WO2014132064A2

WO2014132064A2 - Cells and methods for fatty acid synthesis

Info

Publication number: WO2014132064A2
Application number: PCT/GB2014/050584
Authority: WO
Inventors: Jeff ERRINGTON; Yoshikazu Kawai; Roman MERCIER
Original assignee: University Of Newcastle Upon Tyne
Priority date: 2013-02-27
Filing date: 2014-02-27
Publication date: 2014-09-04
Also published as: WO2014132064A3; GB201303474D0; EP2961837A2

Abstract

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, semi- synthetic and synthetic cells,and reaction vessels suitable for such methods are also provided.

Description

Cells and methods for fatty acid synthesis

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.

Background

The peptidoglycan (PG) cell wall is a major defining structure of bacteria, and is present in all known major bacterial lineages. The rare groups of bacteria that lack a wall, e.g. mycoplasma, have probably lost the structure retrospectively. Therefore the wall was probably present in the last common ancestor of the bacteria, perhaps in the earliest forms of cellular life. 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 β-lactams and glycopeptides.

In light of the pivotal importance of the wall it is intriguing that many bacteria are capable of switching into a wall deficient or "L-form" state (Allan et al., 2009). Most classically described 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). Although mutations acquired by L-forms of several organisms have recently been identified by genome sequencing (Briers et al., 2012a; Leaver et al., 2009; Siddiqui et al., 2006), precisely how they contribute to the L-form state remains poorly understood.

The L-form transition results in dramatic changes in cell physiology and morphology because wall synthesis represents a major drain on cellular resources and the wall is essential in determining cell shape. However, the most dramatic change in cell function recognised so far lies in the mode of L-form proliferation. Almost all bacterial cells use a tubulin homologue, FtsZ, to organise a highly ordered ring structure at the site of cell division. This "Z-ring" then recruits about 10 other essential division factors, which together drive invagination of a division septum, infill this with new PG, and then organise the orderly separation of the daughter cells (Adams and Errington, 2009; Margolin, 2005). Remarkably, this intricate division machinery appears to become completely dispensable in L-forms (Leaver et al., 2009). This is consistent with the finding that L-forms divide not by the precise FtsZ-ring constriction mechanism but by a range of rather poorly regulated shape perturbations, including blebbing, tubulation and vesiculation (Dell'Era et al., 2009; Kandler and Kandler, 1954; Leaver et al., 2009). Cell proliferation in the L-form state therefore has the advantage that it does not require the normally essential cellular division machinery. The L-form has been observed across many diverse bacterial lineages and it is likely that most bacteria are capable of this change (Domingue & Woody, 1997).

Although the L-form state was first observed a number of years ago, the molecular mechanisms driving cell division and proliferation of L-forms remain unclear.

Brief summary of the disclosure

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 applicants have surprisingly found that cells are able to proliferate in the L-form state when they produce excess membrane as a result of over activation of the fatty acid synthetic system. Excess membrane synthesis is sufficient to induce shape modulations that progress to scission of progeny cells from the parent L-form cell. Artificially increasing the surface area in protoplasts of wild type cells is also shown to generate spontaneous L-form-like shape changes and L-form cell division. These findings suggest that the division of L-forms is likely due simply to an imbalance between membrane surface area and cellular volume.

In one aspect, 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) 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; (b) 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; 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.

Preferably, the first nucleic acid molecule, the second nucleic acid molecule and/or the third nucleic acid molecule is part of an expression vector. Preferably, the first nucleic acid molecule encodes a carboxyltransferase subunit of acetyl CoA carboxylase.

Preferably, the first nucleic acid molecule comprises an inducible promoter.

Preferably, 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.

Preferably, 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.

Preferably, the second nucleic acid molecule encodes a polypeptide involved in

peptidoglycan synthesis. More preferably, 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.

Preferably, 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. Preferably, 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.

Preferably, the second nucleic acid molecule comprises a repressible promoter.

Preferably, the second nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.

Preferably, 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. Preferably, the cell further comprises at least one of:

(d) 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; and/or

(e) a fifth 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.

Preferably, 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

Preferably, the fourth nucleic acid molecule comprises a repressible promoter. Preferably, the fourth nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.

Preferably, the fifth nucleic acid molecule encodes a polypeptide selected from the group consisting of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.

Preferably, the fifth nucleic acid molecule comprises a repressible promoter. Preferably, the fifth nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.

Preferably, the cell is a bacterial cell. More preferably, the bacterial cell is B.subtilis. In a further aspect, 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:

(a) increased expression of a carboxyltransferase; (b) decreased expression of at least one polypeptide involved in cell wall synthesis; and/or

(c) modified activity of ribosomal protein S9. Preferably the cell additionally has at least one of:

(d) decreased expression of at least one polypeptide involved in the respiratory chain; and/or

(e) decreased expression of at least one polypeptide involved in the glycolysis pathway. Preferably, the carboxyltransferase is a carboxyltransferase subunit of acetyl CoA

carboxylase.

Preferably, the at least one polypeptide involved in cell wall synthesis is involved in peptidoglycan synthesis. Preferably, 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.

Preferably, the at least one polypeptide involved in cell wall synthesis is involved in wall teichoic acid (WTA) synthesis. Preferably, the polypeptide is selected from the group consisting of tagA, tagB, tagD, tagE, tagF, manA and tagO. More preferably, the polypeptide is tagO.

Preferably, the at least one polypeptide involved in cell wall synthesis is involved in the regulation of peptidoglycan and/or wall teichoic acid synthesis. Preferably, the polypeptide is selected from the group consisting of glmS, glmM, gcaD and mreB. More preferably, the polypeptide is MreB. Preferably, 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.

Preferably, 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. In a further aspect, 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) 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;

(b) 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.

In a further aspect, 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:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or

(c) an agent that modifies ribosomal protein S9 activity.

Preferably, the agent comprises a nucleic acid molecule. Preferably, the nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3 .

Preferably, 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. Preferably, 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. Preferably, 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.

Preferably, 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.

Preferably, the method further comprises providing to the cell at least one of:

(d) an agent that decreases respiratory chain activity; and/or

(e) an agent that decreases the activity of the glycolysis pathway.

Preferably, 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.

Preferably, 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. Preferably, the providing step(s) generate(s) a cell according to the invention.

Preferably, the method further comprises culturing the cell under conditions that support the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.

Preferably, the method further comprises recovering the produced fatty acids, fatty acid derivatives and/or membrane associated molecules. In a further aspect, 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.

In a further aspect, the invention relates to the use of a cell according to the invention in drug or vaccine delivery.

In a further aspect, 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

(c) an agent that modifies protein S9 activity; in the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.

In a further aspect, the invention relates to a reaction vessel containing a cell according to the invention and medium sufficient to support growth of the cell. Preferably, the reaction vessel is a bioreactor or a fermenter.

Preferably, the method of the invention is performed in the reaction vessel of the invention.

In a further aspect, the invention relates to a method of inducing L-form growth in a cell, comprising providing to a cell at least one of:

(a) an agent that increases carboxyltransferase activity; (b) an agent that decreases cell wall synthesis; and/or

(c) an agent that modifies ribosomal protein S9 activity; and treating the cell with an agent that removes the cell wall and/or prevents cell wall synthesis.

Preferably, the agent that removes the cell wall and/or prevents cell wall synthesis is a lysozyme. Preferably, the method further comprises providing to the cell at least one of:

(d) an agent that decreases respiratory chain activity; and/or

(e) an agent that decreases the activity of the glycolysis pathway.

Preferably, the cell is cultured under conditions that support L-form growth.

In a further aspect, the invention relates to a method of preparing a therapeutic composition comprising:

(i) providing to a cell at least one of:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or

(c) an agent that modifies ribosomal protein S9 activity; (ii) treating the cell with an agent that removes the cell wall and/or prevents cell wall synthesis; and (iii) formulating the cell as a therapeutic agent.

In a further aspect, the invention relates to a method of identifying a DNA mutation that supports L-form growth in a cell comprising:

(i) providing to a cell an agent that removes the cell wall to generate a protoplast; (ii) culturing the protoplast under conditions that support L-form growth;

(iii) identifying a cell capable of L-form growth; and

(iv) identifying a DNA mutation in the cell of (iii) that supports L-form growth. Preferably, the cell has previously been provided with at least one of:

(a) an agent that decreases respiratory chain activity; and/or (b) an agent that decreases the activity of the glycolysis pathway.

Preferably, 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.

Preferably, step (iii) further comprises culturing the identified cell under conditions that support cell wall regeneration and identifying a cell with a regenerated cell wall.

In further aspects, 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. Throughout the description and claims of this specification, 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.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

Description of the drawings

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows the effects of ispA and murE-B Mutations on L-form Growth. (A-C) Strains LR2 {ispA P_xyrmurE-B; A), 168CA (wild type; B) and Bs1 15 {P_xyrmurE-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. In 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 μηι. See also Figure 9.

Figure 2 shows that L-form Growth Requires Mutational Lesions Affecting Two Different Pathways. (Figure 2A) Strain LR2 (ispA P_xyrmurE-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_xyrmurE-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 μιτι. (Figures 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). FtsZ levels were also detected as an internal control. (Figure 3F-G) Protoplast growth of an ispA amyE:.P_xyraccDA 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). (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 μιτι. 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)]. (Figure 4B) Quantitative RT-PCR analysis of the relative change expression of several FapR regulated genes in an amyE: P_xyraccDA 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. The following 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_xyrfapR_R106A P_Spac(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. The expression level of 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. Strains with the following mutations were cultured on NA plates at 37°C in the presence (bottom) or absence (top) of 0.5% xylose: left, the wild type (strain 168), middle amyE:.P_xy,-accDA (YK1738), right ispA amyE::P_xy,-accDA (YK1694) . (Figure 4G) Effect of repression of FAS II enzyme synthesis on AccDA overexpression lethality. Strains with the following mutations were cultured on NA plates at 37°C in presence of 0.5% xylose (or without; Figure 10E) and with no (left), 0.05mM (middle) and 0.5 mM (right) IPTG: ispA amyE::P_xyl-accDA with P_spac-plsX (strain YK1707, top), P_spac-fabD (YK1710, middle) or P_spac- fabHA (YK1712, bottom). (Figure 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_Sp_ac-fabHA without (dashed line) or with (dotted line) 0.5 mM IPTG and ispA amyE:.P_xyi-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. (Figure 5A-B) Phase contrast (left) and corresponding epifluorescence micrographs (right) of strain YK1738 (amyE::P_xyraccDA), grown in LB with (B) or without (A) 0.5% xylose and stained with the membrane dye Mitotracker green. Scale bar represents 5 μηι. (Figure 5C-D) N-SIM fluorescence micrographs of wild type (168CA, C) and YK1738 (amyE::P_xyraccDA, 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 μηι. (Figure 5E-F) Transmission electron microscopy images of wild type (168CA, E) and YK1738 {amyE:.P_xyraccDA, 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_xyrmurE-B) was grown in LB with 1 % (left) or 0.1 % (right) xylose at 37°C. Scale bar represents 5 μηι. 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 μηι. (Figure 6C) Exponentially growing L-form culture of YK1694 (ispA amyE P_xyraccDA) in NB/MSM with 0.5% xylose and benzamide. Scale bar represents 3 μηι. (Figure 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 μηι. (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 μηι. 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 μηι. (Figure 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 μηι.

