EP2961837A2 - Zellen und verfahren zur fettsäuresynthese - Google Patents

Zellen und verfahren zur fettsäuresynthese

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Publication number
EP2961837A2
EP2961837A2 EP14709381.9A EP14709381A EP2961837A2 EP 2961837 A2 EP2961837 A2 EP 2961837A2 EP 14709381 A EP14709381 A EP 14709381A EP 2961837 A2 EP2961837 A2 EP 2961837A2
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EP
European Patent Office
Prior art keywords
nucleic acid
cell
acid molecule
polypeptide
agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14709381.9A
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English (en)
French (fr)
Inventor
Jeff ERRINGTON
Yoshikazu Kawai
Roman MERCIER
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Newcastle University of Upon Tyne
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Newcastle University of Upon Tyne
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Publication date
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Publication of EP2961837A2 publication Critical patent/EP2961837A2/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/004—Cytochrome-b5 reductase (1.6.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to methods of producing fatty acids, fatty acid derivatives and membrane associated (e.g. hydrophobic) molecules.
  • the invention also provides methods of inducing L-form growth in a cell. Nucleic acids, expression vectors, recombinant cells, semisynthetic and synthetic cells, and reaction vessels suitable for such methods are also provided.
  • the peptidoglycan (PG) cell wall is a major defining structure of bacteria, and is present in all known major bacterial lineages.
  • Many genes required to make the precursors for cell wall synthesis and assemble them into the meshwork of the growing wall are normally essential for cell viability. This explains why the wall is also such an important target for antibiotics, such as ⁇ -lactams and glycopeptides.
  • L-forms were identified as antibiotic resistant or persistent organisms isolated in association with a wide range of infectious diseases (Domingue and Woody, 1997). Under laboratory conditions, the production of stable L-forms usually requires the inhibition of cell wall synthesis with appropriate antibiotics and long-term passage on osmotically supportive medium to prevent cell lysis (Allan, 1991 ; Leaver et al., 2009). It has long been known that one or more genetic changes from their parent strain are needed for the formation and/or proliferation of stable L-forms (Allan et al., 2009).
  • the applicants have investigated the mechanisms driving cell division and proliferation of L- forms by isolating and studying the effects of mutations that allow cells to proliferate in the L- form state.
  • the invention relates to a recombinant cell with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis, wherein the cell comprises at least one of:
  • the first nucleic acid molecule, the second nucleic acid molecule and/or the third nucleic acid molecule is part of an expression vector.
  • the first nucleic acid molecule encodes a carboxyltransferase subunit of acetyl CoA carboxylase.
  • the first nucleic acid molecule comprises an inducible promoter.
  • the first nucleic acid molecule comprises a mutation that increases expression of the encoded carboxyltransferase. More preferably, the mutation is in the 5' untranslated region (UTR) of the nucleic acid molecule. Most preferably, the mutation in the 5'UTR comprises a single point mutation (C to A) at the equivalent position to the underlined nucleic acid residue of SEQ I D NO: 1.
  • the 5'UTR has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:2. More preferably, the 5'UTR comprises or consists of the nucleic acid sequence of SEQ ID NO: 2.
  • the second nucleic acid molecule encodes a polypeptide involved in
  • the second nucleic acid molecule encodes a polypeptide selected from the group consisting of murF, dapF, racE, yrpC, murAA and murC. Most preferably, the second nucleic acid molecule encodes a polypeptide selected from the group consisting of murAA and murC.
  • the second nucleic acid molecule encodes a polypeptide involved in wall teichoic acid (WTA) synthesis. More preferably, the second nucleic acid molecule encodes a polypeptide selected from the group consisting of tagA, tagB, tagD, tagE, tagF, manA and tagO. Most preferably, the second nucleic acid molecule encodes tagO.
  • the second nucleic acid molecule encodes a polypeptide involved in the regulation of peptidoglycan and/or wall teichoic acid synthesis. More preferably, the second nucleic acid molecule encodes a polypeptide selected from the group consisting of glmS, glmM, gcaD and MreB. Most preferably, the second nucleic acid molecule encodes MreB.
  • the second nucleic acid molecule comprises a repressible promoter.
  • the second nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.
  • the third nucleic acid molecule comprises a mutation that modifies the activity of the encoded ribosomal S9 protein. More preferably, the mutation results in a substitution of glutamic acid to lysine at the equivalent position to the underlined amino acid of SEQ ID NO:4.
  • the cell further comprises at least one of:
  • a fourth nucleic acid molecule encoding a polypeptide involved in the respiratory chain, the fourth nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide;
  • nucleic acid molecule encoding a polypeptide involved in the glycolysis pathway, the fifth nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • the fourth nucleic acid molecule encodes a polypeptide selected from the group consisting of ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS
  • the fourth nucleic acid molecule comprises a repressible promoter.
  • the fourth nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.
  • the fifth nucleic acid molecule encodes a polypeptide selected from the group consisting of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.
  • the fifth nucleic acid molecule comprises a repressible promoter.
  • the fifth nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.
  • the cell is a bacterial cell. More preferably, the bacterial cell is B.subtilis.
  • the invention relates to a semi-synthetic or synthetic cell with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis, the cell having at least one of:
  • the cell additionally has at least one of:
  • the carboxyltransferase is a carboxyltransferase subunit of acetyl CoA
  • the at least one polypeptide involved in cell wall synthesis is involved in peptidoglycan synthesis.
  • the polypeptide is selected from the group consisting of murB, murG, murE, mraY, dal, murF, dapF, racE, yrpC, murAA and murC. More preferably, the polypeptide selected from the group consisting of murB, murG, murE, mraY, dal, murAA and murC.
  • the at least one polypeptide involved in cell wall synthesis is involved in wall teichoic acid (WTA) synthesis.
  • WTA wall teichoic acid
  • the polypeptide is selected from the group consisting of tagA, tagB, tagD, tagE, tagF, manA and tagO. More preferably, the polypeptide is tagO.
  • the at least one polypeptide involved in cell wall synthesis is involved in the regulation of peptidoglycan and/or wall teichoic acid synthesis.
  • the polypeptide is selected from the group consisting of glmS, glmM, gcaD and mreB. More preferably, the polypeptide is MreB.
  • the at least one polypeptide involved in the respiratory chain is selected from the group consisting of ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS.
  • the at least one polypeptide involved in the glycolysis pathway is selected from the group consisting of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.
  • the invention relates to a recombinant cell with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis, wherein the cell comprises at least one of:
  • a first nucleic acid molecule encoding a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule comprises a nucleic acid sequence capable of modifying the activity of the encoded carboxyltransferase;
  • a second nucleic acid molecule encoding a polypeptide involved in cell wall synthesis, the second nucleic acid molecule comprising a nucleic acid sequence capable of modifying the activity of the encoded polypeptide; and/or (c) a third nucleic acid molecule encoding ribosomal protein S9, the third nucleic acid molecule comprising a nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9.
  • the invention relates to a method of producing fatty acids, fatty acid derivatives and/or membrane associated molecules, the method comprising providing to a cell at least one of:
  • the agent comprises a nucleic acid molecule.
  • the nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3 .
  • the agent that decreases cell wall synthesis is not a nucleic acid molecule encoding murB, murG, murE, mraY or dal where the nucleic acid is capable of inhibiting expression of the encoded murB, murG, murE, mraY or dal.
  • the agent that increases carboxyltransferase activity comprises a first nucleic acid molecule encoding a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase.
  • the agent that decreases cell wall synthesis comprises a second nucleic acid molecule encoding a polypeptide involved in cell wall synthesis, wherein the polypeptide is not murB, murG, murE, mraY or dal, the second nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • the agent that modifies ribosomal S9 activity comprises a third nucleic acid molecule encoding ribosomal protein S9, the third nucleic acid molecule comprising a nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9.
  • the method further comprises providing to the cell at least one of:
  • the agent that decreases respiratory chain activity comprises a fourth nucleic acid molecule encoding a polypeptide selected from the group consisting of ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS.
  • the agent that decreases the activity of the glycolysis pathway comprises a fifth nucleic acid molecule encoding a polypeptide selected from the group consisting of ptsH, ptsl, fbaA, gapA, pgk, tpi, pgm and eno.
  • the providing step(s) generate(s) a cell according to the invention.
  • the method further comprises culturing the cell under conditions that support the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • the method further comprises recovering the produced fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • the invention relates to the use of a cell according to the invention in the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • the invention relates to the use of a cell according to the invention in drug or vaccine delivery.
  • the invention relates to the use of at least one of: (a) an agent that increases carboxyltransferase activity; (b) an agent that decreases cell wall synthesis; and/or
  • the invention relates to a reaction vessel containing a cell according to the invention and medium sufficient to support growth of the cell.
  • the reaction vessel is a bioreactor or a fermenter.
  • the method of the invention is performed in the reaction vessel of the invention.
  • the invention relates to a method of inducing L-form growth in a cell, comprising providing to a cell at least one of:
  • the agent that removes the cell wall and/or prevents cell wall synthesis is a lysozyme.
  • the method further comprises providing to the cell at least one of:
  • the cell is cultured under conditions that support L-form growth.
  • the invention relates to a method of preparing a therapeutic composition comprising:
  • the invention relates to a method of identifying a DNA mutation that supports L-form growth in a cell comprising:
  • the cell has previously been provided with at least one of:
  • the agent that decreases respiratory chain activity comprises a fourth nucleic acid molecule encoding a polypeptide selected from the group consisting of ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS.
  • the agent that decreases the activity of the glycolysis pathway comprises a fifth nucleic acid molecule encoding a polypeptide selected from the group consisting of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.
  • step (iii) further comprises culturing the identified cell under conditions that support cell wall regeneration and identifying a cell with a regenerated cell wall.
  • the invention relates to a recombinant cell; a semi-synthetic or synthetic cell; a method of producing fatty acids, fatty acid derivatives and/or membrane associated molecules; a reaction vessel; a method of inducing L-form growth; a method of preparing a therapeutic composition; or a method of identifying a DNA mutation that supports L-form growth in a cell; substantially as described herein with reference to the accompanying drawings.
  • the words "comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.
  • Figure 1 shows the effects of ispA and murE-B Mutations on L-form Growth.
  • A-C Strains LR2 ⁇ ispA P xyr murE-B; A), 168CA (wild type; B) and Bs1 15 ⁇ P xyr murE-B; C) were grown in the walled state then converted to protoplasts, incubated in L-form supporting medium (NB/MSM, no xylose) with benzamide (FtsZ inhibitor) and observed by time-lapse phase contrast microscopy.
  • NB/MSM no xylose
  • FtsZ inhibitor benzamide
  • panel C deformed cells are labelled with arrows, the remains of lysed cells with hashes and a star points to a successful division event. Elapsed time (min) is shown in each panel. Scale bar, 3 ⁇ . See also Figure 9.
  • Figure 2 shows that L-form Growth Requires Mutational Lesions Affecting Two Different Pathways.
  • Figure 2A Strain LR2 (ispA P xyr murE-B) was grown in the walled state then converted to protoplasts and incubated in NB/MSM containing benzamide with (+Xyl, solid line) or without 0.5% xylose (-Xyl, dashed line).
  • Figure 2B Growth of strains with the genotypes indicated on NA/MSM plates (to support L-form growth).
  • strains Bs115 (P xyr murE-B), LR2 (ispA P xyr murE-B), YK1593 (Adal), YK1592 (ispA Adal), RM 119 (AmurC), YK1409 (ispA AmurC).
  • Figure 2C Schematic representation of the chromosomal region deleted in strain RM121 (indicated by the black line).
  • Figure 2D Growth of the reconstructed strain RM121 containing pLOSS-erm-murC (P spac -murC) streaked on NA plates in the presence (left) or absence (right) of IPTG.
  • Figure 2E Growth of protoplasts of strains RM121 (dotted line), AmurC (dashed line) and wild type (solid line) in L-form supporting medium (NB/MSM) with benzamide.
  • Figure 3 shows that upregulation of accDA Supports L-form Growth.
  • Figure 3A Schematic representation of the B. subtilis genomic region containing the accDA genes. The C->A substitution corresponding to the accDA* mutation is shown in a box, the Shine Dalgarno (SD) and the start codon (start). Arrows indicate the putative stem loop.
