNLDE I DLSTEFAGRK DFPFY INAM TGGSNKGRE INQKLAQVAETCGI LFVTGSYSAAL DJPTDDS FSVKS SHPNLLLGTNIGLDKP VELGLQTVEEMNPVLLQWVIWMQELLMPEGERKFRS QSHLADYS Q I PVPIVLKEVGFGM DAKTIE AYEFGV TVDLSG GGTSFAYIENRRSGQRDYL QWGQSTMQALLNAQEWKDKVE LLVSGGVRNPLDMIKCLVFGAKAVGLSRTVLELVETYTVEEVIGIVQG KADLRLIMCSLNC ATIADLQ VDYLLYGKLKEA DQMKKA SEQ ID NO:40

pET1529-IspH up TIR1

CATAGCCATGGCACAGATCCTGTTGG

SEQ ID O:4i

pET 1529-ispH_dowii_ TiRI

CTGAGGATCCTTAATCGACTTCACGAATATC

SEQ ID NO: 42

pET1529-Dxs up TIR1

CTGAGGATCCAGGAGGTAATATATGAGTTTTGATATTGCCAAATAC

SEQ ID NO:43

pET 1529-Dxs_down_TIR 1

CCG G A ATTCT ^' I ATGCC AGC C AGGC C

SEQ ID O:44

pET1529-IspD up TIRi

CCGGAATTCAGGAGGTAATATATGGCAACCACTCATTTG SEQ ID NO:45

pET1529-IspD up TIRi

CGACGAGCTCTCATTTTGTTGCCTTAATGAG

SEQ ID NO:46

pET 1529-IspF_down_TIR 1

CGACGAGCTX:AGGAGGTAATATAT ^' GCAAACGGAACACG

SEQ ID NO:47

pET1529-Ipi- l up TIRI

AAGGAAAAAAGCGGCCGCTTATTTAAGCTGGGTAAATGCAG

SEQ ID NO:48

pET- 1529-IspG_ up_TIRi

CATAGCCATGGCACATAACCAGGCTCCAATTC

SEQ ID NO:49

pET1529-IspG down TIRI

CTGAGGATCCTTATTTTTCAACCTGCTGAAC SEQ ID NO:50

pET 1529-Dxr_up_TlRl

CTGAGGATCCAGGAGGTAATATATGAAGCAACTCACCATTC

SEQ ID O:5 i

pET1529-Dxr_down_mi

CGACGAGCTCTCAGCTTGCGAGACG SEQ ID O:52

pET 1529-IspE_up_TlRl

CGACGAGCTCAGGAGGTAATATATGCGGACACAGTGG

SEQ ID NO:53

pET 1529 spE__down_ TIR 1

CA CG CGTCG ACTT AA AG CATGGCTCTGTG SEQ ID NO:54

pET1529-Dxs up TIR2

CTGAGGATCCAAGGAGATATACCATGAGTTTTGATATTGCCAAATAC

SEQ ID NO: 55

pET 1529-IspD_up_TIR2

CCGGAATTCAAGGAGATATACCATGGCAACCACTCATTTG

SEQ ID NO: : ^" (¾

pET1529-Ipi-l up TIR2

CGACGAGCT ^' CAAGGAGATATACCATGCAAACGGAACACG

SEQ ID NO:57

pET 1529-Dxr_up_TlR2

CTGAGGATCCAAGGAGATATACCATGAAGCAACTCACCATTC

SEQ ID O:5S

pET1529-IspE up TIR2

CGACGAGC CAAGGAGATATACCATGCGGACACAGTGG SEQ ID O:59

pET1529- up TIR2.

CTGACCATGGCAACGACAAATCGTAAGGACG

SEQ ID NO: 60

pET 1529- _down_ T7R2

CGCGGATCCTTACGCCTTTTTCATCTGATC

SEQ ID NO:61

pET1529-mvkl up TIR2

CGCGGATCCAAGGAGATATACCATGACAAAAAAAGTTGGTGTC

SEQ ID NO:62

pET1529-mvkl_down^R2

CGACGAGCTCTCACAGGCTCTCTATCCATG

SEQ ID NO: 63

pET1529-mvdl up TIR2

CGACGAGCTCAAGGAGATATACCATGTATCATAGCCTTGGTAAC SEQ ID NO: 64

pET1529-mvdl down TIR2:

CACGCGTCGACTTAACAGCAATCATCTTGAC SEQ ID O:65

NS Kpn! i ^;

CGGGGTACCCAGATTGCCTTTGACAACAATGTGG

SEQ ID NO: 66

NSJNfspIJR

GGGAATTCACATGTGGACCATTCTCTGGATCATTGCC SEQ ID NO: 67

A2us Eco F

GAGAGAGAATTCAGCGTTCCAGTGGAT

SEQ ID NO:68

A2us_Ndel__Bam_R

GTTGGATCCGTCGTTGTCATATGGTTATAA

SEQ ID NO:69

A2ds Bam F

GAGAGAGAGGATCCTT ^' GGTGTAAT ^' GCC

SEQ ID NO:70

A2ds_SacI_R

GAGAGAGAGAGCTCGATCGCCTTGGCAAAACAA

SEQ ID NO:7i

GlgX EcoRI F

CGTATGAATTCTTCCGCTTTTTAGAGGAArTTTCCC SEQ ID NO: 72

GlgX Sad R

CGTATGAGCTCAAGAATTGGTTAAAGAAGCCGGTCG

SEQ ID NO:73

GlgAus_EcoRI_F

CCGGAATTCGCCATGTCCCAAATTCTTGATCC

SEQ ID NO: 74

GlgAus Ndel R

GGA _^ ATTCCATATGACCGTCGTTATTCCACTAATTGAG

SEQ ID NO:75

GlgAds_BamHl_F

CGCGGATCCCAATTGATGGCCATGCGTTATGG

SEQ ID NO: 76

GlgAds Sad R

GAGAGAGAGAGCT ^' CGAGCGATCAAGACCACCATTAGG SEQ ID NO: 77

Neu usus F

AATTCCCGGTATGGATGGCAC SEQ ID NO: 78

!spS R

CAAGCGGAAACTTAAAGCGGTGG

SEQ ID NO: 79

A2_usus_F

TATCAGAATCCTTGCCCAGATG SEQ ID NO: 80

SRA R

CTCACCGCCAGAATTTAAGCC

SEQ ID NO: 81

GlgX_usus_F

CCCTGCAACTTAAACCCAAGACC

SEQ ID NO: 82.

FKDK R AGGAGGAGATTGGGATGACTAGAC

SEQ ID NO: 83

GlgA_usus_F

GAGTCCAGGGACAAAGCCAG

SEQ ID NO: 84

FKDK Rl

GAGGGCTGCGCTATAAGAACC