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_Xyi-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 9B) Growth of strain YK1410 (P_xyrmurE-B P_spac-ispA) streaked on NA/xylose plates (left) to monitor walled cell growth and on NB/MSM plates with (middle) or without (right) 1 mM IPTG, to monitor L-form growth.

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_xyraccDA, top), YK1738 {amyE::P_xyraccDA, 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_xyraccDA) 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_xyraccDA) stained with Mitotracker green. Cells were grown in LB containing with (bottom) or without (top) 0.5 % xylose to an OD₆oo of 1. Scale bar represents 5 μηι. (Figure 10D) Growth of the following strains: YK1738 {amyE::P_xyl-accDA), YK1730 {amyE::P_xyl-accBC), YK1729 (amyE::P_xyraccA) and YK1728 (amyE::P_xyraccD) cultured on NA plates with (right) or without (left) 0.5% xylose. (Figure 10E) Effect of repression of FAS II synthetic enzymes on the lethality caused by AccDA overproduction. Strains all carrying ispA and amyE::P_xyr accDA mutations and with the following additional mutations were cultured on NA plates at 37°C in absence of xylose (or with 0.5% xylose; see Figure 4G) and with no (left), 0.05mM (middle) and 0.5 mM (right) IPTG: P_spac-plsX (strain YK1707, top), P_spac-fabD (YK1710, middle), P_spac-fabHA (YK1712, bottom). (Figure 10F) Growth of strains 168CA (wild type), YK1738 {amyE::P_xyraccDA) and RM257 {fabHi_108F) cultured on NA plates containing 0.5% xylose, with (right) or without (left) 2 μg/ml cerulenin.

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 11 B-C) Growth of strains with the following mutations P_spac-accDA (strain YK1741), P_spac-fabHA (RM250) and P_spac-plsX (RM237) as L-forms (NA/MSM plates, B) or in the walled state (NA plates with 0.5% xylose, C). All strains also carried an ispA and P_xyrmurE-B mutation to enable L-form growth and plates were supplemented with an intermediate level of IPTG (0.1 mM). (Figure 1 1 D) Effect of sub-MIC levels of cerulenin on L-form growth. An ispA P_xyrmurE-B strain (LR2, left) and isogenic cerulenin resistant mutant (RM227, right) were streaked on L-form growth medium (NA/MSM plates) containing 2 μg/ml cerulenin and with 0.5% xylose (to select for walled cells, top) or without xylose (to select for L-form growth, bottom). (Figure 11 E) Western blot analysis of histidine-tagged AccA levels in BS115 (P_xyrmurE-B, left) and 168CA (wild type, right) strain grown in LB at 37°C with different concentration of xylose. FtsZ levels were also detected in same samples as an internal control for histidine-tagged AccA levels.

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) and RM84 (ispA accDA*, right) after growth in LB at 37°C. (Figure 12B) Membrane imaging of strain YK1706 (amyE:.P_xyraccDA 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 μηι. (Figure 12C) Membrane imaging of strain of the strain YK1736 (P_xyrmurE-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 μηι.

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_xyraccDA, 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 Έ" 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. (A) 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_xyrmurE-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. (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). (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. glutamicum (middle) and E. coli (bottom) L-forms streaked on L-form-supporting medium (MSM) with the antibiotics fosfomycin (S. aureus and E. coli) or D-cycloserine (C. glutamicum) in presence of Penicillin G (S. aureus and C. glutamicum) or Ampicillin (E. coli). The different L-form strains were streaked several times on the same condition every 3 days (left to right).

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. (C) Multiplex PCR of the genes ftsK, murA, ftsZ and mreC on the genomic DNA of the E. coli strains RM323 (1-2), RM345 (3-4), RM350 (5-6) and RM60 (7-8) grown in walled state (1 , 3, 5 and 7) or L-form state (2, 4, 6 and 8) obtained from (B). M represents the 100bp DNA ladder. (D) Growth of the S. 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. (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. (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).

Detailed description

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. Advantageously, 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. Thus, 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.

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). Using the techniques described herein, the applicants have surprisingly found that it is possible to generate cells that have increased membrane and are capable of L-form growth.

(i) Modification of the fatty acid synthesis pathway The applicants have surprisingly found that over-expression of the carboxyltransferase subunit of acetyl CoA carboxylase can increase the propensity of a cell to synthesise fatty acids, fatty acid derivatives and/or membrane associated molecules. Acetyl CoA carboxylase plays a role in fatty acid biosynthesis by converting acetyl CoA to malonyl CoA. Acetyl CoA carboxylase is made up of two subunits; a biotin carboxylase subunit (encoded by the AccBC operon) and a carboxyltransferase subunit (encoded by the AccDA operon). Surprisingly, the applicants have shown that over-expression of the AccDA operon alone (i.e. independently of expression of the AccBC operon) results in an increase in the synthesis of fatty acids, fatty acid derivatives and/or membrane associated molecules. Furthermore, 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). Surprisingly, 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. However, 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). Alternatively, or in addition, 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. 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. Moreover, using the specific protocols provided herein, other genes capable of achieving the desired effect may be identified as a routine procedure.

(ii) Modification of alternative pathways

The applicants have also shown that the synthesis of 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. By way of example, the applicants have shown that 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. By way of example, 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.

Different cell types have been shown to encode one or more mreB homologues. In B subtilis, for example, there are three homologues for mreB (mreB, mbl and mreBH), each of which is located separately within the bacterial chromosome. 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. However, it should be clear to the skilled person that the invention encompasses modifying one or more of these homologues, separately, or in combination. It should also be clear that 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.

As used herein, the term "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.

As used herein, the term "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. However, 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. Alternatively, or in addition, 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. Moreover, using the specific protocols provided herein, other genes capable of achieving the desired effect may be identified as a routine procedure.

The applicants have also surprisingly shown that genetic modification of ribosomal protein S9 (specifically substitution of glutamic acid (E) to lysine (K) at position 112 in ribosomal protein S9 of B. subtilis) 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. As shown herein, 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). However, the skilled person should appreciate that 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 (and corresponding modifications) 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). However, 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.

Modification of ribosomal protein S9 activity may be achieved in a number of ways with which the skilled person will be familiar. By way of example, but not for the purposes of limitation, 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.

A skilled person would readily be able to screen for and identify appropriate alternative modifications and/or alternative polypeptides that are capable of achieving the desired effect. Moreover, using the specific protocols provided herein, other genes capable of achieving the desired effect may be identified as a routine procedure.

L-form growth

The applicants have surprisingly shown that cells with an abnormal cell surface area to volume (A/V) ratio (for example due to excess membrane formation) are capable of L-form growth (also referred to herein as L-form proliferation, which requires cells in the L-form state to undergo division). As discussed above, 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. However, 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.

Means for supporting L form growth

The applicants have demonstrated herein that L-form growth may be supported in a number of ways.

By way of example, the applicants have shown that it is possible to culture the cells of the invention under conditions that support L-form growth. Optionally, 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.

As a further (alternative) example, 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. However, the invention may also be achieved by modifying the expression of other polypeptides involved in the respiratory chain. Alternatively, or in addition, 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)). A skilled person would readily be able to screen for and identify appropriate alternative polypeptides involved in the respiratory pathway and/or suitable modifications of such polypeptides that are capable of achieving the desired effect. Moreover, using the specific protocols provided herein, other genes capable of achieving the desired effect may be identified as a routine procedure. As a further (alternative) example, L-form growth may be supported by modifying the glycolysis pathway of the cell of interest. 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.

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. However, the invention may also be achieved by modifying the expression of other polypeptides involved in the glycolysis pathway. Alternatively, or in addition, 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)). A skilled person would readily be able to screen for and identify appropriate alternative polypeptides involved in the glycolysis pathway and/or suitable modifications of such polypeptides that are capable of achieving the desired effect. Moreover, using the specific protocols provided herein, other genes capable of achieving the desired effect may be identified as a routine procedure. Aspects of the invention will be discussed in more detail below.

Recombinant cell

In one aspect, 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) 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;

(b) 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; 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. As used herein, 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", as used herein 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. Preferably, the cell is a bacterial cell. Alternatively, 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. Alternatively, the host cell may be of the genus Lactococcus, for example Lactococcus lactis. Alternatively, the bacterial cell is of the actinomycetes family, more particularly from the genus Streptomyces, Rhodococcus, Corynebacterium, Mycobacterium. More particularly, Streptomyces lividans, Streptomyces ambofaciens, Streptomyces fradiae, Streptomyces griseofuscus, Rhodococcus erythropolis, Corynebacterium gluamicum, Mycobacterium smegmatis may be used. Alternatively, 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. E.coli BL21 , BL21 (DE3), BL21 (DE3) pLysS, pLysE, DH1 , DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DHIOB/p3, DH1 IS, C600, HB101 , JM101 , JM105, JM 109, JM 110, K38, RR1 , Y1088, Y1089, CSH18, ER1451 , ER1647 are particularly suitable for expression. E. 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)).

Alternatively, 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.