  • Figure 3B Effect of the accDA* mutation (strain RM84) on growth of protoplasts in L-form supporting medium (NB/MSM) with benzamide, visualized by time-lapse phase contrast microscopy. Elapsed time (min) is shown in each panel. Scale bar, 3 ⁇ .
  • FIG. 3C-D Growth profiles of 168CA (wild type), RM81 ⁇ ispA) and RM84 ⁇ ispA accDA*) strains in the walled state (NA plate incubated at 30°C for 24h; C) or under L-form conditions (protoplasts incubated 30°C in NB/MSM with benzamide; D).
  • Figure 3E Western blot analysis of histidine-tagged AccA levels in 168CA (wild type, lane 1), YK1731 ⁇ accA-his, lane 2), YK1732 ⁇ ispA accA-his, lane 3) and YK1733 (ispA accDA* accA-his, lane 4).
  • FIG. 3F-G Protoplast growth of an ispA amyE:.P xyr accDA strain (YK1694, dotted and dashed line) or an isogenic ispA + strain (YK1738, line) in L-form supporting medium (NB/MSM) containing benzamide with (dotted line and solid line) or without 0.5% xylose (dashed line) (F), or with several different xylose concentrations (YK1694, G): 1 % (dotted line), 0.5% (dashed line) and 0.1 % or no (solid line).
  • NB/MSM L-form supporting medium
  • Figure 3H-I Two typical examples of L- form proliferation by strain YK1694 in L-form supporting medium (NB/MSM) with 0.5% xylose and benzamide, visualized by time-lapse phase contrast microscopy. Elapsed time (min) is shown in each panel. Scale bar, 3 ⁇ .
  • Figure 4 shows the roles for the FapR Regulator and FAS II Enzyme System in L-form Growth Promoted by Overexpression of accD.
  • Figure 4A Schematic representation of the B. subtilis FAS II system and genes regulated by the FapR protein (underlined) [after (Rock and Cronan, 1996) and (Schujman et al., 2003)].
  • FIG. 4B Quantitative RT-PCR analysis of the relative change expression of several FapR regulated genes in an amyE: P xyr accDA strain (YK1738) grown in LB with 1 % xylose at 37°C. The expression level of each gene is expressed relative to that of a parallel culture without xylose (assigned a value of 1). Mean and SD values (error bars) were calculated using values generated from three independent cultures.
  • Figure 4C Effect of FapR overexpression on growth in the walled state and its rescue by AccDA overexpression.
  • strains were cultured on NA without (top) or with 0.5% xylose and 0.5mM IPTG (bottom) and incubated for 20h at 37°C: amyE::P xyl -fapR (strain RM208, left), amyE::P xyl -fapR P spac(hy) -accDA (YK1726, middle), amyE:.P xy rfapR R106 A P S pac(hy ) -3CcDA (YK1735, right).
  • Figure 4D Quantitative RT-PCR analysis of the relative change in expression of several FapR regulated genes in the AfapR mutant (RM258) grown in LB at 37°C.
  • each gene is expressed relative to that of the wild type (168CA) grown in LB at 37°C as described above (B).
  • Figure 4E Protoplast growth in NB/MSM with benzamide of strains carrying the following mutations: AfapR (strain RM258, triangles), ispA AfapR (RM259, squares), ispA accDA* (RM84, dashed line), ispA accDA* AfapR (RM260, dotted line).
  • Figure 4F Effect of overproduction of AccDA on growth in the walled state.
  • FIG. 4H Protoplast growth in NB/MSM with 0.5 % xylose and benzamide of strains with the following markers and culture supplements: ispA amyE::P xyr accDA P S p ac -fabHA without (dashed line) or with (dotted line) 0.5 mM IPTG and ispA amyE:.P xy i-accDA P spac -plsX (solid line) or P spac -fabD (triangles) with 0.05 mM IPTG. See also Figures 10 and 1 1.
  • Figure 5 shows that overexpression of AccDA Results in Excess Membrane Synthesis in Walled B. subtilis Cells.
  • FIG. 5A-B Phase contrast (left) and corresponding epifluorescence micrographs (right) of strain YK1738 (amyE::P xyr accDA), grown in LB with (B) or without (A) 0.5% xylose and stained with the membrane dye Mitotracker green. Scale bar represents 5 ⁇ .
  • Figure 5C-D N-SIM fluorescence micrographs of wild type (168CA, C) and YK1738 (amyE::P xyr accDA, D1-4), grown in LB with 0.5% xylose and stained with the membrane dye Mitotracker green. Enlarged images are shown in D2-4. Scale bar represents 5 ⁇ .
  • FIG. 5E-F Transmission electron microscopy images of wild type (168CA, E) and YK1738 ⁇ amyE:.P xyr accDA, F1-3), grown in LB with 0.5% xylose. Enlarged images are shown in F2-3. Arrows indicate internal membrane like structures. Scale bar represents 200 nm.
  • Figure 5G Epifluorescence micrographs of cell membranes stained with the Mitotracker green. Bs115 (P xyr murE-B) was grown in LB with 1 % (left) or 0.1 % (right) xylose at 37°C. Scale bar represents 5 ⁇ . For all of Figures 5A-G B. subtilis strains were grown in LB at 37°C. See also Figure 12.
  • Figure 6 shows that excess Membrane Promotes Shape Changes and Membrane Scission in Protoplasts.
  • Figure 6A Theoretical relationship between surface area and volume in rod shaped (dashed line) or spherical (solid line) cells.
  • Figure 6B Epifluorescence microscopy of rod shape cells of the strain 168CA (wild type), stained with the membrane dye Nile red, after treatment with benzamide for various time periods (30, 60 and 90 min) or without (no). Scale bar represents 3 ⁇ .
  • Figure 6C Exponentially growing L-form culture of YK1694 (ispA amyE P xyr accDA) in NB/MSM with 0.5% xylose and benzamide. Scale bar represents 3 ⁇ .
  • FIG. 6D Phase contrast and corresponding epifluorescence microscopy of cells with increased volume, corresponding to panel B, immediately after treatment with lysozyme, leading to their conversion into protoplasts. Cells were stained with the membrane dye Nile red. Scale bar represents 3 ⁇ .
  • Figure 6E Effect of the increased surface area of wild type protoplast on membrane scission, visualized by time-lapse phase contrast microscopy. Exponentially growing wild type (168CA) cells in LB were treated with benzamide for 60 min and then obtained filamentous rod cells were treated with lysozyme. Elapsed time (min) after the period of treatment with lysozyme is shown in each panel. Scale bar, 3 ⁇ . See also Figure 13.
  • Figure 7 provides a Model for Proliferation of L-form Cells.
  • Newborn L-forms (A) grow in an unbalanced manner with excess surface area (membrane) synthesis.
  • the excess surface area (B) drives shape deformation (C). Scission of lobes or blebs of cytoplasm generates smaller progeny cells in which the Area/Volume ratio is normalized by simple geometric effects.
  • Figure 8 shows that a mutation affecting the C-terminal tail of Rpsl triggers excess membrane formation and L-form division.
  • Figure 8A Protoplast growth in NB/MSM with benzamide of strains ispA accDA* (RM84, circles) and ispA rpsl* (RM85, triangles).
  • Figure 8B Epifluorescence micrographs of strain wild type (Bs168, left) and ispA rpsl* (RM85, right) grown in LB and stained with the membrane dye Mitotracker green. Scale bar represents 5 ⁇ .
  • FIG. 8C Protoplast growth in NB/MSM with benzamide of strains ispA rpsl* P spac - fabHA supplemented without (circles) or with (triangles) 0.5 mM IPTG.
  • Figure 8D Membrane imaging of strain ispA rpsl* P spac -fabHA, grown in LB at 37°C in the absence (no) or presence (0.5 mM IPTG) of 0.5 mM IPTG. Scale bars represent 5 ⁇ .
  • Figure 9 shows that repression of ispA expression supports L-form growth.
  • Figure 9A Growth of strains Bs1 15 ⁇ P xyl -murE-B, left), LR2 ⁇ ispA P xyl -murE-B, right) and RM82 ⁇ ispA P X yi-murE-B, amyE xseB-ispA + ' bottom) cultured on NB/MSM plates to support L-form growth. Provision of an ectopic copy of ispA + prevents growth in the L-form state.
  • Figure 10 shows that overproduction of AccDA results in cell lysis in walled cells and its rescue by cerulinin.
  • Figure 10A Growth of the strains YK1694 (ispA amyE::P xyr accDA, top), YK1738 ⁇ amyE::P xyr accDA, middle) and 168CA (wild type, bottom) cultured on NA plates with different concentrations of xylose, as indicated.
  • Figure 10B Growth of the strains 168CA (wild type, dotted line) and YK1738 (amyE::P xyr accDA) with (triangles) or without (squares) 0.5% xylose, in LB at 37°C.
  • Figure 10C Phase contrast and corresponding epifluorescence microscopy of the cell membranes of the strain YK1738 (amyE::P xyr accDA) stained with Mitotracker green. Cells were grown in LB containing with (bottom) or without (top) 0.5 % xylose to an OD 6 oo of 1. Scale bar represents 5 ⁇ .
  • Figure 11 shows that repression of the FAS II activity inhibits L-form growth promoted by repression of PG precursor synthesis.
  • Figure 11A Effects of repression of FAS II enzyme synthesis and PG precursor synthesis on growth of protoplasts in NB/MSM. All strains carried an ispA mutation to enable L-form growth and P xyi -murE-B. Additional mutations and supplements were as follows: P spac -plsX (strain RM237, dotted line), P spac -accDA (YK1741 , squares) and P spac -fabHA (RM250, triangles) with 0.1 mM IPTG, and P spac -fabHA (RM250, diamonds) without IPTG.
  • Figure 12 shows excess membrane synthesis by overproduction of AccDA or repression of the PG precursor synthesis.
  • Figure 12A Phase contrast and corresponding epifluorescence microscopy of the cells stained with the membrane dye Mitotracker green.
  • B. subtilis strains RM81 ispA, left
  • RM84 ispA accDA*, right
  • Figure 12B Membrane imaging of strain YK1706 (amyE:.P xyr accDA P spac -fabHA), grown in LB with 0.5 % xylose at 37°C in absence (no IPTG) or in presence (+IPTG) of 0.5 mM IPTG. Scale bars represent 5 ⁇ .
  • FIG. 12C Membrane imaging of strain of the strain YK1736 (P xyr murE-B Pspac- sbHA) stained with the Mitotracker green. Cells were grown in LB containing 0.1 % xylose with (left) or without (right) 0.5 mM IPTG. Scale bar represents 5 ⁇ .
  • Figure 13 shows absence of vesicle structures in a round mutant of B.
  • Subtilis. Phase contrast and corresponding epifluorescence micrographs of strains YK1824 (P spac -rodA, A) and YK1825 (P spac -rodA amyE: :P xyr accDA, B), stained with the membrane dye Nile red. rodA mutants are spherical, and viable in NB/MSM medium (Kawai et al., 2011). The cells were grown in NB/MSM for 180 min in the absence of IPTG to generate round cells. The cultures were further diluted into fresh NB/MSM containing 0.5% xylose and benzamide, and incubated for 0, 60 or 90 min.
  • Figure 14 provides the nucleotide sequence for Bacillus subtilis carboxyltransferase; Genbank: http://www.ncbi.nlm.nih.gov/genbank/, ACCESSION: AL009126.3 (SEQ ID NO: 1). Nucleic acid residue "C” that may be replaced with nucleic acid residue "A” within the context of the invention is underlined.
  • Figure 15 provides the nucleotide sequence for the 5'UTR of Bacillus subtilis carboxyltransferase; Genbank: http://www.ncbi.nlm.nih.gov/genbank/, ACCESSION: AL009126.3 (SEQ ID NO:2). Nucleic acid residue "C” that may be replaced with nucleic acid residue "A” within the context of the invention is underlined.
  • Figure 16 provides the nucleotide sequence for yeast ACC1 ; Genbank: http://www.ncbi.nlm.nih.gov/genbank/, ACCESSION: BK006947 (SEQ ID NO:3).
  • Figure 17 provides the amino acid sequence for Bacillus subtilis ribosomal protein S9; genbank: http://www.ncbi.nlm.nih.gov/genbank/, ACCESSION: AL009126.3 (SEQ ID N04).