Other groups of bacteria that have been converted to L-form bacteria include members of the Fusobacteria, such as Streptobacillis, and Spirochaetes (Domingue & Woody, 1997). Such cells may therefore also be suitable hosts in the context of the invention. 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. has not been transformed, transfected or transduced with the nucleic acid molecule(s) of interest). By way of example, but not for the purposes of limitation, 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. It will be further appreciated that 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).

The applicants have shown herein that the 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. As used herein, the phrase "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. By way of example, but not for the purposes of limitation, 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. By way of example (but not for the purposes of limitation) this may include membrane proteins (integral or peripheral), glycoproteins, isoprenoids, cholesterol-type lipids, and amphipathic lipids (e.g. glycolipids).

As used herein, the phrase "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.

As used herein, the phrase "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. Accordingly, as an example, a recombinant cell of the invention may comprise a third nucleic acid molecule, without comprising a first or a second nucleic acid molecule etc. By way of a further example, 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:

- a 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;

- a 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; and/or

- a 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.

Nucleic acid molecules

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. In one aspect, 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).

As used herein, the term "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.

With regard to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, 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. Moreover, 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. As used herein, 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). As used herein, 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.

First nucleic acid molecule

In one aspect of the invention, 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. In a suitable aspect of the invention, 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.

In a suitable aspect of the invention, 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.

In a suitable aspect of the invention, 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.

In a suitable aspect of the invention, 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. In a suitable aspect of the invention, 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). In one embodiment, and by way of example, the carboxyltransferase is the carboxyltransferase subunit of acetyl CoA carboxylase. In one embodiment, the carboxyltransferase is encoded by the AccDA operon in nature.

The phrase "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.

As used herein, the phrase "wherein the nucleic acid molecule comprises a nucleic acid sequence capable of increasing expression of the encoded carboxyltransferase" is intended to cover, for example, a nucleic acid sequence that comprises a promoter capable of increasing expression of the encoded carboxyltransferase (e.g. an inducible promoter), and (as an alternative or additional embodiment) 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.

As used herein, 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. increase) the relative ratio of carboxyltransferase: biotin carboxylase (expression) compared to the ratio of carboxyltransferase: biotin carboxylase observed in an equivalent cell that lacks the "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" ("control"). By way of example, but not for the purposes of limitation, 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.

By way of example, 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. By way of example, 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. However, the skilled person should appreciate that 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.

Calculations of 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). In a preferred embodiment, 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. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The 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. In a preferred embodiment, 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 (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) 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.

The 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). BLAST nucleotide searches can be performed with the N BLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http://www.ncbi.nlm.nih.gov>.

As used herein, the phrase "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". By way of example, but not for the purposes of limitation, "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.

Second nucleic acid molecule

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). In one embodiment, and by way of example, the polypeptide may be involved in peptidoglycan synthesis, wall teichoic acid synthesis, or the regulation of either (or both) of these synthetic pathways.

Examples of particular 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). One or a combination of these 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). In one embodiment of the invention, 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. Accordingly, 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. 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). As used herein, the phrase "nucleic acid molecule comprises a nucleic acid sequence capable of inhibiting expression of the [encoded polypeptide]" is intended to cover, for example, a nucleic acid sequence that comprises a promoter capable of inhibiting expression of the encoded polypeptide (e.g. a repressible promoter), and (as an alternative or additional embodiment) a nucleic acid sequence that comprises a mutation within (or outside) the polypeptide encoding region, where the mutation is capable of inhibiting expression of the encoded polypeptide.

As used herein, the phrase "a 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". The term "inhibit" is used herein to encompass partial inhibition as well as total inhibition. By way of example, but not for the purposes of limitation, 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.

Third nucleic acid molecule

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. As used herein, the phrase "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]". The definitions provided elsewhere herein with respect to the meaning of, for example, "increased propensity" apply equally here. By way of example, a 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).

Fourth nucleic acid molecule

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. One or a combination of these polypeptides may be present within the recombinant cell of the invention. 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. Fifth nucleic acid molecule

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).

Examples of particular polypeptides generally encompassed by this aspect of the invention include: 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.

Any of the 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. Preferably, 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). Rewriting of larger stretches of DNA may require up to three consecutive rounds of recursive PCR, Hale, RS and Thomas G. Protein Exper. Purif. 12, 185-188 (1998), Te'o et al, FEMS Microbiol. Lett. 190: 13-19, (2000). Alternatively, 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.

Any of the 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). Preferably, 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. One example of 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

In one aspect, the invention provides the expression vectors discussed herein per se.

One or a combination of the 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).

As used herein, 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. Preferably the vector is suitable for expression in a cell (i.e. the vector is an "expression vector"). Preferably, the expression vector is suitable for expression in any appropriate cell. Most preferably, the vector is suitable for expression in bacteria. Preferably, the vector is a bacterial expression vector. The terms "expression vector", "expression construct", "construct" and "vector" are used interchangeably herein.

Preferably the vector is capable of propagation in a host cell and is stably transmitted to future generations.

"Operably linked" as used herein, 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" as used herein, refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of 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. Preferably 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", as used herein, 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. Alternatively, the promoter is under the control of a repressor or stimulatory protein. Preferably the promoter is a T7, T3, lac, lac UV5, tac, trc, [lambda]PL, Sp6 or a UV-inducible promoter.

"Transcriptional terminator" as used herein, 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", as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, 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" as used herein, 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. Generally this may be a protein that confers resistance to an antibiotic such as ampicillin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate. Further examples of antibiotics are Penicillins; Ampicillin HCI, Ampicillin Na, Amoxycillin Na, Carbenicillin sodium, Penicillin G, Cephalosporins, Cefotaxim Na, Cefalexin HCI, Vancomycin, Cycloserine. Other examples include 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.

Expression of proteins in prokaryotes is most often carried out in a bacterial host cell with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. 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. Often, 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.

Preferably 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.

Preferably, the vector is suitable for expression in the target host cell. Preferably, the vector is suitable for integration into a host chromosome.

Preferably, when B subtilis is the host cell, plasmid pMutin4 or pSG1 154 (or their derivatives) may be suitable. By way of example, the plasmid pMutin4 or derivatives (Vagner et al, 1998) may be used to construct IPTG-inducible or inactivation B. subtilis mutants. For the IPTG-inducible mutants described herein, 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.

By way of a further example, the plasmid pSG1 154 or derivatives (Lewis and Marston, 1999) 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.

Preferably, 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. Preparation of Expression Vectors

A person of skill in the art will be aware of the molecular techniques available for the preparation of expression vectors.

The nucleic acid molecule for incorporation into the expression vector of the invention, as described above, 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. In one embodiment, 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.

In an alternative embodiment, synthetic linkers containing one or more restriction sites are used to operably link the nucleic acid molecule to the expression vector. In one embodiment, the nucleic acid molecule is generated by restriction endonuclease digestion. Preferably, 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. Thus, 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.

Alternatively, 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).

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.

In a preferred embodiment 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. In a preferred embodiment the amplification primers contain restriction endonuclease recognition sites which allow the amplified sequence product to be cloned into an appropriate vector.

Preferably, 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. Alternatively, 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.

Host Cell Transformation

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", "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, MgS0₄, salts mix, Casamino acids, Tryptophan and other supplements if appropriate. The cells are incubated with shaking at 37°C until OD₆oonm of 1. Then an equal volume of Spizizen minimal medium with glucose, MgS0₄ 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.

Successfully transformed cells, that is, those cells containing the expression vector of the present invention, can be identified by techniques well known in the art.

In a preferred embodiment the invention comprises a culture of recombinant cells. Preferably 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.

Standard techniques for propagating vectors in prokaryotic hosts are well-known to those of skill in the art (see, for example, Ausubel et al. Short Protocols in Molecular Biology 3rd Edition (John Wiley & Sons 1995)).

To maximize recombinant protein expression, 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). Alternatively, 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.

Semi-synthetic or synthetic cell

In one aspect of the invention, 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:

(a) increased expression of a carboxyltransferase;

(b) decreased expression of at least one polypeptide involved in cell wall synthesis; and/or

(c) modified activity of ribosomal protein S9. The results provided herein show that complex division machinery can be dispensed with, provided that the level of membrane synthesis is sufficiently high. This work therefore shows how artificial cells (e.g. a semi-synthetic cell or synthetic cell) could be made to proliferate without needing to incorporate the complex division machinery.

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. There are also many situations in which it is convenient to avoid the presence of bacterial cell wall material, especially in medical, veterinary or cosmeceutical applications. The present invention thus provides a way of generating cell wall free cells that will proliferate efficiently.

All terms defined above in respect of other aspects of the invention apply equally here.

As used herein, 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.

By way of example, 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.

There is considerable interest in synthesis of artificial genomes with minimal gene content from a technological view point. Such cells have reduced DNA content and thus devote less metabolic resource to synthesising nucleic acids. Reduced gene number reduces the level of complexity of regulatory pathways and networks. Finally, it simplifies the potential problems and complexity of metabolic reprogramming to optimise commercial production of valuable biomolecules. The term "decreased expression" is to be interpreted herein in an equivalent manner to the term "inhibit expression" defined above in respect of the recombinant cell of the invention (for example in the context of the second nucleic acid molecule). By way of example, but not for the purposes of limitation, "decreased expression" occurs 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 control. In the context of a semi-synthetic or synthetic cell of the invention, the control may be expression observed in an equivalent semi-synthetic or synthetic cell that lacks one, more than one, or all of:

• increased expression of a carboxyltransferase;

· decreased expression of at least one polypeptide involved in cell wall synthesis;

• modified activity of ribosomal protein S9; or

• an agent (such as a nucleic acid) present in 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) increased expression of a carboxyltransferase;

(c) modified activity of ribosomal protein S9

due to the presence of a first, second or third nucleic acid molecule of the invention. These aspects of the invention may thus be combined.

A method of producing fatty acids, fatty acid derivatives and/or membrane associated molecules

In a further aspect of the invention, a method of producing fatty acids, fatty acid derivatives and/or membrane associated molecules is provided. The method comprises providing to a cell at least one of:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or

(c) an agent that modifies ribosomal protein S9 activity.

Optionally, the method further comprises providing to the cell at least one of:

(d) an agent that decreases respiratory chain activity; and/or

(e) an agent that decreases the activity of the glycolysis pathway. All terms defined above in respect of other aspects of the invention apply equally here.

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. By way of example, but not for the purposes of limitation, an agent may be an inhibitor of cell wall synthesis; or an activator of carboxyltransferase activity. By way of example, but not for the purposes of limitation, 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. A skilled person would readily be able to screen for and identify putative agents that would be appropriate for use in the method of the invention.

In one embodiment, an agent comprises (or is) a nucleic acid molecule in accordance with the invention. By way of example, an agent (e.g. nucleic acid molecule) may thus be provided to a cell by transformation (e.g. by transformation of an expression vector comprising a nucleic acid molecule according to the invention). The cell may be a recombinant cell in accordance with the invention. Alternatively, 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.

As used herein, 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). By way of example, but not limitation, 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. As used herein, 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). By way of example, but not limitation, 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. As used herein, 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"). By way of example, but not for the purposes of limitation, 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.

Methods of inducing L-form growth in a cell

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:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or

(c) an agent that modifies ribosomal protein S9 activity; and

treating the cell with an agent that removes the cell wall and/or prevents cell wall synthesis.

Optionally, the method further comprises providing to the cell at least one of:

(d) an agent that decreases respiratory chain activity; and/or

An agent that "removes the cell wall and/or prevents cell wall synthesis" can readily be screened for and identified by the skilled person. By way of example, the agent may be a lysozyme. However, 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. In addition, or alternatively, inhibitors of cell division, such as benzamides, may also be suitable (see Haydon et al., 2008). 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

In a further aspect, a method of identifying a DNA mutation that supports L-form growth in a cell is provided comprising

(i) providing to a cell an agent that removes the cell wall to generate a protoplast;

(ii) culturing the protoplast under conditions that support L-form growth;

(iii) identifying a cell capable of L-form growth; and

(iv) identifying a DNA mutation in the cell of (iii) that supports L-form growth.

Optionally, the cell has previously been provided with at least one of:

(a) an agent that decreases respiratory chain activity; and/or

(b) an agent that decreases the activity of the glycolysis pathway.

Optionally, 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.

As used herein, 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.

Culture conditions 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.

Cells are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. The culture medium (also called "growth medium", "medium" or "media" herein) to be used must suitably meet the requirements of the strains in question. Preferably, 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.

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.

With regard to B subtilis optimal growth, as an example, sucrose is a suitable carbon source for cells in the L-form state, whereas glucose is an optimal carbon source for cells in the normal walled state. However, other 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. Examples of 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. 53-73, ISBN 0 19 963577 3). 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

continuously. Batch fermentation may prove particularly useful when large scale production of fatty acids, fatty acid derivatives and/or membrane associated molecules is required. Alternatively, 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. To this end, the host cells can advantageously be disrupted beforehand. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

An overview of known cultivation methods can be found in the textbook by Chmiel (BioprozeBtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

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. To maintain the stability of vector it is possible to add to the medium 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. In certain embodiments of the invention, cells may be grown in medium that supports L-form growth. Suitable medium can readily be identified by the skilled person. By way of example, the medium may comprise an osmoprotectant (such as sucrose) medium that supports growth of the host cell. Optionally, 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. In one embodiment, the osmoprotective medium may comprise 20 mM MgCI₂, 500 mM sucrose and 20 mM maleic acid in nutrient broth (NB, Oxoid).

In certain embodiments of the invention, 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. By way of a general example, 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. By way of a specific example, the medium may comprise 0.5 M succinate, 0.5% casamino acids, 0.5% yeast extract, 0.5% glucose, 0.35% K₂HP0₄, 0.15% KH₂P0₄, 20 mM MgCI₂, 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 ("broth") 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. Preferably, the fatty acids, fatty acid derivatives and/or membrane associated molecules are secreted directly into the culture medium. Preferably, 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. In 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. Typically, cell based bioreactors comprise the cells of interest and include all the nutrients and/or co- factors necessary to carry out the reactions.

In yet another embodiment, 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.

Drug and vaccine delivery

In one aspect, the cells of the invention (e.g. recombinant cells, semi-synthetic or synthetic cells) may be used in drug or vaccine delivery.

By way of example only, 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.

Furthermore, 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 is also provided, comprising:

(i) providing to a cell at least one of:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or

(c) an agent that modifies ribosomal protein S9 activity;

(ii) treating the cell with an agent that removes the cell wall and/or prevents cell wall synthesis; and

(iii) formulating the cell as a therapeutic agent.

All terms defined above in respect of other aspects of the invention apply equally here. Aspects of the invention are demonstrated by the following non-limiting examples. EXAMPLES

1. Materials and methods

1.1 Bacterial Strains, Plasmids, and Growth Conditions

The bacterial strains and plasmid constructs used in this study are shown in Table 1 below. DNA manipulations and E. coli DH5a transformation were carried out using standard methods (Sambrook, 1989). Transformation of competent B. subtilis cells was performed by the two-step starvation procedure as previously described (Anagnostopoulos and Spizizen, 1961 ; Hamoen et al., 2002). E. coli and normal B. subtilis cells were grown on nutrient agar (NA, Oxoid) and in Luria-Bertani broth (LB). B. subtilis L-forms were grown in osmoprotective medium composed of 2 x MSM media pH 7 (40 mM MgCI₂, 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 % K₂HP0₄, 0, 15% KH₂P0₄, 20 mM MgCI₂, 0.01 % BSA and 1 % agar) (Bourne and Dancer, 1986) was used to regenerate cell wall from B. subtilis L-forms. Supplements, xylose and IPTG, were added as needed at the concentration indicated. When necessary, antibiotics were added to media at the following concentrations: cerulenin, 2 μg/ml or 10 μg/ml; ampicillin, 100 μg/ml; chloramphenicol, 5 μg/ml; kanamycin, 5 μg/ml; spectinomycin, 50 μg/ml; erythromycin, 1 μg/ml or 0.2 μg/ml; and tetracycline, 10 μg/ml. 1 μg/ml benzamide [FtsZ inhibitor 8J (Adams et al., 2011)] was used for protoplast growth experiments to prevent the growth of walled cells. Table 1. Bacillus subtilis strains and plasmids used in this study

Strain Relevant Genotype Reference and plasmid

168CA trpC2 (Kunst et al., 1997)

Bs1 15 168CA QspoVD::cat P_xyl-murE QamyE::(tet xylR) (Leaver et al., 2009)

LR2 Bs115 xseB* (Frameshift 22T>-)^a R. Mercier, unpublished

OBS20 cer20 BGSC^C

RM80 Bs115 amyE::P_xseB-xseB-ispA spc R. Mercier, unpublished pSG1 154-xseB-ispA

RM81 168CA xseB/. Tn (kan R. Mercier, unpublished RM82 LR2 QamyE::P_xseB-xseB-ispA spc pSG1 154-xseB-/spA RM84 RM81 accDA* This study

RM121 †Q8Ck A 18::tet pLOSS-P_spac-murC erm pLOSS-erm-murC

R. Mercier and P.

Dominguez-Cuevas,

RM119 168CA AmurCr.spc pLOSS-P_spac-murC erm

unpublished, pLOSS- erm-murC

RM198 Bs115 QplsX::pMulm4-erm-P_spac-plsX This study

RM208 168CA QamyE::P_xyi-fapR spc pSG1 154-/apR

RM227 LR2 cer20 This study

RM237 LR2 plsX: :pM

pMUTIN4-p/sX5'

RM250 LR2 Qfabh Ά: :pM ut\n4-erm-P_spac-fabHA pMUTIN4-fa )H \5'

RM257 168CA cer20 This study

RM258 168CA AfapR::erm This study

RM259 RM81 AfapR::erm This study

RM260 RM84 AfapR::erm This study

YK1387 Bs115 xseB/. Tn (kan) This study

YK1409 RM119 xseB/. Tn (kan) This study

YK1410 BS1 15 DispA::pMuX\r4-erm-P_spac-ispA pMUTIN4-/sp \5'

YK1592 RM81 Adal::spc This study

YK1593 168CA Adal::spc (Leaver et al., 2009)

YK1694 RM81 QamyE::P_xyi-accDA spc pSG1 154-accOA

YK1706 YK1738 fabHA::p uWn4-erm-P_spac-fabHA pMUTIN4-fa )H \5'

YK1707 YK1694 QplsX .p ut\n4-erm-P_spac-plsX pMUTIN4-p/sX5'

YK1710 YK1694

pMUTIN4-fa )D5'

YK1712 YK1694 QfabH A: :pM v \r\4-erm-P_spac-fabHA pMUTIN4-fa )H \5'

YK1726 RM208 P_spac(hy)-accDA cat pPL82-accD \ YK1728 RM81 DamyE::P_xyi-accD spc pSG1 154-accD

YK1729 RM81 QamyE::P_xyi-accA spc pSG1 154-accA

YK1730 RM81 QamyE::P_xyi-accBC spc pSG1 154-accBC

YK1731 168CA f.accA;pMutinHis erm pMUTinHis-accA3'

YK1732 RM81 -T-accA.pMutinHis erm pMUTinHis-accA3'

YK1733 RM84 iDaccA/pMutinHis erm pMUTinHis-accA3'

YK1734 168CA namyE::P_xyi-fapRR106A spc pSG1 154-/apft_{Ri06 (}

YK 735 YK 734 P_spac(hy)-accDA cat pPL82-accDA

YK1738 168CA QamyE::P_xyi-accDA spc pSG1 154-accOA

YK1736 Bs115 nfabHA::pMut\n4-erm-P_spac-fabHA pMUTIN4-/a )HA5'

YK1741 LR2 QaccDA::pMut\r4-erm-P_spac-accDA pMUTIN4-accDA5'