  • Amino acid ⁇ " that may be replaced with amino acid "K” within the context of the invention is underlined.
  • Figure 18 shows inhibition of PG precursor synthesis induce L-form proliferation in bacteria.
  • FIG. 1 Schematic model of PG precursor (lipid II) synthesis in bacteria and its inhibition by the antibiotics fosfomycin and D-cycloserine.
  • the protein MurA inhibited by the antibiotic fosfomycin, and MurB catalyse the transformation of N-acetylglucosamine (GlcNAc) into N- acetylmuramic acid (MurNAc).
  • the protein Dal inhibited by the antibiotic D-cycloserine, transformed L-ala into D-ala prior its incorporation in the MurNAc-pentapeptide, synthesised by the proteins MurC, MurD, MurE, MurF.
  • MurNAc-pentapeptide is translocated to the membrane compartment via covalent bound formation with undecaprenyl pyrophosphate molecule by MraY and the transfer of GlcNAc is catalysed by MurG to form lipid II.
  • B Growth of B. subtilis strain LR2 (ispA P xyr murE-B) streaked on L-form-supporting medium (MSM) or nutrient agar (NA) plates in presence (lipid II ON) or in absence (lipid II OFF) of 0.5% xylose.
  • MSM L-form-supporting medium
  • NA nutrient agar
  • C Phase contrast microscopy of B. subtilis LR2 cells grown on L-form- supporting medium (MSM) plates in presence (left) or in absence (right) of 0.5% xylose.
  • D-l Growth on plates (D, F, H) and corresponding phase contrast microscopy (E, G, I) of bacterial strains S. aureus ATCC2913 (D-E), C. glutamicum ATCC13032 (F-G) and E. coli MG1655 (H-l).
  • D, F, H The different bacterial strains were streaked on L-form-supporting medium (MSM) or nutrient agar (NA) plates in absence (lipid II ON) or in presence (lipid II OFF) of the antibiotics fosfomycin (D, H) or D-cycloserine (F).
  • MSM L-form-supporting medium
  • NA nutrient agar
  • E, G, I Phase contrast microscopy of the different bacterial cells grown on L-form-supporting medium (MSM) plates in absence (left) or in presence (right) of the antibiotics fosfomycin (E, I) or D-cycloserine (G).
  • Figure 19 shows Lipid II targeting antibiotics induced L-forms proliferate on b-lactamase.
  • A Growth of S. aureus (left), C. glutamicum (middle) and E. coli (right) walled strains streaked on L-form-supporting medium (MSM) in presence of Penicillin G (S. aureus and C. glutamicum) or Ampicillin (E. coli).
  • B Growth of S. aureus (top), C.
  • L-form-supporting medium MSM
  • MSM L-form-supporting medium
  • Penicillin G S. aureus and C. glutamicum
  • Ampicillin E. coli
  • Figure 20 shows Lipid II targeting antibiotics induced L-forms proliferate in absence of the cell wall and cell division machineries -1-.
  • A Growth of the E. coli strains TB28 (top) and RM345 (murA-, bottom) containing the unstable plasmid pOU82-Amp-mi//"/A streaked on NA plates in the presence of X-gal.
  • B L-form colonies of the E.
  • coli strains RM345 (murA-, pOU82-Amp-mi/fiA, top left), RM323 (ftsZ-, pOU82-Amp-ffsZ, top right), RM350 (murA- ftsZ-, pOU82-Amp-ffsZ, pSK122-Cm-mt/rA bottom left) and RM60 (ftsK-, pSK122-Cm-ftsK, bottom right) on L-form-supporting medium (MSM) plates in presence of fosfomycin and X- gal, after several repeated streaking on L-form-supporting medium (MSM) plates in presence of fosfomycin.
  • MSM L-form-supporting medium
  • aureus strain RNpFtsZ-1 (erm-pSPAC-ffsZ, (Pinho MG and Errington J, 2003) streaked on L-form-supporting medium (MSM) plates in absence (Lipid II ON, left) or in presence (Lipid II OFF, middle and right) of fosfomycin, with (+FtsZ, middle) or without (-FtsZ, left and right) IPTG.
  • E Growth profiles of C. glutamicum strain in L-form-supporting medium (MSM) in walled (left, Lipid II ON) or in L-form (right, Lipid II OFF) state in absence (red) or in presence (blue) of cephalexin.
  • Figure 21 shows Lipid II targeting antibiotics induced L-forms proliferate in absence of the cell wall and cell division machineries -2-.
  • A L-form colonies of the E. coli strain RM359 (mreBCD-, pHM82-Kn-mreSCD) on L-form-supporting medium (MSM) plates in presence of fosfomycin and X-gal, after several repeated streaking on L-form-supporting medium (MSM) plates in presence of fosfomycin.
  • MSM L-form-supporting medium
  • B Multiplex PCR of the genes ftsK, murA, ftsZ and mreC on the genomic DNA of the E. coli strain RM359 grown in walled state (1) or L-form state (2) obtained from (A).
  • M represents the 100bp DNA ladder.
  • C Cell wall reversion, on L-form- supporting medium (MSM) plates with IPTG (+FtsZ), of S. aureus strain RNpFtsZ-1 L-forms grown on L-form-supporting medium (MSM) plates with fosfomycin and without IPTG obtained from Figure3D, right.
  • D Growth of the S. aureus RNpFtsZ-1 L-form reverted strain from (C) on nutrient agar (NA) plates with (+FtsZ) or without (-FtsZ) IPTG.
  • E Growth of the S.
  • aureus strain ATCC2913 ftsZ R191p (Haydon DJ et al., 2008) streaked on L-form- supporting medium (MSM) plates with (Lipid II ON, left and middle) or without (Lipid II OFF, right) of fosfomycin in presence (left) or in absence (middle and right) of benzamide.
  • MSM L-form- supporting medium
  • F Growth of the C. glutamicum strain streaked on nutrient agar plates (NA) with (left) or without (middle) cephalexin or on L-form-supporting medium (MSM) plates with fosfomycin and cephalexin (right).
  • Fatty acids are essential components of cell membranes and are important sources of metabolic energy in all organisms.
  • the regulation of fatty acid degradation and biosynthesis is essential to maintain membrane lipid homeostasis.
  • the applicants have surprisingly shown that modification of particular biosynthetic pathways in a cell can result in increased fatty acid synthesis and excess membrane production.
  • the production of excess membrane is shown to induce shape modulations that induce proliferation and cell division of a cell in the L-form state.
  • L-form division does not require the sophisticated, highly conserved and normally essential cellular machinery required for classical cell division. Instead, cells in the L-form state are able to divide via a membrane blebbing or tubulation process.
  • the data provided herein suggests that the division of L-forms is likely due simply to an imbalance between membrane surface area and cellular volume.
  • the invention provides novel cells and methods for producing fatty acids and/or fatty acid derivatives and also provides a means for cell division that does not require the normally essential cell division machinery.
  • the ability to stimulate the generation of fatty acids and/or fatty acid derivatives means that the invention provides a means for producing excess membrane. This in turn may allow for excess production of membrane associated molecules (for example in the case of production of membrane associated molecules the synthesis of which is limited by the amount of available membrane in the cell).
  • the novel cells and methods of the invention may therefore be used to produce membrane associated (e.g. hydrophobic) molecules as well as stimulating the production of fatty acids and/or fatty acid derivatives.
  • the cells and methods provided herein for the production of fatty acids and/or fatty acid derivatives may thus equally be applied to the production of membrane associated molecules. References to the production of fatty acid and/or fatty acid derivatives herein can therefore be construed to apply equally to production of membrane associated molecules (unless the context clearly indicates otherwise).
  • the invention is based on the surprising and unexpected finding that an increase in membrane synthesis may be promoted directly (e.g. by increasing fatty acid synthesis) or indirectly (e.g. by (partial) repression of competing pathways). Furthermore, it is surprisingly shown that modifying ribosomal activity also results in an increase in membrane synthesis.
  • Each of the mechanisms for promoting increased membrane synthesis discussed below has been shown by the applicants to provide cells capable of L-form growth.
  • Novel mechanisms for increasing the synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules are novel mechanisms for increasing the synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • One or more of the mechanisms discussed below may be used within the context of the invention to increase the synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules (and thus promote membrane synthesis).
  • the applicants have surprisingly found that it is possible to generate cells that have increased membrane and are capable of L-form growth.
  • over-expression of the AccDA operon alone results in an increase in the synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • the inventors have shown that over-expression of both genes within the AccDA operon is required for the desired effect (over-expression of either AccD or AccA is not sufficient).
  • over-expression of the AccBC operon alone does not result in an increase in the synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • the invention is exemplified herein using over-expression of carboxyltransferase.
  • the invention may also be achieved by modifying the expression of other polypeptides involved in fatty acid synthesis (for example polypeptides that have an indirect or direct effect on carboxyltransferase activity).
  • the invention may also be achieved by modifying carboxyltransferase activity within the cell in any other suitable way (e.g. by increasing carboxyltransferase efficiency within the cell or increasing carboxyltransferase stability (thereby decreasing carboxyltransferase degradation)), or by decreasing biotin carboxylase activity (e.g.
  • biotin carboxylase expression by decreasing biotin carboxylase expression, the activity of expressed biotin carboxylase, or by modifying biotin carboxylase activity).
  • a skilled person would readily be able to screen for and identify appropriate alternative polypeptides within the fatty acid synthetic pathway and/or suitable modifications of carboxyltransferase that are capable of achieving the desired effect.
  • other genes capable of achieving the desired effect may be identified as a routine procedure.
  • fatty acids, fatty acid derivatives and/or membrane associated molecules can also be increased indirectly, for example by modifying competing biosynthetic pathways within the cell.
  • modification of cell wall synthesis specifically inhibition of cell wall synthesis, can increase a cell's propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis.
  • the applicants have surprisingly demonstrated that a number of different pathways involved in cell wall synthesis may be modified to achieve the desired effect.
  • the applicants have shown that inhibiting, in a cell, expression of one or more of the polypeptides involved in peptidoglycan synthesis (for example murB, murG, murE, mraY, murAA, dal and murC, or inhibition of the murE-B operon); inhibiting expression of a polypeptide involved in wall teichoic acid (WTA) synthesis (for example tagO); or inhibiting expression of a polypeptide involved in the regulation of peptidoglycan and wall teichoic acid synthesis (for example mreB) results in an increase in the cell's propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis. In each case it is successfully demonstrated that the resultant cells have increased fatty acid and membrane synthesis and are capable of L- form growth.
  • WTA wall teichoic acid
  • mreB inhibiting expression of a polypeptide involved in the regulation of peptidoglycan and wall teichoic acid synthesis
  • mreB mreB
  • mreB mreB
  • mbl mreB
  • mreBH mreB
  • Other bacteria are known to have two or a single mreB gene.
  • the inventors show herein the deletion of all three of mreB, mbl and mreBH in B subtilis results in cells with increased fatty acid and membrane synthesis that are capable of L-form growth.
  • the invention encompasses modifying one or more of these homologues, separately, or in combination.
  • the invention encompasses equivalent changes to the mreB homologues (separately, or in any combination) of different cell types and is not limited to the examples provided herein.
  • murE-B refers to an operon that comprises nucleic acid molecule(s) encoding the murB, murG, murE and mraY polypeptides.
  • the murB, murG, murE and mraY encoding genes may be present in any order within the operon.
  • mreB refers to one or more mreB homologues.
  • the term mreB therefore encompasses, in the context of B subtilis for example, one or more of the homologues mreB, mbl and mreBH (in any combination).
  • the term may equally be used to refer to one or more (if present) homologues of mreB present in a particular (suitable) cell type.
  • An operon that comprises nucleic acid molecule(s) encoding the mreB, mreC and mreD polypeptides is refered to herein as a "mreB operon".
  • the mreB, mreC and mreD encoding genes may be present in any order within the operon.
  • the invention is exemplified herein by inhibiting expression of one or more of murB, murG, murE, mraY, murAA, dal, murC, tagO and mreB.
  • the invention may also be achieved by modifying the expression of other polypeptides involved in cell wall synthesis (e.g. polypeptides involved in peptidoglycan synthesis, wall teichoic acid synthesis or the regulation of peptidoglycan and/or wall teichoic acid synthesis) e.g.