YK1824 168CA P_Sp_ac-rodA kan R.A. Daniel, unpublished

YK1825 YK1824 namyE::P_Xyi-accDA spc This study

Plasmid Relevant Genotype Reference/Origin pMUTIN4 bla erm P_spac mcs lacZ lacl (Vagner et al., 1998) pMUTIN4-accDA5' bla erm P_spac-accDA5' lacZ lacl This study

pMUTIN4-p/sX5' bla erm P_spac-plsX5' lacZ lacl This study

pMUTIN4-fa )D5' bla erm P_spac-fabD5' lacZ lacl This study

pMUTIN4-fa0HA5' bla erm P_spac-fabHA lacZ lacl This study

pMUTIN4-/^'spA5' bla erm P_spac-ispA lacZ lacl This study

pPL82 bla amyE3' cat P_spaC( y) amyE5' (Quisel et al., 2001) pPL82-accDA bla amyE3' cat P_spac(hy)-accDA amyE5' This work

pLOSS-erm bla erm P_soac mcs lacZ (Claessen et al., 2008)

R. Mercier and P. p OSS-erm-murC bla erm P_spac-murC lacZ Dominguez-Cuevas, unpublished

(Lewis and Marston, pSG1 154

bla amyE3' spc P_xyi-'gfp amyE5' 1999) pSG1 154-accDA bla amyE3' spc P_xyraccDA amyE5' This study

pSG1 154-accD bla amyE3' spc P_xyraccD amyE5' This study

pSG1 154-accA bla amyE3' spc P_xyraccA amyE5' This study

pSG1 154-accBC bla amyE3' spc P_xyraccBC amyE5' This study

pSG1 154-fap bla amyE3' spc P_xyrfapR amyE5' This study

PSG1 154- bla amyE3' spc P_xyl-fapRR106A amyE5' This study fapRR106A

pSG1 154-xseB-

This study

ispA bla amyE3' spc P_xseB-xseB-ispA amyE5'

pMUTinHis bla erm P_spac mcs 12xhis lacZ lacl (Ishikawa et al., 2006) pMUTinHis-acc/A3' bla erm P_spac-accA3' 12xhis lacZ lad This work

spc, spectinomycin; kan, kanamycin; erm, erythromycin; neo, neomycin; cat, chloramphenicol; fef.tetracyclin; mcs, multiple cloning site; bla, β-lactamase; lacZ, β-galactosidase

a and ^b these mutations inhibit IspA expression, ^c Bacillus Genetic Stock Center

5

1.2 Protoplast Preparation and Growth and Growth conditions of L-forms from protoplast

Protoplasts were prepared as described by (Dominguez-Cuevas et al., 2012). Briefly, an exponential cell culture (OD₆oonm of 0.2) was harvested and re-suspended in NB/MSM medium containing lysozyme (500 μg/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. A protocol has been developed within this study in which cells of a defined genotype were grown in the rod state, then converted to protoplasts by stripping the cell wall with lysozyme and cultured in osmoprotective medium, composed of: 20 mM MgCI2, 500 mM sucrose and 20 mM maleic acid in nutrient broth (NB, Oxoid). Several other carbon sources, i.e. glucose, fructose, glycerol, malate and succinate, were also tested in place of sucrose. Strains LR2 (ispA P_Xyi-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.

1.3 Selection of Mutations Promoting L-form Proliferation

For selection of the RM121 mutant, 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 (as judged by phase contrast microscopy) was extracted and the mutations were identified by whole genome sequencing. For the selection of the accDA* mutant, 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 (as judged by phase contrast microscopy) were diluted at 10^"3 into fresh medium and incubated at 30°C for 3 days. After several dilution cycles, purified 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.

1.4 Genome Sequencing and Identification of SNPs Whole genome sequencing was performed with the illumina HiSeq 2000 System (GATC- Biotech, Germany). Sequencing samples were prepared as described previously (Dominguez-Cuevas et al., 2012). Sequence reads were aligned with SeqMan Ngen (DNASTAR^®, USA) software using the NCBI B. subtilis 168 genome (GenBank: AL009126.3) as reference. SNPs and nucleotides deletion/insertion were analysed with SeqMan Pro (DNASTAR^®, USA) software.

1.5 Western Blot and Quantitative Real Time PCR

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 OD₆₀₀ 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. Cell 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 μΙ of cDNA were added to 10 μΙ of MESA Blue qPCR Master Mix Plus (Eurogentec), 2 μΙ of each primer (1 μΜ stock) and 2 μΙ of H₂0. 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. 1.6 Microscopy and Image Analysis

For time-lapse microscopy, B. subtilis L-form cells were imaged in ibiTreat adherent, 35 mm sterile glass bottom microwell dishes (ibidi GmbH, Munich, 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.

For fluorescence microscopy, a late exponential phase culture of B. subtilis grown on LB at 37°C was stained with Mitotracker Green. After several washings with fresh LB, the cells were mounted on microscope slides covered with an agarose pad. The cells were imaged on a Zeiss Axiovert 200 M microscope equipped with a Sony Cool-Snap HQ cooled CCD camera or on a Nikon N-SIM microscope equipped with a Nikon APO TIRF ^χ 100/1.49 lens in both EPI and 2D-SIM modes with 488-nm solid state lasers.

For electron microscopy, a late exponential phase culture of B. subtilis grown on LB at 37°C was fixed with 2% gluteraldehyde in sodium cacodylate buffer and secondarily fixed with 1 % osmium tetroxide and potassium ferricyanide. The dehydration, embedding, sectioning and staining were performed by the Electron Microscopy Research Services Unit (Newcastle University). The grids were examined on a Philips CM 100 Compustage (FEI) Transmission Electron Microscope and digital images were collected using an AMT CCD camera (Deben). Pictures were prepared for publication using ImageJ (http://rsb.info.nih.gov/ij) and Adobe Photoshop.

1.7 Theoretical Cell Volume and Surface Area Considerations

As described in Bendezu and de Boer (Bendezu and de Boer, 2008), the volume (V) and surface area (S) of a rod shaped cell were calculated using V_r = 4/3ΤΤ/·³ + π η, S_c = r² + 2 rh, with r the radius and h the cell length, r was measured from B. subtilis cells grown in NB/MSM. The volume and surface area of a spherical cell were calculated using V_s = 4/3π?, S_s = 4π . 2. Results

2. 1. Contrasting Effects of ispA and P_xyrmurE-B Mutations on L-form Growth

It has previously been shown that repression of the peptidoglycan (PG) precursor pathway using a repressible P_xyrmurE-B construct, together with a single point mutation of the ispA gene encoding a polyisoprenoid synthase, results in L-form growth of B. subtilis 168 (Leaver et al., 2009). Consistent with this, complementation of the ispA mutation prevented L-form growth in a similar strain (Figure 9A). A strain with P_xyrmurE-B, and a repressible allele of ispA was also built and it was shown that this strain also grew well in the L-form state when both promoters were repressed (Figure 9B). Investigations of L-form phenotypes are complicated by several factors: i) the heterogeneity of the population in terms of cell shape and size; ii) strong selection for compensating mutations that enhance the growth rate or cell stability; iii) requirement for "escape" mutations that facilitate the emergence of L-forms from a population of rods (Dominguez-Cuevas et al., 2012). To help assess the effects of the different mutations (e.g. repressible ispA and P_xyrmurE-B) on L-form growth a protocol was developed in which cells of a defined genotype were grown in the rod state, then converted to protoplasts by stripping the cell wall with lysozyme and cultured in our standard L-form medium. 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. For reasons that are not understood, reversion of protoplasts or L-forms to the walled state (regeneration) occurs at a very low frequency, even if they are capable of synthesising wall material. Benzamide is added to prevent the rare regenerated rods, which grow much more rapidly than L-forms, from overrunning the cultures. Figure 1A shows the transition from protoplasts to proliferating L-forms, for strain LR2 (ispA P_xyrmurE-B). In contrast, wild type cells or ispA mutant cells showed only a slow increase in size over many hours (Figure 1 B). Remarkably, even though they showed very little growth and no detectable division, the cells remained intact for many days under these conditions (not shown). In contrast, after a limited amount of growth, the P_xyrmurE-B protoplasts frequently initiated L-form-like pulsating shape changes but the cells then lysed. Figure 1C 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. An asterisk highlights one cell that successfully produced at least one smaller progeny cell (Figure 1C, 395 min). It was evident from these and similar time lapses that these cells are capable of initiating L-form-like shape perturbations and occasionally producing progeny cells, but they do not undergo prolonged proliferative increase because the shape changes are almost always a prelude to cell lysis. In section 2.10 below, it is shown that the ispA mutation can be substituted by mutations in many genes on different metabolic pathways, albeit generally giving less rapid culture growth than ispA. Each mutation presumably works at least in part to prevent the cell lysis described for the P_xyrmurE-B construct and thereby increases the frequency of successful cell division events.

2.2 Repression of the PG Precursor Pathway Promotes L-form Growth

In the original experiments of Leaver et al. (2009) the P_xyrmurE-B construct was used to repress PG precursor synthesis so as to provide an efficient means of converting cells to a wall deficient state, from which growing L-form variants could be isolated. However, the experiments illustrated in Figure 2 show that repression of PG synthesis is required continuously for the efficient growth of L-form cells (in the presence of a lysis suppressing mutation, in this case, ispA). As shown in Figure 2A, repression of the murE-B operon allowed vigorous L-form growth, whereas in the presence of inducer, growth was abolished. Figure 2B shows that repression of 2 other genes in the PG precursor pathway, murC or dal, also allowed L-form growth. The applicants wished to test whether inhibition of PG precursor synthesis together with an ispA (or equivalent) mutation was the only way to generate efficient L-form growth. They used an improved selection regime (see 1.3 above) to isolate L-form mutants in a single step, starting from wild type cells. One such mutant was isolated successfully. Genome sequencing revealed that the mutation responsible was an 18 kbp deletion. This deletion removed the murC gene, together with 17 other coding regions (Figure 2C). The 18 kbp deletion was reconstructed in the presence of an IPTG-inducible ectopic copy of murC on a plasmid (strain RM121) and it was shown that murC is the only essential gene in the deletion (Figure 2D). The ability of this strain to grow as an L-form was then confirmed (Figure 2E). Although not wishing to be bound to this theory, it is likely that deletion of murC blocks the PG precursor pathway in a manner similar to depleting MurE-B and that one or more of the nearby deleted genes (within the 18 kbp deletion) operates like an ispA mutation. Thus, optimal L-form growth under the conditions used herein probably requires at least 2 mutations, and it appeared important that a least one of the mutations blocks the PG precursor pathway. i) Candidate gene approach in the PG precursor pathway

As mentioned above, 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. 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. These results suggest that repression of any gene required for PG precursor synthesis might support L-form growth and excess membrane production. ii) Candidate gene approach in the wall teichoic acid synthesis

In B. subtilis, the cell wall has two major components: peptidoglycan (PG) and the PG- attached anionic cell wall polymer, wall teichoic acid (WTA). 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

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.

2.3 Overproduction of AccDA Supports L-form Growth

To understand better the mechanisms underlying the PG precursor effect the applicants attempted to isolate L-form promoting mutations that do not affect the PG precursor pathway. Since mutations blocking this pathway, such as deletion of murC, prevent L-forms from reverting to the walled state, L-form variants that retained the ability to grow as rods were screened for (see 1.3 above). Cells containing an ispA mutation were used to eliminate the need for two mutational events. Revertable mutants were rare but as shown in Figure 3 one such mutant was isolated, which was able to grow in the walled state, irrespective of the presence or absence of the ispA mutation (panel C), and which grew in the L-form state, in the presence of the ispA mutation (panels B, D). Whole genome sequencing revealed that the mutant strain RM84 had a single point mutation ("accDA*"; Figure 3A) in the 5'UTR of the operon containing the genes accD and accA, which together encode the carboxyltransferase subunit of acetyl CoA carboxylase (Cronan and Waldrop, 2002). Since the mutation lay in an inverted repeat just upstream of the accDA coding region, it was possible that it works by altering the rate of AccDA synthesis. This was tested by making an accA-his fusion gene. 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).

To test whether overexpression of AccDA was responsible for the L-form growth phenotype, a strain was constructed that carried an extra copy of accDA under the control of xylose inducible promoter (P_xyj) at the ectopic amyE locus. The ability of various protoplasts to grow in L-form medium was then tested. As shown in Figure 3F, accDA overexpression did indeed induce L-form proliferation in the presence of ispA mutation. Figure 3G shows that the rate of growth was dependent on the level of induction of the ectopic copy of accDA. In addition, in cells overexpressing AccDA in a wild type background, time lapse microscopy revealed shape changes and lysis similar to those observed following PG precursor gene repression. In the presence of the ispA mutation, the process of L-form proliferation was also similar to that observed for repression of PG precursor synthesis (Figures 3H, I) (Leaver et al., 2009). These results demonstrated that accDA overexpression supports L-form growth in much the same way as inhibition of PG precursor synthesis. Control experiments showed that L-form growth was not promoted by overexpression of either gene separately, nor by overexpression of the accBC operon, encoding the biotin carboxylase subunit of acetyl CoA carboxylase (not shown).

2.4 Overproduction of AccDA Increases the Intracellular Levels of Malonyl-CoA and Fatty Acid Synthase Enzymes

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). In bacteria, 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). The later steps of the FAS II cycle are carried out by proteins almost all of which, in B. subtilis, are transcriptionally regulated by the FapR repressor (Figure 4A) (Schujman et al., 2003). Binding of malonyl CoA to FapR prevents binding of FapR to its target sequences thereby inducing expression of the fapR regulon (Schujman et al., 2006; Schujman et al., 2003). Therefore, malonyl CoA is not only an essential molecular intermediate in the FAS II system, but it also plays a key role as a signalling molecule regulating FapR activity to control the synthesis of FAS II components.

It seemed possible that overproduction of AccDA might increase the intracellular levels of malonyl CoA, which would in turn increase the expression of FAS II genes via relief of FapR repression. To test this possibility, the applicants first examined the effect of AccDA overproduction on expression of the FapR regulon using qPCR. As shown in Figure 4B, the expression of various genes in the FapR regulon (but notably not plsY, an example of a downstream gene not regulated by FapR), were significantly induced by the overproduction of AccDA. To further investigate the role of FapR a P_xyrfapR fusion placed at the amyE locus was made. Xylose-induced ectopic expression of fapR strongly inhibited cell viability (normal walled cells) presumably due to the repression of FAS II components (Figure 4C, left). However, when AccDA was simultaneously over-produced (via an IPTG-inducible construct), the lethality was suppressed (Figure 4C, middle), consistent with increased malonyl CoA levels relieving the FapR repression. A fapR point mutant encoding a protein, FapR_{R1 0}6A, with greatly decreased affinity for malonyl CoA but normal DNA binding activity (Schujman et al., 2006) was then made. Synthesis of FapR_{R1 0}6A inhibited cell viability similarly to wild-type FapR, but in this case the growth defect was not suppressed by AccDA overproduction (Figure 4C, right).

These results were consistent with the notion that overproduction of AccDA results in increased levels of malonyl CoA and that this in turn leads to enhanced FAS II activity via the lifting of FapR repression. To test whether overexpression of the fapR regulon was the reason why the overproduction of AccDA promoted L-form growth, the effects of a deletion of fapR on protoplast growth was examined. Figure 4D shows that various genes in the fapR regulon (but not p/sY) were, as expected, highly induced in the AfapR mutant. However, this did not allow protoplasts to grow as L-forms, either in the presence or absence of an ispA mutation (Figure 4E). In contrast, L-form growth occurred normally independent of fapR status in cells overproducing AccDA (Figure 4E).

2.5 L-form Growth Promotion Requires Fatty Acid Synthase Activity

During the course of above experiments, it was found that the ectopic overexpression of AccDA (amyE::P_xyraccDA) that supported L-form growth was lethal in the walled state, in both wild type and ispA mutant backgrounds (Figures 4F and 10A). In liquid medium lethality was manifested by culture lysis in the late exponential or early stationary phase (Figures 10B and 10C). These phenotypic effects were again specific for AccDA overproduction and were not seen in the cells overexpressing AccA, AccD, or AccBC (Figure 10D). The applicants wondered whether the lytic phenotype and potentially also the L-form growth capability might be due to excessive fatty acid and or membrane lipid synthesis. If so, 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. Note that the plsX and fabD strains failed to grow at zero IPTG because fatty acid synthesis is of course an essential process in all cells. In contrast, fabHA repression was not lethal because it is partially redundant to the fabHB gene (Choi et al., 2000). In a complementary experiment the applicants took advantage of the antibiotic cerulenin, which is a specific inhibitor of FabF (Figure 4A) (Moche et al., 1999). As shown in Figure 10F, the growth inhibition of walled cells caused by overproduction of AccDA was rescued in the presence of sub-MIC levels of cerulenin, which did not affect growth of the wild type strain. The effects of FAS II enzyme repression or inhibition on L-form growth promoted by overproduction of AccDA or inhibition of PG precursor synthesis were then examined. Importantly, the same levels of IPTG that suppressed the lethal effects of AccDA overexpression in walled cells were incompatible with growth in the L-form state (Figures 4H and 11A-C). A low concentration of cerulenin also blocked growth in the L-form but not the walled state, and this cerulenin effect was overcome by a resistant allele [cer-20; (Schujman et al., 1998)] (Figure 11 D). These results strongly suggested that L-form growth is dependent on a threshold level of flux through the fatty acid synthetic pathway.

2.6 AccDA Overproduction Results in Excess Membrane Synthesis

Given the above results the applicants wished to test more directly for the effects of AccDA overproduction on fatty acid or membrane synthesis. A strain containing the ectopic xylose- inducible copy of accDA was grown and induced with xylose, then stained with a membrane dye (Mito Tracker) and examined the cells by fluorescence microscopy. Non-induced cells exhibited the typical rod shape morphology with a highly regular and smooth fluorescence only associated with the cell surface (Figure 5A). However, in the xylose-treated cells overexpressing AccDA, large irregular patches of staining were evident within many cells (Figure 5B). Interestingly, cell division was slightly impaired for reasons that are not yet clear. A similar phenotype was observed in the accDA* strain (Figure 12A). By higher resolution structured illumination microscopy (typical examples and controls in Figures 5C and D), the abnormal intracellular staining resolved into what appeared to be closed vesicular structures. Sections of the cells were also examined by transmission electron microscopy and again abnormal intracellular vesicular structures were found in almost all cells (typical examples and controls in Figures 5E and F). Importantly, formation of the abnormal vesicular structures was abolished when FAS II synthesis was down regulated by repression of the P_spac-fabHA construct (Figure 12B), as in the experiments described above.

These results strongly support the idea that the upregulation of malonyl CoA synthesis and its increased utilization by the FAS II system, resulting from accDA overexpression, leads to excess membrane synthesis.

The effects of reduced PG precursor synthesis on membrane morphology was also examined. Although complete repression of the P_xyrmurE-B construct is lethal, a low level of xylose (0.1 %) allowed a limited degree of growth. As shown in Figure 5G, an excess membrane phenotype similar to that generated by overproduction of AccDA was observed. This phenotype was again suppressed by co-repression of fabHA (Figure 12C). 2.7 Excess Membrane Surface Area is Sufficient to Drive L-form-Like Division in Wild Type Protoplasts

The above results lead to a simple model in which L-form proliferation is driven by excess membrane synthesis, leading to an abnormal cell surface area to volume (A/V) ratio. If so, artificially increasing the surface area of protoplasts of wild type (i.e. accDA⁺) cells should lead to spontaneous L-form-like proliferation. The applicants reasoned that a simple way to generate protoplasts with excess surface area was to derive the protoplasts from cells that had been treated with a division inhibitor thereby generating elongated filaments. When rod shaped cells elongate they maintain an almost constant A/V ratio (Figure 6A, dashed line). However, when such cells are converted to protoplasts, simple geometry dictates that, while V should remain more or less constant, A will reduce as the cell tends towards an energy minimizing spherical shape (line). The applicants therefore cultured a wild type strain for several time periods (0, 30, 60 and 90 min) in the presence of benzamide to generate increasingly elongated cells (Figure 6B). The cells were then treated with lysozyme to generate protoplasts. As shown in Figure 6D1 , protoplasts from normal length rods generated protoplasts with the expected uniform spherical shape. Presumably, the membrane is sufficiently elastic to accommodate relatively small changes in surface area. However, protoplasts with increased cell surface area immediately showed a range of abnormal shapes (Figure 6D2-4) reminiscent of L-forms (Figure 6C). Time lapse imaging revealed that many of the cells went on to divide, generating two or more smaller "progeny" cells. Figure 6E shows still images from a typical time course. Undulating shape changes continued for 15 or so minutes, during which smaller cells pinched off. Eventually (32 min), the shape changes subsided and relatively stable, smaller and more or less spherical cells remained. The shape changes were most intense in the filaments treated with benzamide for 60 min. In the 90 minutes sample, the very long filaments were prone to lysis or spontaneous fission during the protoplasting process. Nevertheless, L-form like shape changes and cell fission were very prominent in all three samples. These proliferative events differed from those of L-forms in occurring over a shorter time period and in soon subsiding. This presumably reflects the fact that the experiment begins with an abrupt and large change in A/V but this soon terminates because cell fission generates increased numbers of smaller cells that "use up" the excess surface area (note that small spheres have a greater A/V ratio than larger spheres; Figure 6A). 2.8 Mutations that support L-form growth by generating excess membrane

The applicants have found that increasing membrane surface area leads to L-form division, and that at least three different genetic changes (accDA*, P_xyraccDA [presence of xylose], P_Xyi-murE-B [absence of xylose]) that lead to L-form growth also promote excess membrane synthesis. Other mutations that have been shown to promote L-form growth may also lead to excess membrane synthesis (see Table 2).