  • polypeptides that have an indirect effect on the activity of one or more of murB, murG, murE, mraY, murAA, dal, murC, tagO and mreB.
  • the invention may also be achieved by modifying cell wall synthesis within the cell in any other suitable way (e.g. by decreasing the efficiency of cell wall synthesis within the cell, or by decreasing the efficiency of or stability of one or more of the polypeptides involved (thereby increasing protein degradation)).
  • a skilled person would readily be able to screen for and identify appropriate alternative polypeptides involved in the cell wall biosynthetic pathway and/or suitable modifications of such polypeptides that are capable of achieving the desired effect.
  • other genes capable of achieving the desired effect may be identified as a routine procedure.
  • ribosomal protein S9 can increase a cell's propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis.
  • the ribosomal protein S9 is encoded by the rpsl gene.
  • substitution of glutamic acid to lysine at position 112 in the ribosomal protein S9 polypeptide of B subtilis modifies ribosomal protein S9 activity and results in cells with increased synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules, where the cells are capable of L-form growth.
  • the invention is exemplified herein by modifying ribosomal protein S9 activity in B. subtilis (and specifically by genetically altering the ribosomal protein S9 polypeptide sequence at position 112 from E to K).
  • B. subtilis and specifically by genetically altering the ribosomal protein S9 polypeptide sequence at position 112 from E to K.
  • an equivalent modification can be made to the ribosomal protein S9 of any other suitable cell type to achieve the desired effect.
  • the invention is therefore not limited to the specific mutation of B. subtilis ribosomal protein S9 but may be applied equally to mutation (and thus modification) of ribosomal protein S9s of other suitable cell types.
  • Suitable mutations may be those that bring about a change in ribosomal protein S9 activity that is comparable to that brought about by the E to K change at position 112 of SEQ ID NO:4. Such a change in activity may, for example, be assessed by means that are well known to the person skilled in the art.
  • the invention thus encompasses, for example, a third nucleic acid molecule that comprises a mutation that modifies the activity of the encoded ribosomal S9 protein, wherein the mutation results in a substitution of glutamic acid to lysine at the equivalent position to the underlined amino acid of SEQ ID NO:4.
  • the invention is exemplified herein by modifying ribosomal protein S9 activity (and specifically by genetically altering the ribosomal protein S9 polypeptide sequence at position 112 from E to K in B.subtilis).
  • the invention may also be achieved by modifying other amino acids within ribosomal protein S9, or by modifying other polypeptides (e.g. other ribosomal proteins), wherein the modification affects ribosomal protein S9 activity in the cell.
  • ribosomal protein S9 activity may be achieved in a number of ways with which the skilled person will be familiar.
  • the ribosomal protein S9 of B subtilis can be acetylated on its N-terminus. This may reflect a regulatory mechanism of ribosomal protein S9 activity.
  • the skilled person should appreciate that there is a high degree of homology between B.subtilis and other eubacterial ribosomal proteins (see Lauber et al., 2009 J Proteome Res. Sep;8(9):4193-206) and thus the modifications exemplified herein within the context of B subtilis are equally applicable to other (suitable) cell types.
  • L-form growth also referred to herein as L-form proliferation, which requires cells in the L-form state to undergo division.
  • A/V abnormal cell surface area to volume
  • the applicants have generated a number of different genetically modified cells to demonstrate this capability. In order to induce L-form growth and division in such cells it is necessary to remove (or partially remove) the peptidoglycan cell wall (if present).
  • (Partial) removal of the peptidoglycan cell wall may be achieved in a number of ways that are well known to the skilled person, including treating the cells with a lysozyme or genetically modifying the cell such that the peptidoglycan wall is (partially) absent.
  • the cells in the absence (or partial absence) of the cell wall, when cell surface area is abnormally increased compared to cell volume, the cells become more vulnerable to cell lysis. This is because although protoplasts (generated by e.g. treatment with lysozyme) are relatively stable, they become more unstable (e.g. vulnerable to cell lysis) when excess membrane synthesis is induced.
  • an osmoprotectant medium may be used to provide optimal conditions for L-form growth and cell viability. Appropriate conditions and media are discussed in more detail below.
  • L-form growth may be supported by modifying the respiratory pathway (also called the respiratory chain herein) of the cell of interest.
  • the applicants have surprisingly demonstrated that inhibition of one or more polypeptides involved in the respiratory chain (for example ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS) in cells capable of L-form growth supports cell viability and proliferation in the L-form state.
  • This aspect of the invention is exemplified herein by inhibiting expression of one or more of ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS.
  • the invention may also be achieved by modifying the expression of other polypeptides involved in the respiratory chain.
  • the invention may also be achieved by modifying the activity of the respiratory chain within the cell in any other suitable way (e.g. by decreasing the efficiency of the respiratory pathway within the cell, or by decreasing the efficiency of or stability of one or more of the polypeptides involved (thereby increasing protein degradation)).
  • the applicants have surprisingly demonstrated that inhibition of one or more polypeptides involved in the glycolysis pathway (for example ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm, and eno) in cells capable of L-form growth supports cell viability and proliferation in the L-form state.
  • one or more polypeptides involved in the glycolysis pathway for example ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm, and eno
  • This aspect of the invention is exemplified herein by inhibiting expression of one or more of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm, and eno.
  • the invention may also be achieved by modifying the expression of other polypeptides involved in the glycolysis pathway.
  • the invention may also be achieved by modifying the activity of the glycolysis pathway within the cell in any other suitable way (e.g. by decreasing the efficiency of the glycolysis pathway within the cell, or by decreasing the efficiency of or stability of one or more of the polypeptides involved (thereby increasing protein degradation)).
  • the invention provides a recombinant cell with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis, wherein the cell comprises at least one of:
  • a first nucleic acid molecule encoding a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3, wherein the first nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase;
  • a second nucleic acid molecule encoding a polypeptide involved in cell wall synthesis, wherein the polypeptide is not murB, murG, murE, mraY or dal, the second nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide;
  • a third nucleic acid molecule encoding ribosomal protein S9 comprising a nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9.
  • the term "recombinant” refers to a biomolecule, for example a gene or a protein that (1) has been removed (e.g. isolated) from its naturally occurring environment, (2) is not associated with all or a portion of a nucleic acid molecule as it is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • a recombinant cell refers to a cell that has been transformed, transfected or transduced with a nucleic acid molecule of the invention.
  • the term refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the terms "host cell” and “recombinant cell” are used interchangeably herein.
  • the host cell may be an aerobic cell or alternatively a facultative anaerobic cell.
  • the cell is a bacterial cell.
  • the cell may be a yeast cell (e.g. Saccharomyces, Pichia), an algae cell, an insect cell, or a plant cell.
  • Bacterial host cells include Gram-positive and Gram-negative bacteria.
  • Suitable bacterial host cells include, but are not limited to the Gram-positive bacteria, for example a bacterium of the genus Bacillus, for example Bacillus brevis, Bacillus subtilis, or Bacillus thuringienesis.
  • the host cell may be of the genus Lactococcus, for example Lactococcus lactis.
  • the bacterial cell is of the actinomycetes family, more particularly from the genus Streptomyces, Rhodococcus, Corynebacterium, Mycobacterium.
  • Streptomyces lividans Streptomyces ambofaciens, Streptomyces fradiae, Streptomyces griseofuscus, Rhodococcus erythropolis, Corynebacterium gluamicum, Mycobacterium smegmatis may be used.
  • the host cell may be of the genus Acinetobacter, for example A.baylyi ADP1.
  • Alternative suitable bacterial host cells include, but are not limited to the Gram-negative bacteria, for example a bacterium of the family Enterobacteria, most preferably Escherichia coli. Expression in E. coli offers numerous advantages, particularly low development costs and high production yields. Cells suitable for high protein expression include, for example, E.CO// W3110, and the B strains of E.coli.
  • coli K12 strains are also preferred as such strains are standard laboratory strains, which are non-pathogenic, and include NovaBlue, JM109 and DH5a (Novogen®), E. coli K12 RV308, E. coli K12 C600, E. coli HB101 , see, for example, Brown, Molecular Biology Labfax (Academic Press (1991)).
  • Enterobacteria from the genera Salmonella, Shigella, Enterobacter, Serratia, Proteus and Erwinia may be suitable.
  • Other prokaryotic host cells include Serratia, Pseudomonas, Caulobacter, or Cyanobacteria, for example bacteria from the genus Synechocystis or Synechococcus.
  • the recombinant cell of the invention has an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis.
  • the phrase "increased propensity" is used herein to indicate that the recombinant cell is more likely (i.e. has an increased tendency) to synthesize fatty acids, fatty acid derivatives and/or membrane associated molecules compared to an equivalent cell that is not recombinant (e.g.
  • an "increased propensity" within this context may be represented by a 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more increase in propensity towards fatty acid, fatty acid derivative and/or membrane associated molecule synthesis.
  • the increases in propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis described above may be represented by a 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more increase in fatty acid, fatty acid derivative and/or membrane associated molecule synthesis in a cell exhibiting such an increased propensity (these increases also suitably being compared to a relevant equivalent cell of the sort described above).
  • fatty acid synthetic pathway can be upregulated to generate cells capable of L-form growth and that this results in increased amounts of membrane (shown using general membrane stains).
  • the membrane comprises hydrophobic material, including fatty acids. Accordingly, excess fatty acids are produced within the context of the invention.
  • fatty acid refers to a carboxylic acid with a long aliphatic tail, which is either saturated or unsaturated.
  • Excess fatty acids produced within the context of the invention may be converted in the cell to fatty acid derivatives of various kinds.
  • excess fatty acids may be converted to phospholipids, including saturated or unsaturated phospholipids; branched or straight chain phospholipids.
  • the produced phospholipids may also have various head groups (for example, but not limited to triglycerides with phosphatidyl-glycerol, -serine, - choline, -ethanolamine etc).
  • Other fatty acid derivatives that may be produced in the context of the present invention include fatty acid alcohols, fatty esters, fatty aldehydes, triglycerides, amphipathic lipids (e.g. glycolipids) and hydrocarbons (e.g. an alkane, alkene or alkyne).
  • the excess hydrophobic material produced within the context of the invention may also support increased accumulation of any other normal constituents of membrane (e.g. membrane associated molecules) that are normally limited by membrane surface area.
  • membrane proteins e.g. membrane associated molecules
  • this may include membrane proteins (integral or peripheral), glycoproteins, isoprenoids, cholesterol-type lipids, and amphipathic lipids (e.g. glycolipids).
  • fatty acid derivatives therefore includes phospholipids of various kinds (e.g. saturated or saturated phospholipids; branched or straight chain phospholipids; phospholipids with various head groups (for example, but not limited to triglycerides with phosphatidyl-glycerol, -serine, - choline, -ethanolamine etc); fatty acid alcohols, fatty esters, fatty aldehydes, triglycerides, amphipathic lipids (e.g. glycolipids) and hydrocarbons (e.g. an alkane, alkene or alkyne) or any other fatty acid derivative with which the skilled person is familiar.
  • phospholipids of various kinds e.g. saturated or saturated phospholipids; branched or straight chain phospholipids; phospholipids with various head groups (for example, but not limited to triglycerides with phosphatidyl-glycerol, -serine, - choline, -ethanol
  • membrane associated molecule includes membrane proteins (integral or peripheral), glycoproteins, isoprenoids, cholesterol-type lipids, and amphipathic lipids (e.g. glycolipids).
  • the recombinant cell of the invention comprises at least one of a first nucleic acid molecule, a second nucleic acid molecule and/or a third nucleic acid molecule (as defined herein).
  • the terms “first”, “second” and “third” are not limiting in any way and, are only used herein for the ease of distinguishing the nucleic acid molecules when, for example, they are presented in a list. It should be clear, therefore, that the effects of the invention can be achieved using only a first nucleic acid molecule, a second nucleic acid molecule or a third nucleic acid molecule.
  • a recombinant cell of the invention may comprise a third nucleic acid molecule, without comprising a first or a second nucleic acid molecule etc.
  • the invention may be seen to provide a recombinant cell with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis, wherein the cell comprises at least one of:
  • nucleic acid molecule encoding a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase;
  • nucleic acid molecule encoding a polypeptide involved in cell wall synthesis, wherein the polypeptide is not murB, murG, murE, mraY or dal, the nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide;
  • nucleic acid molecule encoding ribosomal protein S9, the nucleic acid molecule comprising a nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9.