Table 2. Genetic changes shown to promote L-form growth and where known, confirmation that they generate excess membrane synthesis. L-form growth was examined in an ispA mutant background on sucrose containing L-form selective plates and / or liquid medium. N.d. Not determined.

Furthermore, mutations in genes that may be expected to promote L-form growth may also promote excess membrane synthesis (see table 3).

dapF tagB glmM

racE tagD gcaD

yrpC tagE

tagF

manA

Table 3. List of genes expected to promote L-form growth and potentially excess membrane synthesis but as yet untested

2.9 Genetic screen to identify other L-form promoting mutations that do not affect the PG precursor pathway

Many 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. Using this system, another mutant was also isolated, with a single point mutation in the rpsl gene (E112K), encoding ribosomal protein S9. This mutation also generated excess membrane production (Figure 8A and B). As for the other L-form mutants described, the excess membrane phenotype was suppressed when fatty acid synthesis was down regulated by repression of FabHA (Figure 8C and D). The mechanism whereby the rpsl mutation generates these effects is not yet clear, but these results show that it should be possible to isolate additional L-form mutants with excess membrane synthesis by this or similar mutational approaches.

2.10 Identification of mutations enabling L-form growth in P_xyrmurE-B

The applicants have found that under the standard experimental conditions described herein two genetic changes are needed for optimal L-form proliferation in B. subtilis. One mutation is needed to oversynthesize membrane fatty acids which drive shape modifications and cell division. The second mutation, such as ispA is needed to sustain long-time L-form growth, but the molecular basis for this effect is not yet understood. IspA catalyses the formation of farnesyl pyrophosphate in the polyprenoid synthetic pathway (Julsing et al., 2007). This pathway leads to the formation of two essential lipid molecules: menaquinone, involved in the respiratory chain, and bactoprenol, required for synthesis of both peptidoglycan and teichoic acids. To understand better the mechanisms underlying the ispA mutation in L-form growth further L-form promoting mutations that might act in a similar way to ispA were isolated by the applicants as described below. i) Genetic screen by transposon mutagenesis

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_Xyi-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). A second screen was carried out similarly but using a tn10 transposon delivery system. From these experiments, 9 mutants were newly identified (Table 4), that were capable of growth as L- forms when PG precursor synthesis was repressed (culture in the absence of xylose).

Table 4. Transposon-induced mutations resulting in stable L-form growth promoted by repression of P_xyrmurE-B. Selection for L-form growth in a P_xyrmurE strain on selective plates containing sucrose and benzamide (No xylose).

Most of mutants isolated were, directly or indirectly, related to the respiratory chain. 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. To test this further, an IPTG-dependent promoter was inserted in front of hepS gene, encoding heptaprenyl diphosphate synthase required for menaquinone synthesis, in the P_xyrmurE-B background. The repression of hepS indeed allowed L-form growth on L-form selection medium (and absence of xylose). The results suggest that the ispA mutational pathway works by reducing activity of the respiratory chain. It can be envisaged that other mutations affecting this pathway could be isolated by this approach. ii) Candidate gene approach in carbon utilization and metabolism pathways

High concentrations of sucrose (0.5 M) 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. To test this idea, several mutations were introduced into the P_xyrmurE-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. Thus, the effects of mutations disrupting genes used for utilization and uptake of glucose and sucrose (ptsG, fruA, fruK, fruC and levDEFG operon) were also examined, but no L-form growth was observed. The uptake of certain sugars including glucose, fructose and sucrose, is coupled to its phosphorylation by the phosphotransferase system (PTS). The phosphotransferase reaction requires proteins encoded by the ptsl and ptsH genes. Interestingly, ptsHI mutations were able to support L- form growth (the ptsH and ptsl mutations were present within one operon in these experiments). These results suggest that the uptake of excess sugar through the PTS system interferes with cell growth in the L-form state.

fruA No Fructose uptake

fruC No Fructose utilization

fruK No Fructose utilization

Table 5. Ability of mutants affected in pathways for sugar uptake and utilization to promote stable L-form growth following repression of P_xyrmurE-B. Selection for L-form growth in a P_Xyi-murE strain on selective plates containing sucrose and benzamide (No xylose). To understand the problem underlying the uptake of excess sugar in L-form growth, the effects of several mutations affecting carbon metabolism was examined. Several mutants of genes in glycolysis, gluconeogenesis and certain other pathways associated with glycolysis, such as the pentose phosphate pathway and the TCA cycle, were introduced into P_xyrmurE- B strain and its ability to grow in the L-form state was tested on sucrose containing L-form selective plates (Table 6). Repression of genes in the glycolysis pathway (fbaA, gapA and the pgk-tpiA-pgm-eno operon) supported L-form growth, but mutations affecting genes in the gluconeogenesis pathway and certain other pathways did not (Table 6).

Table 6. Ability of mutants affected in carbon metabolism pathways to promote stable L-form growth following repression of P_xyrmurE-B. Selection for L-form growth in a P_xyrmurE strain on selective plates containing sucrose and benzamide (No xylose).

These results suggest that the uptake of excess sugar increases flux through the glycolytic pathway, which in some way leads to inhibition of cell growth in the L-form state. Growth can be supported by any one of a range of mutations described herein. Similar methods could be used to isolate other mutations similarly allowing L-form growth affecting sugar transport, glycolytic activity, or allied processes. 3. General discussion

3.1 Two Genetic Changes Required for L-form Growth

The natural history of bacterial L-forms has been extensively described in the literature (Allan et al., 2009; Domingue and Woody, 1997), but until recently almost nothing was known about the molecular mechanisms underlying L-form proliferation. The applicants show herein that under the standard experimental conditions used herein two genetic changes are needed for optimal L-form growth in B. subtilis. One mutation is needed to oversynthesize membrane fatty acids and can occur directly, by upregulating the FAS II system via AccDA overproduction, or indirectly, via inhibition of the PG precursor pathway or inhibition of ribosomal protein S9 expression. A single point mutation in a stem-loop structure just upstream of accD coding region increased intracellular levels of AccDA, suggesting that a regulatory mechanism controlling AccDA synthesis may exist in B. subtilis.

Acetyl CoA carboxylase (ACC) 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. Similarly, in Escherichia coli, overproduction of ACC increases the rate of fatty acid synthesis (Davis et al., 2000), but overexpression of other phospholipid synthetic genes has little affect on membrane synthesis (Cronan, 2003). Thus, Intracellular levels of ACC might have a widespread role as a rate-limiting step in membrane phospholipid synthesis. Acetyl CoA is important not only for fatty acid metabolism, but also for energy production via carbohydrate metabolism. Therefore, the intracellular levels of acetyl CoA and/or AccDA might be important factors in coordinating membrane synthesis with cell mass increase. Indeed, although little is known about the regulation of accDA expression in B. 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). Interestingly, 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.

Little is known about how PG synthesis is coordinated either with membrane synthesis or cell growth. Interestingly, it was found that inhibition of PG precursor synthesis also promotes excess membrane synthesis. This suggests that membrane lipid synthesis is negatively regulated by some element of the PG precursor synthesis pathway. However, such regulation seems indirect, since no significant effects on intracellular levels of AccDA were seen following the inhibition of PG precursor synthesis (Figure 11 E). Given that L- forms are viable in the absence of PG precursor synthesis, they may provide an interesting experimental vehicle for studying the apparent regulatory interplay between membrane and wall synthesis.

Interestingly, Bendezu and de Boer (2008) recently showed that when E. coli cultures are mutationally induced to switch from rods to spheres they fail to compensate for the changes in A/V ratio and continue to produce membrane at the higher rate required for rod shaped cells. These mutants then generate intracellular vesicles when cell division is inhibited and cell size increases, presumably due to the excess membrane (Bendezu and de Boer, 2008). However, no such excess membrane phenotype was seen when normal sized B. subtilis rods were converted to spherical protoplasts (Figure 6D1). Moreover, no vesicles were observed in an equivalent B. subtilis round (rodA) mutant after inhibition of cell division (Figure 13A). However, vesicles did appear in the rodA mutant when membrane synthesis was elevated by AccDA overproduction (Figure 13B). Therefore, it seems that, unlike E. coli, B. subtilis can at least partially adapt its rate of membrane synthesis to accommodate changes in A/V.

It now seems that the ispA mutation previously reported to sustain L-form growth Leaver et al. (2009) works by somehow stabilising L-form cells undergoing shape modulation. IspA catalyses the formation of farnesyl pyrophosphate in the polyprenoid synthetic pathway (Julsing et al., 2007). This pathway leads to the formation of two essential lipid molecules: menaquinone, involved in the respiratory chain, and bactoprenol, required for synthesis of both peptidoglycan and teichoic acids. Recent papers have described the isolation and characterization of L-forms of Listeria monocytogenes, a close relative of B. 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.