  • the recombinant cell of the invention comprises at least one of a first nucleic acid molecule, a second nucleic acid molecule and/or a third nucleic acid molecule.
  • the invention also provides the nucleic acid molecules discussed herein per se (i.e. as an isolated nucleic acid molecule or as part of an expression vector).
  • nucleic acid molecule includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., a mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.
  • the nucleic acid molecule can be single- stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and/or 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the term “gene” refers to nucleic acid molecules which includes an open reading frame encoding protein, and can further include non-coding regulatory sequences and introns.
  • the first nucleic acid molecule of the invention encodes a polypeptide comprising a carboxyltransferase, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase.
  • the first nucleic acid molecule of the invention encodes a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule does not comprise a naturally occurring acetyl CoA carboxylase-encoding nucleic acid sequence, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase.
  • the first nucleic acid molecule of the invention encodes a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase.
  • the first nucleic acid molecule of the invention encodes a polypeptide comprising a carboxyltransferase, wherein the first nucleic acid molecule does not comprise a nucleic acid sequence encoding a biotin carboxylase, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase.
  • the first nucleic acid molecule of the invention encodes a polypeptide comprising a carboxyltransferase, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing the expression of the encoded carboxyltransferase within a cell relative to the expression of biotin carboxylase within the cell.
  • the first nucleic acid molecule of the invention encodes a polypeptide consisting of a carboxyltransferase, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase.
  • any of these aspects of the invention may be combined with the other aspects of the invention described herein. Accordingly, any reference to a specific first nucleic acid molecule of the invention may be replaced with reference to a different first nucleic acid molecule of the invention; the first nucleic acid molecules discussed above are therefore interchangeable.
  • Carboxyltransferases their function, amino acid sequence and the nucleotide sequence that encodes them are known in the art (see for example Cronan and Waldrop, 2002). The invention is therefore not limited to the specific carboxyltransferase used in the examples, but encompasses all polypeptides with carboxyltransferase activity, irrespective of source (e.g. cell type).
  • the carboxyltransferase is the carboxyltransferase subunit of acetyl CoA carboxylase.
  • the carboxyltransferase is encoded by the AccDA operon in nature.
  • phrases "consisting of a carboxyltransferase” indicates that the first nucleic acid molecule does not encode a functionally related polypeptide, for example, it does not encode the biotin carboxylase subunit of acetyl CoA carboxylase.
  • nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase
  • a nucleic acid sequence that comprises a promoter capable of increasing expression of the encoded carboxyltransferase e.g. an inducible promoter
  • a nucleic acid sequence that comprises a mutation within (or outside) the carboxyltransferase encoding region where the mutation is capable of increasing expression of the encoded carboxyltransferase.
  • the phrase "increasing the expression of the encoded carboxyltransferase within a cell relative to the expression of biotin carboxylase within the cell” is intended to cover, for example, a nucleic acid sequence that specifically increases the expression of the encoded carboxyltransferase relative to the amount of biotin carboxylase expression within the same cell.
  • the presence of a nucleic acid sequence capable of increasing the expression of the encoded carboxyltransferase within a cell relative to the expression of biotin carboxylase in a cell may alter (i.e.
  • an increase in the relative ratio of carboxyltransferase: biotin carboxylase (expression) may be a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more difference compared to the control.
  • the first nucleic acid molecule may comprise a mutation in the 5' untranslated region (UTR) of the molecule, where the mutation increases expression of the encoded carboxyltransferase.
  • Appropriate mutations in this region include the single point mutation (C to A) at the equivalent position to the underlined nucleic acid residue of SEQ ID NO: 1.
  • the 5'UTR may have at least 70% 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:2.
  • Suitable first nucleic acid molecules with the percentage identities referred to above may retain the C to A mutation set out in SEQ ID NO: 1 and the degrees of variation referred to may arise as a result of changes in other nucleic acid residues.
  • the invention is exemplified herein by modifying the 5'UTR of the carboxyltransferase encoding nucleic acid molecule in B.subtilis.
  • an equivalent modification can be made to the 5'UTR of any nucleic acid molecule that encodes carboxyltransferase in a suitable cell type to achieve the desired effect.
  • the invention is therefore not limited to the specific mutation of the 5'UTR of B.subtilis carboxyltransferase but may be applied equally to mutation of the 5'UTR of B.subtilis carboxyltransferase of other suitable cell types.
  • the invention thus encompasses, for example, a first nucleic acid molecule that comprises a mutation that increases expression of an encoded carboxyltransferase, wherein the mutation is in the 5' untranslated region (UTR) of the nucleic acid molecule and comprises a single point mutation (C to A) at the equivalent position to the underlined nucleic acid residue of SEQ ID NO: 1.
  • UTR 5' untranslated region
  • C to A single point mutation
  • sequence homology or identity (the terms are used interchangeably herein) between sequences may be performed as follows. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”
  • percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410).
  • gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See ⁇ http://www.ncbi.nlm.nih.gov>.
  • a nucleic acid sequence capable of increasing expression refers to a nucleic acid sequence that is capable of increasing the expression of the encoded polypeptide (e.g. a carboxyltransferase) compared to the level of expression observed using an equivalent nucleic acid molecule ("control") that lacks the "nucleic acid sequence capable of increasing expression”.
  • control an equivalent nucleic acid molecule
  • “increased expression” within this context may be represented by a 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more increase in expression compared to the control.
  • the second nucleic acid molecule of the invention encodes a polypeptide involved in cell wall synthesis, wherein the polypeptide is not murB, murG, mraY or dal, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • Polypeptides involved in cell wall synthesis, their function, amino acid sequence and the nucleotide sequence that encodes them are known in the art (see for example Typas et al 201 1 ; see also Leaver et al, 2009).
  • the invention is therefore not limited to the specific polypeptides used in the examples, but encompasses all polypeptides with the equivalent activity, irrespective of source (e.g. cell type).
  • the polypeptide may be involved in peptidoglycan synthesis, wall teichoic acid synthesis, or the regulation of either (or both) of these synthetic pathways.
  • polypeptides generally encompassed by the invention include: any one (or more) of murB, murG, murE, mraY, murF, dapF, racE, yrpC, murAA, dal and murC (involved in peptidoglycan synthesis); one (or more) of tagA, tagB, tagD, tagE, tagF, manA and tagO (involved in wall teichoic acid synthesis); and/or one (or more) of glmS, glmM, gcaD and mreB (involved in the regulation of one or both of these synthetic pathways).
  • polypeptides may be present within the recombinant cell of the invention (if there is more than one, it may be any combination, and is not limited to combinations of polypeptides involved in the same synthetic pathway or different pathways).
  • the particular polypeptides encompassed by the invention include a polypeptide involved in cell wall synthesis, wherein the polypeptide is not murB, murG, murE, mraY or dal.
  • the second nucleic acid molecule may encode a polypeptide involved in cell wall synthesis, wherein the polypeptide is not murB, murG, murE, mraY or dal, the second nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • the polypeptide is not murB, murG, murE, mraY or dal
  • the second nucleic acid molecule comprising a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • Each of the polypeptides discussed above (and their function) is well known in the art. The invention is therefore not limited to the specific polypeptides used in the examples, but encompasses all polypeptides with the same functional activity, irrespective of source (e.g. cell type).
  • nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the [encoded polypeptide]
  • a nucleic acid sequence that comprises a promoter capable of inhibiting expression of the encoded polypeptide e.g. a repressible promoter
  • nucleic acid sequence capable of inhibiting expression refers to a nucleic acid sequence that is capable of inhibiting the expression of the encoded polypeptide compared to the level of expression observed using an equivalent nucleic acid molecule ("control") that lacks the "nucleic acid sequence capable of inhibiting expression".
  • control an equivalent nucleic acid molecule
  • inhibitor is used herein to encompass partial inhibition as well as total inhibition.
  • expression is inhibited within this context when there is a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more (up to 100%) reduction in expression compared to the control.
  • the third nucleic acid molecule of the invention encodes ribosomal protein S9, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9.
  • Ribosomal protein S9, its function, amino acid sequence and the nucleotide sequence that encodes it are known in the art (see for example Brodersen et al., 2002). The invention is therefore not limited to the specific ribosomal protein S9 used in the examples, but encompasses all polypeptides with the equivalent activity, irrespective of source (e.g. cell type).
  • the third nucleic acid molecule comprises a nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9.
  • a nucleic acid sequence capable of modifying the activity of [the encoded polypeptide] is intended to cover, for example, any nucleic acid sequence that modifies the activity of the encoded polypeptide in the cell such that it provides the cell with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis compared to the propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis observed using an equivalent nucleic acid molecule ("control") that lacks the "nucleic acid sequence capable of modifying the activity of [the encoded polypeptide]".
  • nucleic acid sequence capable of modifying the activity of the encoded ribosomal protein S9 of B.subtilis may comprise a mutation that results in substitution of the amino acid glutamic acid (E) at position 1 12 in the encoded polypeptide with lysine (K).
  • the fourth nucleic acid molecule of the invention encodes a polypeptide involved in the respiratory chain, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • Polypeptides involved in the respiratory chain, their function, amino acid sequence and the nucleotide sequence that encodes them are known in the art (see for example Zamboni et al., 2003).
  • the invention is therefore not limited to the specific polypeptides used in the examples, but encompasses all polypeptides with the equivalent activity, irrespective of source (e.g. cell type).
  • Examples of particular polypeptides generally encompassed by this aspect of the invention include: any one (or more) of ispA, ispC, aroB, aroC, ndh, qoxB, ctaB, mhqR, nsr, speA and hepS.
  • the fourth nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • the definitions provided above with respect to the equivalent feature of the second nucleic acid molecule apply equally here.
  • the fifth nucleic acid molecule of the invention encodes a polypeptide involved in the glycolysis pathway, wherein the nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • Polypeptides involved in the glycolysis pathway, their function, amino acid sequence and the nucleotide sequence that encodes them are known in the art (see for example Fujita Y 2009).
  • the invention is therefore not limited to the specific polypeptides used in the examples, but encompasses all polypeptides with the equivalent activity, irrespective of source (e.g. cell type).
  • polypeptides generally encompassed by this aspect of the invention include: any one (or more) of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.
  • ptsH any one (or more) of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.
  • One or a combination of these polypeptides may be present within the recombinant cell of the invention.
  • the fifth nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the encoded polypeptide.
  • the definitions provided above with respect to the equivalent feature of the second nucleic acid molecule apply equally here.
  • nucleic acid molecules described herein may comprise specific changes in the nucleotide sequence so as to optimize codons and mRNA secondary structure for translation in the host cell.
  • the codon usage of the nucleic acid is adapted for expression in the host cell, for example codon optimisation can be achieved using Calcgene, Hale, RS and Thomas G. Protein Exper. Purif. 12, 185-188 (1998), UpGene, Gao, W et al. Biotechnol. Prog. 20, 443-448 (2004), or Codon Optimizer, Fuglsang, A. Protein Exper. Purif. 31 , 247- 249 (2003).
  • Amending the nucleic acid according to the preferred codon optimization can be achieved by a number of different experimental protocols, including, modification of a small number of codons, Vervoort et al. Nucleic Acids Res. 25: 2069-2074 (2000), or rewriting a large section of the nucleic acid sequence, for example, up to 1000 bp of DNA, Hale, RS and Thomas G. Protein Exper. Purif. 12, 185-188 (1998). Rewriting of the nucleic acid sequence can be achieved by recursive PCR, where the desired sequence is produced by the extension of overlapping oligonucleotide primers, Prodromou and Pearl, Protein Eng. 5: 827- 829 (1992).
  • the level of cognate tRNA can be elevated in the host cell. This elevation can be achieved by increasing the copy number of the respective tRNA gene, for example by inserting into the host cell the relevant tRNA gene on a compatible multiple copy plasmid, or alternatively inserting the tRNA gene into the expression vector itself.
  • nucleic acid molecules described herein may comprise specific changes in the nucleotide sequence so as to optimize expression, activity or functional life of the encoded polypeptide(s).
  • the nucleic acids described previously are subjected to genetic manipulation and disruption techniques.
  • Various genetic manipulation and disruption techniques are known in the art including, but not limited to, DNA Shuffling (US 6, 132,970, Punnonen J et al, Science & Medicine, 7(2): 38-47, (2000), US 6,132,970), serial mutagenesis and screening.
  • mutagenesis is error-prone PCR, whereby mutations are deliberately introduced during PCR through the use of error-prone DNA polymerases and reaction conditions as described in US 2003152944, using for example commercially available kits such as The GeneMorph ® II kit (Stratagene ® , US). Randomized DNA sequences are cloned into expression vectors and the resulting mutant libraries screened for altered or improved protein activity.
  • Expression vector
  • the invention provides the expression vectors discussed herein per se.
  • nucleic acid molecules of the invention may be present within an expression vector of the invention (if there is more than one, it may be any combination, and is not limited to combinations that encode polypeptides involved in the same synthetic pathway or different pathways).
  • the term “vector” or “construct” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the terms “vector” and “construct” are used interchangeably herein.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • the vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers.
  • the vector can be a nucleic acid in the form of a plasmid, a bacteriophage or a cosmid.
  • the vector is suitable for expression in a cell (i.e. the vector is an "expression vector”).
  • the expression vector is suitable for expression in any appropriate cell.
  • the vector is suitable for expression in bacteria.
  • the vector is a bacterial expression vector.
  • the vector is capable of propagation in a host cell and is stably transmitted to future generations.
  • Operaably linked refers to a single or a combination of the below-described control elements together with a coding sequence in a functional relationship with one another, for example, in a linked relationship so as to direct expression of the coding sequence.
  • Regulatory sequences refers to, DNA or RNA elements that are capable of controlling gene expression.
  • expression control sequences include promoters, enhancers, silencers, Shine Dalgarno sequences, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites for transcription factors, transcriptional terminators, polyadenylation sites, RNA transporting signals or sequences important for UV-light mediated gene response.
  • the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissue-specific regulatory and/or inducible sequences.
  • Promoter refers to the nucleotide sequences in DNA or RNA to which RNA polymerase binds to begin transcription.
  • the promoter may be inducible or constitutively expressed.
  • the promoter is under the control of a repressor or stimulatory protein.
  • the promoter is a T7, T3, lac, lac UV5, tac, trc, [lambda]PL, Sp6 or a UV-inducible promoter.
  • Transcriptional terminator refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA.
  • Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.
  • Translational control element refers to DNA or RNA elements that control the translation of mRNA.
  • Preferred translational control elements are ribosome binding sites.
  • the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site.
  • Preferred ribosome binding sites are T7 or T3 ribosome binding sites.
  • Restriction enzyme recognition site refers to a motif on the DNA recognized by a restriction enzyme.
  • Selectable marker as used herein, refers to proteins that, when expressed in a host cell, confer a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene.
  • this may be a protein that confers resistance to an antibiotic such as ampicillin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate.
  • antibiotics include Penicillins; Ampicillin HCI, Ampicillin Na, Amoxycillin Na, Carbenicillin sodium, Penicillin G, Cephalosporins, Cefotaxim Na, Cefalexin HCI, Vancomycin, Cycloserine.
  • Bacteriostatic Inhibitors such as: Chloramphenicol, Erythromycin, Lincomycin, Tetracyclin, Spectinomycin sulfate, Clindamycin HCI, Chlortetracycline HCI.
  • the design of the expression vector depends on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such vectors are within the scope of the present invention.
  • the vector comprises those genetic elements which are necessary for expression of the desired polypeptide in a host cell.
  • the elements required for transcription and translation in the host cell include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.
  • Expression vectors of the invention can be bacterial expression vectors, for example recombinant bacteriophage DNA or plasmid DNA.
  • the vector is suitable for expression in the target host cell.
  • the vector is suitable for integration into a host chromosome.
  • plasmid pMutin4 or pSG1 154 may be suitable.
  • the plasmid pMutin4 or derivatives (Vagner et al, 1998) may be used to construct IPTG-inducible or inactivation B. subtilis mutants.
  • the first 300 bp of the gene, containing the Shine-Dalgarno (SD) sequence may amplified by PCR using the specific primers and then cloned into plasmid pMutin4 or derivatives. The resulting plasmid may used to transform B.
  • subtilis strains with selection for, for example, erythromycin.
  • the gene is then expressed from the IPTG inducible promoter P spac on the B. subtilis chromosome.
  • An internal segment (150-300 bp) of the gene may be cloned to construct the inactivation B. subtilis mutants.
  • the plasmid pSG1 154 or derivatives may be used to construct the xylose inducible gene expression system described herein at the amyE locus.
  • the gene may be amplified by PCR and then cloned into plasmid pSG1154 or derivatives.
  • the resulting plasmid may be used to transform B. subtilis strains, with selection for, for example, spectinomycin resistance.
  • the gene is then expressed from the xylose inducible promoter P xy , at the amyE locus on the B. subtilis chromosome.
  • a suitable plasimd may be integrated into the B. subtilis chromosome by homologous reconbination.
  • the expression vector is a high-copy-number expression vector; alternatively, the expression vector is a low -copy-number expression vector, for example, a Mini-F plasmid.
  • the nucleic acid molecule for incorporation into the expression vector of the invention can be prepared by synthesizing nucleic acid molecules using mutually priming oligonucleotides and the nucleic acid sequences described herein.
  • a number of molecular techniques have been developed to operably link DNA to vectors via complementary cohesive termini.
  • complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA.
  • the vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • synthetic linkers containing one or more restriction sites are used to operably link the nucleic acid molecule to the expression vector.
  • the nucleic acid molecule is generated by restriction endonuclease digestion.
  • the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3'-single-stranded termini with their 3'-5'- exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities, thereby generating blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the product of the reaction is a nucleic acid molecule carrying polymeric linker sequences at its ends.
  • These nucleic acid molecules are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the nucleic acid molecule.
  • a vector comprising ligation-independent cloning (LIC) sites can be employed.
  • the required PCR amplified nucleic acid molecule can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, Nucl. Acid. Res. 18, 6069-6074, (1990), Haun, et al, Biotechniques 13, 515-518 (1992).
  • PCR In order to isolate and/or modify the nucleic acid molecule of interest for insertion into the chosen plasmid, it is preferable to use PCR.
  • Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.
  • a nucleic acid molecule for incorporation into an expression vector of the invention is prepared by the use of the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491 , using appropriate oligonucleotide primers.
  • the coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product.
  • the amplification primers contain restriction endonuclease recognition sites which allow the amplified sequence product to be cloned into an appropriate vector.
  • the nucleic acid molecule of interest is obtained by PCR and introduced into an expression vector using restriction endonuclease digestion and ligation, a technique which is well known in the art.
  • the nucleic acid molecule of interest is introduced into an expression vector by yeast homologous recombination (Raymon et al., Biotechniques. 26(1): 134-8, 140-1 , 1999).
  • the expression vectors of the invention can contain a single copy of a nucleic acid molecule described previously, or multiple copies of the nucleic acid molecule described previously.
  • a host cell can be transformed, transduced or transfected with an expression vector of the invention, comprising a nucleic acid molecule as described previously.
  • the expression vector of the present invention can be introduced into the host cell by conventional transformation, transduction or transfection techniques.
  • Transformation transformation
  • transduction and “transfection” are used interchangeably herein to refer to a variety of techniques known in the art for introducing foreign nucleic acids into a cell.
  • Transformation of appropriate cells with an expression vector of the present invention is accomplished by methods known in the art and typically depends on both the type of vector and cell. Said techniques include, but are not limited to calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, chemoporation or electroporation. Techniques known in the art are disclosed in for example, Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637-646. All such methods are incorporated herein by reference.
  • Transformations of competent B. subtilis cells may be performed by the two-step starvation procedure as previously described (Anagnostopoulos and Spizizen, 1961 ;Hamoen et al., 2002). Briefly, cells are inoculated in Spizizen minimal medium with glucose, MgS0 4 , salts mix, Casamino acids, Tryptophan and other supplements if appropriate. The cells are incubated with shaking at 37°C until OD 6 oonm of 1. Then an equal volume of Spizizen minimal medium with glucose, MgS0 4 is added to the culture. After incubation for an hour with shaking at 37°C, The plasmid or genomic DNA is added to the cell and incubated with shaking prior plating on appropriate selection plate.
  • the invention comprises a culture of recombinant cells.
  • the culture is clonally homogeneous.
  • the recombinant cell can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.
  • the expression vectors of the invention may express the nucleic acid molecule incorporated therein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California, 1 19-128).
  • the nucleic acid molecule incorporated into an expression vector of the invention can be altered so that the individual codons for each amino acid are those preferentially utilized in the chosen host cell (Wada et al., (1992) Nucleic Acids Res. 20:21 11-2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • a semi-synthetic or synthetic cell is provided with an increased propensity for fatty acid, fatty acid derivative and/or membrane associated molecule synthesis, the cell having at least one of:
  • Semi-synthetic or synthetic cells according to the invention could serve many purposes.
  • the types of semi-synthetic or synthetic cells that could be generated using the invention would be particularly suited to applications aimed at production of various membrane associated factors, such as fatty acids, fatty acid derivatives and/or membrane associated molecules. They could also be used as delivery vehicles for drugs which could be designed to fuse with other cells, or to deliver vaccines.
  • membrane associated factors such as fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • drugs could be designed to fuse with other cells, or to deliver vaccines.
  • the present invention thus provides a way of generating cell wall free cells that will proliferate efficiently.
  • a "semi-synthetic cell or synthetic cell” refers to membrane bound components that are not found in nature. A skilled person would readily be able to identify semi-synthetic and synthetic cells that fall within the invention.
  • the phrase "semi-synthetic cell or synthetic cell” is intended to encompass organisms and cells with reduced genomes and/or organisms and cells that have been "re-booted” from in vitro synthesised DNA (see Leprince et al. 2012; Gibson et al. 2010).
  • Such cells and organisms could undergo further genome reduction if induced to grow in the L-form state because all genes associated with cell wall synthesis (including peptidoglycan, teichoic acids, capsular polysaccharide, etc) or cell division (including divlB, divlVA, ftsA, ftsB, ftsE, ftsl, ftsK, ftsL, ftsN, ftsQ, ftsZ and others) could be deleted from (genome reduction) or omitted from (re-booting) the genome sequences.
  • all genes associated with cell wall synthesis including peptidoglycan, teichoic acids, capsular polysaccharide, etc
  • cell division including divlB, divlVA, ftsA, ftsB, ftsE, ftsl, ftsK, ftsL, ftsN, ftsQ, f
  • control may be expression observed in an equivalent semi-synthetic or synthetic cell that lacks one, more than one, or all of:
  • an agent such as a nucleic acid
  • a semi-synthetic or synthetic cell of the invention that provides such increases or decreases in expression, or modified activity.
  • the semi-synthetic or synthetic cell of the invention may have at least one of:
  • a method of producing fatty acids, fatty acid derivatives and/or membrane associated molecules comprises providing to a cell at least one of:
  • the method further comprises providing to the cell at least one of:
  • An agent may be any chemical, compound, small molecule, composition, protein, drug, nucleic acid, expression vector etc capable of carrying out the desired function of increasing carboxyltransferase activity, decreasing cell wall synthesis or modifying ribosomal protein S9 activity.
  • an agent may be an inhibitor of cell wall synthesis; or an activator of carboxyltransferase activity.
  • an agent may be an antibiotic, for example an antibiotic that is an inhibitor of cell wall synthesis, such as fosfomycin, D-cycloserine, penicillin G, and/or ampicillin.
  • an antibiotic for example an antibiotic that is an inhibitor of cell wall synthesis, such as fosfomycin, D-cycloserine, penicillin G, and/or ampicillin.
  • an agent comprises (or is) a nucleic acid molecule in accordance with the invention.
  • an agent e.g. nucleic acid molecule
  • the cell may be a recombinant cell in accordance with the invention.
  • the cell may be any other cell. Appropriate cell types are discussed above in the context of different aspects of the invention, but apply equally here.
  • an "agent that increases carboxyltransferase activity” encompasses agents that are capable of increasing expression of carboxyltransferase, or are capable of increasing carboxyltransferase functionality within the cell in any other way.
  • An agent increases carboxyltransferase activity when an increase in carboxyltransferase activity is observed in the presence of agent compared to carboxyltransferase activity observed in the absence of agent (control).
  • increased activity may be represented by a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more increase in activity compared to the control.
  • an agent that "decreases" cell wall synthesis, respiratory chain activity or activity of the glycolysis pathway encompasses agents that are capable of decreasing expression of polypeptides involved in the relevant pathway or activity (as detailed above), or are capable of decreasing functionality of the relevant pathway or activity within the cell in any other way.
  • An agent decreases cell wall synthesis, respiratory chain activity or activity of the glycolysis pathway when a decrease in cell wall synthesis, respiratory chain activity or activity of the glycolysis pathway is observed in the presence of agent compared to the level of cell wall synthesis, respiratory chain activity or activity of the glycolysis pathway observed in the absence of agent (control).
  • decreased activity may be represented by a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more (up to 100%) reduction in activity compared to the control.
  • an agent that "modifies" ribosomal protein S9 activity encompasses agents that are capable of modifying the functionality of ribosomal protein S9 within the cell.
  • An agent modifies ribosomal protein S9 activity when a change in activity is observed in the presence of agent compared to the activity observed in the absence of agent ("control").
  • modified activity may represent a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more difference in activity compared to the control.
  • Another aspect of the invention provides a method of inducing L-form growth in a cell.
  • the method comprises providing to a cell at least one of:
  • the method further comprises providing to the cell at least one of:
  • an agent that "removes the cell wall and/or prevents cell wall synthesis” can readily be screened for and identified by the skilled person.
  • the agent may be a lysozyme.
  • any one of a large number of cell wall active antibiotics e.g. beta- lactam antibiotics (e.g. penicillins, cephalosporins, monobactams), glycopeptides (e.g. vancomycin, teicoplanin), bacitracin, moeonomycin) may be used within the context of the invention as an agent that removes the cell wall and/or prevents cell wall synthesis.
  • inhibitors of cell division such as benzamides
  • Penicillin G may also be used.
  • Agents such as Penicillin G or benzamide (8j) (FtsZ inhibitor) prevent reverse mutation in cell wall defective mutants and/or re-growth of cell wall from L-form or protoplast. Any aspects of the invention discussed above in the context of other methods of the invention may equally be applied here.
  • a method of identifying a DNA mutation that supports L-form growth in a cell comprising
  • the cell has previously been provided with at least one of:
  • step (iii) of the method further comprises culturing the identified cell under conditions that support cell wall regeneration and identifying a cell with a regenerated cell wall.
  • An agent that is capable of removing the cell wall is described elsewhere herein. All terms defined above in respect of other aspects of the invention apply equally here.
  • a "protoplast” refers to a cell that (substantially) lacks a cell wall. Identification of cells capable of L-form growth (or identification of cells with a regenerated cell wall) may be carried out using standard procedures known in the art. For example, L- form growth may be observed in medium containing cell wall synthesis or cell division inhibitors, using time-lapse phase contrast, dark field or fluorescence microscopy. Identification of DNA mutation(s) that support L-form growth in a cell may be carried out using standard procedures known in the art. For example, a genetic screen by spontaneous, chemical or transposon mutagenesis may be used. By way of example, the sites of the mutations can be determined by sequencing the end junctions of the transposon insertions or by whole genome sequencing.
  • the methods of the invention may comprise culturing the cell under conditions that support the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.
  • the culture medium also called “growth medium”, “medium” or “media” herein
  • the culture media is sufficient to support the growth of the host cell. Descriptions of suitable culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
  • Cells may be grown in a liquid medium comprising one or more of a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, inorganic salts, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100°C, preferably between 10°C and 60°C, while gassing in oxygen.
  • a carbon source usually in the form of sugars
  • a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, inorganic salts, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100°C, preferably between 10°C and 60°C, while gassing in oxygen.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • Examples of carbon sources are glucose, carbon dioxide, sodium bicarbonate, bicarbonate, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • sucrose is a suitable carbon source for cells in the L-form state
  • glucose is an optimal carbon source for cells in the normal walled state
  • suitable carbon sources may also be used such as sucrose, fructose, glycerol, succinate and malate etc.
  • Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining.
  • the addition of mixtures of a variety of carbon sources may also be advantageous.
  • Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds.
  • nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.
  • Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.
  • Chelating agents may be added to the medium in order to keep the metal ions in solution.
  • Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.
  • the culture media used according to the invention may also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
  • growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium.
  • the exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach” (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp.
  • Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
  • the pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not.
  • the cultures can be grown batchwise, semi-batchwise or
  • a fed batch and/or continuous culture can be used to generate the required yield of fatty acid, fatty acid derivative and/or membrane associated molecule.
  • Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously.
  • the products produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography.
  • the host cells can advantageously be disrupted beforehand.
  • the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.
  • All media components are sterilized, either by heat (20 min at 1.5 bar and 121 °C) or by filter sterilization.
  • the components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
  • the culture temperature will vary depending on the particular experiment and the host cell.
  • the culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40°C, more preferably at from 25 to 37 °C and may be kept constant or may be altered during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid.
  • Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters.
  • suitable substances having a selective effect for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture.
  • the temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C.
  • cells may be grown in medium that supports L-form growth.
  • Suitable medium can readily be identified by the skilled person.
  • the medium may comprise an osmoprotectant (such as sucrose) medium that supports growth of the host cell.
  • the medium may further comprise an inhibitor of cell division (such as benzamide; Adams et al., 201 1) that efficiently kills rods but not L-forms.
  • the osmoprotective medium may comprise 20 mM MgCI 2 , 500 mM sucrose and 20 mM maleic acid in nutrient broth (NB, Oxoid).
  • cells that are in the L-form state may be grown under conditions (e.g. in medium) that support cell wall regeneration.
  • Suitable medium can readily be identified by the skilled person.
  • cell wall regeneration can be induced when excess membrane synthesis is inhibited under conditions where there is no cell wall synthesis inhibitor and inhibitor of cell division.
  • the medium may comprise 0.5 M succinate, 0.5% casamino acids, 0.5% yeast extract, 0.5% glucose, 0.35% K 2 HP0 4 , 0.15% KH 2 P0 4 , 20 mM MgCI 2 , 0.01 % BSA and 1 % agar at pH7.3.
  • the culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 24 to 96 hours.
  • the resultant media can then be processed further (e.g. to recover the produced fatty acids, fatty acid derivatives and/or membrane associated molecules).
  • the fatty acids, fatty acid derivatives and/or membrane associated molecules may, according to requirement, be removed completely or partially from the broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It may be advantageous to process the fatty acids, fatty acid derivatives and/or membrane associated molecules after its separation.
  • the methods of the invention may include culturing the cells in conditions that promote direct product (i.e. fatty acids, fatty acid derivatives and/or membrane associated molecules) secretion for easy recovery without the need to extract biomass.
  • direct product i.e. fatty acids, fatty acid derivatives and/or membrane associated molecules
  • the fatty acids, fatty acid derivatives and/or membrane associated molecules are secreted directly into the culture medium.
  • the secreted products are easily recovered and can be used directly or used with minimal processing.
  • Product recovery efficiency is an important determinant of the total production cost. Techniques known in the art for the large scale culture of host cells are disclosed in for example, Bailey and Ollis (1986) Biochemical Engineering Fundamentals, McGraw-Hill, Singapore; or Shuler (2001) Bioprocess Engineering: Basic Concepts, Prentice Hall. All such techniques are incorporated herein by reference.
  • Transformed host cells can be cultured in aerobic or anaerobic conditions.
  • aerobic conditions preferably, oxygen is continuously removed from the culture medium, by for example, the addition of reductants or oxygen scavengers, or, by purging the reaction medium with neutral gases.
  • the host cells of the invention can be cultured in a vessel, for example a bioreactor.
  • Bioreactors for example fermenters, are vessels that comprise cells or enzymes and typically are used for the production of molecules on an industrial scale.
  • the molecules can be recombinant proteins or compounds that are produced by the cells contained in the vessel or via enzyme reactions that are completed in the reaction vessel.
  • cell based bioreactors comprise the cells of interest and include all the nutrients and/or co- factors necessary to carry out the reactions.
  • the method comprises culturing the host cell in the presence of an antibiotic, where said antibiotic selects for the presence of a corresponding "selectable marker" on the expression vector of the invention in the host cell.
  • the cells of the invention may be used in drug or vaccine delivery.
  • the L-form cells of the invention could be engineered to express genes encoding immunogenic proteins, peptides or peptide derivatives.
  • Immunogenic molecules could be displayed on the cell surface or be cytosolic. Cell surface display would enable direct interaction with the surfaces of immune cells. This would have the advantage that the L-form cells would be deficient in various immunogenic molecules that are normally associated with bacterial cell surfaces, including peptidoglycans of various types, anionic polymers of teichoic acids, teichuronic acids, capsular polysaccharides, flagella, curli, pili or fimbrae, and which might cause inappropriate or noon-specific immune reactions.
  • L-form cells could be engineered to express therapeutic molecules which would be delivered by fusion with eukaryotic cells bearing particular cell surface receptors. Such fusion would not normally occur with walled forms of bacteria in which the cell cytoplasmic membrane is covered by the wall and other envelope layers.
  • a method of preparing a therapeutic composition comprising:
  • subtilis L-forms were grown in osmoprotective medium composed of 2 x MSM media pH 7 (40 mM MgCI 2 , 1 M sucrose and 40 mM maleic acid) mixed 1 : 1 with 2 x nutrient broth (NB, Oxoid) or 2 x NA. DM3 medium pH 7.3 (0.5 M succinate, 0.5% casamino acids, 0.5% yeast extract, 0.5% glucose, 0.35 % K 2 HP0 4 , 0, 15% KH 2 P0 4 , 20 mM MgCI 2 , 0.01 % BSA and 1 % agar) (Bourne and Dancer, 1986) was used to regenerate cell wall from B. subtilis L-forms.
  • 2 x MSM media pH 7 40 mM MgCI 2 , 1 M sucrose and 40 mM maleic acid
  • NB x nutrient broth
  • DM3 medium pH 7.3 0.5 M succinate, 0.5% casamino acids, 0.5% yeast extract, 0.5% glucose, 0.
  • antibiotics were added to media at the following concentrations: cerulenin, 2 ⁇ g/ml or 10 ⁇ g/ml; ampicillin, 100 ⁇ g/ml; chloramphenicol, 5 ⁇ g/ml; kanamycin, 5 ⁇ g/ml; spectinomycin, 50 ⁇ g/ml; erythromycin, 1 ⁇ g/ml or 0.2 ⁇ g/ml; and tetracycline, 10 ⁇ g/ml.
  • spc spectinomycin
  • kan kanamycin
  • erm erythromycin
  • neo neomycin
  • cat chloramphenicol
  • fef.tetracyclin fef.tetracyclin
  • mcs multiple cloning site
  • bla ⁇ -lactamase
  • lacZ ⁇ -galactosidase
  • Protoplasts were prepared as described by (Dominguez-Cuevas et al., 2012). Briefly, an exponential cell culture (OD 6 oonm of 0.2) was harvested and re-suspended in NB/MSM medium containing lysozyme (500 ⁇ g/ml) and benzamide. After incubation at 37°C with shaking for 1 h, the cell cultures were diluted at 10 "3 into fresh NB/MSM containing benzamide and supplements, if required. The cell cultures were incubated at 30°C without shaking and samples were removed about every 12h for measurement.
  • osmoprotective medium composed of: 20 mM MgCI2, 500 mM sucrose and 20 mM maleic acid in nutrient broth (NB, Oxoid).
  • osmoprotective medium composed of: 20 mM MgCI2, 500 mM sucrose and 20 mM maleic acid in nutrient broth (NB, Oxoid).
  • NB nutrient broth
  • Strains LR2 (ispA P X yi-murE) and RM84 (ispA accDA*) were grown in the rod state, then converted to protoplasts with lysozyme treatment and cultured in osmoprotective medium containing various carbon sources, respectively. In both strains, significant L-form growth was seen in medium containing glucose, although the growth rate was slower than sucrose containing medium.
  • a protoplast suspension of strain 168CA were diluted at 10 "2 into fresh NB/MSM containing benzamide and incubated at 30°C without shaking for several days.
  • Genomic DNA of a proliferating L-form culture was extracted and the mutations were identified by whole genome sequencing.
  • protoplasts of strain RM81 (ispA) were diluted at 10 "2 into fresh NB/MSM containing benzamide and incubated at 30°C without shaking for several days.
  • Proliferating L-form cultures were diluted at 10 "3 into fresh medium and incubated at 30°C for 3 days.
  • L-form cultures were diluted at 10 "3 in the protoplast regeneration DM3 medium and incubated at 30°C for 3 days.
  • Regenerated (walled) rod shape cell cultures were chosen and the intrinsic ability of these mutants to grow as L-forms was monitored by protoplasting and transfer back into L-form medium (NB/MSM).
  • Genomic DNA of the selected mutants was extracted and the mutations were identified by whole genome sequencing.
  • the intracellular concentrations of FtsZ or histidine-tagged AccA were determined using Western blot analyses as previously described (Ishikawa et al., 2006).
  • B. subtilis cells were grown in LB medium at 37°C, and at an OD 600 of 0.5 a 10 ml sample was taken. After centrifugation, the cells were lysed by lysozyme, mixed with SDS sample buffer, heat denatured them, and loaded onto a SDS-PAGE gel for western blot analysis. For quantitative real time PCR, cultures were grown in 5 ml of LB medium with or without appropriate supplements, at 37°C.
  • RNA samples were harvested at an OD600nm of 0.8, and total RNA was isolated and retro-transcribed ( ⁇ g) as previously described (Dominguez- Cuevas et al., 2012).
  • cDNA samples were diluted 1 :80.
  • Four ⁇ of cDNA were added to 10 ⁇ of MESA Blue qPCR Master Mix Plus (Eurogentec), 2 ⁇ of each primer (1 ⁇ stock) and 2 ⁇ of H 2 0.
  • qPCR was performed on a Rotor-Gene Q cycler (Qiagen) with 40 cycles of 5s at 95°C and 60s at 60°C. Cycle and threshold were obtained according to the manufacturer instruction. Control genes (noc and soj) were used as references for comparison with the genes of interest. Changes in expression given are the average of three biological replicates.
  • B. subtilis L-form cells were imaged in ibiTreat adherent, 35 mm sterile glass bottom microwell dishes (ibidi GmbH, Kunststoff, Germany). Cells were prepared as previously described (Mercier et al., 2012). The cells were imaged on a DeltaVision® RT microscope (Applied Precision, Washington, USA) controlled by softWoRx (Applied Precision) with a Zeiss *100 apo fluor oil immersion lens. A Weather Station environmental chamber (Precision Control) regulated the temperature of the stage.
  • V r 4/3 ⁇ / ⁇ 3 + ⁇ ⁇
  • the medium contains an osmoprotectant (sucrose) and an inhibitor of cell division (benzamide; Adams et al., 201 1) that efficiently kills rods but not L-forms.
  • an osmoprotectant sucrose
  • benzamide an inhibitor of cell division
  • reversion of protoplasts or L-forms to the walled state regeneration
  • Figure 1A shows the transition from protoplasts to proliferating L-forms, for strain LR2 (ispA P xyr murE-B).
  • Figure 1 B shows a detailed time lapse of a small group of cells over a period of 395 min in L-form medium. Hashes point to the remains of cells that had undergone lysis at some point after the preceding frame. Arrowheads point to these cells in previous frames during which they exhibited L-form like shape changes.
  • the Pxyl-murE-B construct allows repression of an operon containing 4 different genes encoding enzymes of the peptidoglycan (PG) precursor synthetic pathway: murE, mraY, murG and murB.
  • PG peptidoglycan
  • Several other genes from this pathway were also tested to see if they also promoted L-form growth. Repression of all three other genes tested, murAA, murC, and dal, also allowed L-form growth on L-form selective plate, suggesting that these mutations also induce excess membrane production.
  • PG peptidoglycan
  • the cell wall has two major components: peptidoglycan (PG) and the PG- attached anionic cell wall polymer, wall teichoic acid (WTA).
  • PG peptidoglycan
  • WTA wall teichoic acid
  • TagO protein carries out the first step in the WTA biosynthetic pathway.
  • the repression of tagO in the ispA mutant allowed L- form growth on an L-form selective plate, suggesting that the repression of tagO also induces excess membrane production. This result suggests that repression of later steps in the WTA biosynthetic pathway might also support L-form growth and excess membrane production.
  • iii) Candidate gene approach in mreB cytoskeletal proteins
  • MreB cytoskeleton In B. subtilis, the MreB cytoskeleton (MreB, Mbl and MreBH) somehow spatially regulates the synthesis of PG and WTA.
  • the triple mreB mutant is lethal in walled state, but in the presence of ispA mutation the cells were able to grow as L-form on L-form selective plate. Therefore, mutation in mreB genes might induce excess membrane synthesis.
  • Figure 3E shows that the concentration of AccA-His was substantially raised in the presence of the accDA* mutation (lane 4), and that this was not affected by presence or absence of an ispA mutation (lanes 2 and 3). As expected, no signal was seen in the absence of the his tag (lane 1). FtsZ was used as an internal control, and its concentration was not affected by any of the mutations (Figure 3E).
  • Acetyl-CoA carboxylase comprising biotin carboxylase (AccBC) and carboxyltransferase (AccDA), carries out the first committed step of fatty acid synthesis, the conversion of acetyl CoA to malonyl CoA (Cronan and Waldrop, 2002).
  • fatty acids are synthesized by a repeated cycle of reactions catalyzed by the fatty acid synthase type II enzyme (FAS II) system ( Figure 4A) (Rock and Cronan, 1996).
  • the first enzyme in the pathway, FabD converts malonyl CoA to malonyl ACP, the key substrate for the initiation and elongation cycles ( Figure 4A) (Rock and Cronan, 1996).
  • both phenotypes should be dependent on activity of the various FAS II enzymes.
  • An IPTG-dependent promoter was inserted in front of several genes encoding FAS II enzymes. As shown in Figures 4G and 10E, at low levels of IPTG all three constructs supported growth in the walled state on plates, despite the normally lethal effects of AccDA overproduction, presumably due to the reduction in fatty acid synthesis. Indeed, suppression was obtained even at saturating levels of IPTG, for the plsX and fabD constructs (not shown), suggesting that high levels of their protein products are required for the lethal effect. In the case of the fabHA construct suppression of lethality was only seen in the fully repressed (no IPTG) state.
  • mutations in genes that may be expected to promote L-form growth may also promote excess membrane synthesis (see table 3).
  • L-form mutants are unable to revert to the walled state because they have defects in components of the pathways leading to PG or WTA.
  • the applicants have developed a system to isolate L-form mutants in other pathways by looking for L-form mutants (starting with a strain containing an ispA mutation) that retained the ability to revert to the rod state (see 1.3 and 2.3 above).
  • the first mutant (described in 2.3) had a single point mutation in the 5'UTR of the operon containing the genes accD and accA. This mutation leads excess membrane synthesis and L-form proliferation.
  • another mutant was also isolated, with a single point mutation in the rpsl gene (E112K), encoding ribosomal protein S9.
  • the tnYLB-1 transposon delivery system was first introduced into B. subtilis strain 168ca. Transposition was induced and mutants bearing random transposon insertions were selected. About 40,000 individual colonies were obtained and used as an initial mutant library. Mixed chromsomal DNA extracted from the mutant library was then introduced into a P X yi-murE-B strain carrying second copy of ispA at amyE locus (to avoid selection of ispA mutation) growing in walled state (presence of xylose) (strain RM80). About 20,000 individual colonies were then picked and transferred to plates selecting for L-forms (no xylose but containing sucrose as an osmoprotectant and 8j FtsZ inhibitor).
  • IspC works in the isoprenoid biosynthetic pathway leading to menaquinone.
  • AroB and AroC work in the biosynthetic pathway of chorismate, also used for menaquinone synthesis.
  • NADH dehydrogenase (Ndh) cytochrome aa3 quinol oxidase (QoxB) and heme O synthase (CtaB) have roles in the synthesis of other components of the respiratory chain.
  • MhqR is a transcriptional repressor for genes required for quinone detoxification. All these mutants including the ispA mutation could work by reducing activity of the respiratory chain.
  • sucrose used as an osmoprotectant in the L-form medium used herein could affect cellular metabolism.
  • the ispA mutational pathway might therefore work to adapt cells to such a growth condition.
  • several mutations were introduced into the P xyr murE-B strain and the effects on growth of L-forms (absence of xylose) was examined (Table 5). Mutations disrupting genes required for sucrose utilization and uptake (sacB, sacC, sacX and levB) did not support L-form growth. Glucose and fructose are generated from sucrose.
  • Acetyl CoA carboxylase carries out the first committed step of the FAS II synthetic pathway, the conversion from acetyl CoA to malonyl CoA.
  • the applicants have found that the overexpression of the carboxyltransferase (AccDA) subunit of ACC is lethal in walled cells and leads to the formation of large membranous vesicles in B. subtilis. Such vesicles were not produced by overexpression of other genes encoding enzymes of the FAS II system, nor plsX, which governs the first step in phospholipid synthesis (data not shown), suggesting that the effect is specific for accDA overexpression.
  • AccDA carboxyltransferase
  • subtilis the separated accA and accD genes of Escherichia coli are both negatively regulated at the translational level by binding of the AccDA complex to the accA and accD mRNAs (Meades et al., 2010).
  • the RNA-binding and catalytic functions of AccDA are dependent on the metabolic state of the cell via the intracellular level of acetyl CoA (Meades et al., 2010).
  • the RNA binding domain of the AccDA complex appears to be conserved in Staphylococcus aureus (Bilder et al., 2006), a close relative of B. subtilis.
  • the location of the accDA* mutation in a stem-loop structure just upstream of accD coding region suggests that a translational regulatory mechanism may also exist in B. subtilis.
  • subtilis (Briers et al., 2012a; Dell'Era et al., 2009). Although the proliferation of L. monocytogenes L-forms appears different in morphological detail to B. subtilis, it is noted firstly that the culture conditions used in the Listeria experiments were very different (cells embedded in soft agar) from those used here (liquid medium), and secondly that the genome sequence of a Listeria L-form isolate apparently contained a mutation in the gene encoding HMG-CoA synthase, which participates in polyprenoid precursor synthesis, and which might therefore operate in a similar manner to ispA of B. subtilis.
  • L-forms proliferate by an unusual membrane deformation and scission process that is completely independent of the normally essential FtsZ based cell division machinery in B. subtilis (Leaver et al., 2009), and they also do not require any of the currently known cytoskeletal systems (Mercier et al., 2012).
  • Chen Chen (Chen, 2009) has pointed out that L-form division might occur by purely biophysical processes and in Mercier (Mercier et al., 2012) it was shown that a late stage in proliferation is dependent on a particular membrane composition, probably associated with high membrane fluidity.
  • the results described here strongly suggest that an imbalance between cell membrane and volume growth drives the cell shape deformations leading to scission and, thus, L-form proliferation.
  • the deformed cell resolves spontaneously (scission) into discrete progeny cells.
  • the total surface area of several small cells is > that of a single cell of equal total volume and similar shape, so the disequilibrium between surface area and volume can be corrected by progeny formation (iii). Repetition of this cycle leads to indefinite L-form proliferation.
  • L-forms might represent a useful model system for the study of bacterial evolution and ancestry (Briers et al., 2012a; Leaver et al., 2009; Mercier et al., 2012).
  • Several in vitro studies have demonstrated proliferation in relatively simple vesicle systems without the intervention of protein-based mechanisms (Hanczyc et al., 2003; Peterlin et al., 2009; Terasawa et al., 2012; Zhu and Szostak, 2009).
  • the in vitro replication methods mentioned above all rely in one way or another on achieving an imbalance between vesicle surface area and internal volume.
  • Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus structure of the proteins and their interactions with 16 S RNA. J Mol Biol 316(3):725-68.
  • Listeria monocytogenes L-forms respond to cell wall deficiency by modifying gene expression and the mode of division. Mol Microbiol 73, 306-322.
  • the rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus. Mol Microbiol 83, 52-66.
  • FapR a bacterial transcription factor involved in global regulation of membrane lipid biosynthesis. Dev Cell 4, 663-672.

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D'ELIA MICHAEL A ET AL: "Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis", JOURNAL OF BACTERIOLOGY, vol. 188, no. 23, December 2006 (2006-12-01), pages 8313 - 8316, XP055437197, ISSN: 0021-9193 *
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