3.2 Excess Membrane as a Mechanistic Driver for L-form Division

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, 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. This conclusion is based on the following key observations; (i) overproduction of AccDA, leading to excess membrane synthesis, is sufficient for L-form growth and proliferation; (ii) a partial decrease in flux through the FAS II system can block L-form proliferation without affecting growth of walled cells; (iii) artificially increasing cell surface area by the conversion of elongated rods to protoplasts is sufficient to produce L-form-like shape changes and scission in wild type cells. Figure 7 summarises how the applicants currently view the process of L-form proliferation in B. subtilis in light of the findings presented herein. In step one (i), unbalanced growth generates an increase in cell surface area relative to cytoplasmic volume. The resultant torsional stress then leads to spontaneous shape deformation (ii). The deformed cell then 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.

3.3 L-forms as a Model for Proliferation in Primitive Cell

It has been suggested that 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. Furthermore, the theoretical basis for generation of shape changes, pinching and budding of vesicles, by this mechanism, has been well documented (Bozic and Svetina, 2007; Luisi et al., 2008; Svetina, 2009). Similarly, L-form proliferation in B. subtilis does not require the mechanisms that are pivotal for regulation of cell division, cell shape, elongation, coordinated chromosome segregation and balanced membrane lipid synthesis of walled cells (Leaver et al., 2009; Mercier et al., 2012) but it does require excess membrane production to generate an imbalance between growth of cell surface area and volume. In addition, although various modes of cell division have been described for proliferation of L-forms, including membrane extrusion, blebbing and vesiculation (reviewed recently; (Briers et al., 2012b; Errington., 2013)), all of these events seem to be achievable in relatively simple in vitro lipid vesicle systems (Hanczyc et al., 2003; Peterlin et al., 2009; Terasawa et al., 2012; Zhu and Szostak, 2009). The results provided herein provide direct support for the notion that purely biophysical effects could have supported an efficient mode of proliferation in primitive cells, before the invention of the cell wall, and provide an extant model for exploration of the possible properties of early forms of cellular life.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. REFERENCES Adams, D.W., and Errington, J. (2009). Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nat Rev Microbiol 7, 642-653.

Adams, D.W., Wu, L.J., Czaplewski, L.G., and Errington, J. (2011). Multiple effects of benzamide antibiotics on FtsZ function. Mol Microbiol 80, 68-84.

Allan, E.J. (1991). Induction and cultivation of a stable L-form of Bacillus subtilis. J AppI Bacteriol 70, 339-343.

Allan, E.J., Hoischen, C, and Gumpert, J. (2009). Bacterial L-forms. Adv AppI Microbiol 68, 1-39.

Anagnostopoulos, C, and Spizizen, J. (1961). Requirements for transformation in Bacillus subtilis J Bacteriol 81,

Bendezu, F.O., and de Boer, P. A. (2008). Conditional lethality, division defects, membrane involution, and endocytosis in mre and mrd shape mutants of Escherichia coli. J. Bacteriol 190, 1792-181 1.

Bilder, P., Lightle, S., Bainbridge, G., Ohren, J., Finzel, B., Sun, F., Holley, S., Al-Kassim, L, Spessard, C, Melnick, M., et al. (2006). The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme. Biochemistry 45, 1712-1722.

Bourne, N., and Dancer, B.N. (1986). Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains. J Gen Microbiol 132, 251-255.

Bozic, B., and Svetina, S. (2007). Vesicle self-reproduction: the involvement of membrane hydraulic and solute permeabilities. Eur Biophys J 24, 79-90.

Briers, Y., Staubli, T., Schmid, M.C., Wagner, M., Schuppler, M., and Loessner, M.J. (2012a). Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria. PloS One 7, e38514.

Briers, Y., Walde, P., Schuppler, M., and Loessner, M.J. (2012b). How did bacterial ancestors reproduce? Lessons from L-form cells and giant lipid vesicles: Multiplication similarities between lipid vesicles and L-form bacteria. Bioessays 34, 1078-1084.

Brodersen, DE., demons, WM Jr., Carter, AP., Wimberly, BT., Ramakrishnan, V. (2002).

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.

Chen, I.A. (2009). Cell division: breaking up is easy to do. Curr Biol 19, R327-328. Choi, K.H., Heath, R.J., and Rock, CO. (2000). beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol 182, 365-370.

Claessen, D., Emmins, R., Hamoen, L.W., Daniel, R.A., Errington, J., and Edwards, D.H. (2008). Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis. Mol Microbiol 68, 1029-1046.

Cronan, J.E., Jr., and Waldrop, G.L. (2002). Multi-subunit acetyl-CoA carboxylases. Prog Lipid Res 41, 407-435.

Cronan, J.E. (2003). Bacterial membrane lipids: where do we stand? Annu Rev Microbiol 57, 203-224.

Davis, M.S., Solbiati, J., and Cronan, J.E., Jr. (2000). Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli. J Biol Chem 275, 28593-28598.

Dell'Era, S., Buchrieser, C, Couve, E., Schnell, B., Briers, Y., Schuppler, M., and Loessner, M.J. (2009). Listeria monocytogenes L-forms respond to cell wall deficiency by modifying gene expression and the mode of division. Mol Microbiol 73, 306-322.

Domingue, G.J., Sr., and Woody, H.B. (1997). Bacterial persistence and expression of disease. Clin Microbiol Rev 10, 320-344.

Dominguez-Cuevas, P., Mercier, R., Leaver, M., Kawai, Y., and Errington, J. (2012). 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.

Errington, J. (2013) L-form bacteria, cell walls and the origins of life. Open Biol 3(1): 120143 Fujita, Y. (2009). Carbon catabolite control of the metabolic network in Bacillus subtilis. Biosci Biotechnol Biochem. 73(2):245-59

Gibson et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 2010 Jul 2;329(5987):52-6

Hamoen, L.W., Smits, W.K., de Jong, A., Holsappel, S., and Kuipers, O.P. (2002). Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach. Nucleic Acids Res 30, 5517-5528.

Hanczyc, M.M., Fujikawa, S.M., and Szostak, J.W. (2003). Experimental models of primitive cellular compartments: encapsulation, growth, and division. Science (New York, NY) 302, 618-622.

Haydon et al., 2008, Science Sep 19;321 (5896):1673-5

Ishikawa, S., Kawai, Y., Hiramatsu, K., Kuwano, M., and Ogasawara, N. (2006). A new FtsZ- interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis. Mol Microbiol 60, 1364-1380. Julsing, M.K., Rijpkema, M., Woerdenbag, H.J., Quax, W.J., and Kayser, O. (2007). Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis. Appl Microbiol Biotechnol 75, 1377-1384.

Kandler, G., and Kandler, O. (1954). Studies on morphology and multiplication of pleuropneumonia-like organisms and on bacterial L-phase, I. Light microscopy. Arch Mikrobiol 21, 178-201.

Kawai, Y., Maries-Wright, J., Cleverley, R.M., Emmins, R., Ishikawa, S., Kuwano, M., Heinz, N., Bui, N.K., Hoyland, C.N., Ogasawara, N., et al. (2011). A widespread family of bacterial cell wall assembly proteins. EMBO J 30, 4931-4941.

Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., et al. (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249-256.

Leaver, M., Dominguez-Cuevas, P., Coxhead, J.M., Daniel, R.A., and Errington, J. (2009). Life without a wall or division machine in Bacillus subtilis. Nature 457, 849-853.

Leprince et al. Streamlining genomes: toward the generation of simplified and stabilized microbial systems. Curr Opin Biotechnol. 2012 Oct;23(5):651-8;

Lewis, P.J., and Marston, A.L. (1999). GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis Gene. 227, 101-110.

Luisi, P.L., de Souza, T.P., and Stano, P. (2008). Vesicle behavior: in search of explanations. J Phys Chem B 112, 14655-14664.

Margolin, W. (2005). FtsZ and the division of prokaryotic cells and organelles. Nat Rev Mol Cell Biol 6, 862-871.

Meades, G., Jr., Benson, B.K., Grove, A., and Waldrop, G.L. (2010). A tale of two functions: enzymatic activity and translational repression by carboxyltransferase. Nucleic Acids Res 38, 1217-1227.

Mercier, R., Dominguez-Cuevas, P., and Errington, J. (2012). Crucial role for membrane fluidity in proliferation of primitive cells. Cell Rep 1, 417-423.

Moche, M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. (1999). Structure of the complex between the antibiotic cerulenin and its target, beta-ketoacyl-acyl carrier protein synthase. J Biol Chem 274, 6031-6034.

Peterlin, P., Arrigler, V., Kogej, K., Svetina, S., and Walde, P. (2009). Growth and shape transformations of giant phospholipid vesicles upon interaction with an aqueous oleic acid suspension. Chem Phys Lipids 159, 67-76.

Quisel, J.D., Burkholder, W.F., and Grossman, A.D. (2001). In vivo effects of sporulation kinases on mutant SpoOA proteins in Bacillus subtilis. J. Bacteriol 183, 6573-6578. Rock, CO., and Cronan, J.E. (1996). Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis. Biochim Biophys Acta 1302, 1-16.

Sambrook, J., Fritsch, E.F., and Maniatis, T (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Schujman, G.E., Grau, R., Gramajo, H.C., Ornella, L., and de Mendoza, D. (1998). De novo fatty acid synthesis is required for establishment of cell type-specific gene transcription during sporulation in Bacillus subtilis. Mol Microbiol 29, 1215-1224.

Schujman, G.E., Guerin, M., Buschiazzo, A., Schaeffer, F., Llarrull, L.I., Reh, G., Vila, A.J., Alzari, P.M., and de Mendoza, D. (2006). Structural basis of lipid biosynthesis regulation in Gram-positive bacteria. EM BO J 25, 4074-4083.

Schujman, G.E., Paoletti, L., Grossman, A.D., and de Mendoza, D. (2003). FapR, a bacterial transcription factor involved in global regulation of membrane lipid biosynthesis. Dev Cell 4, 663-672.

Siddiqui, R.A., Hoischen, C, Hoist, O., Heinze, I., Schlott, B., Gumpert, J., Diekmann, S., Grosse, F., and Platzer, M. (2006). The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli. FEMS Microbiol Lett 258, 305-31 1.

Svetina, S. (2009). Vesicle budding and the origin of cellular life. Chemphyschem 10, 2769- 2776.

Terasawa, H., Nishimura, K., Suzuki, H., Matsuura, T., and Yomo, T. (2012). Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules. Proc Natl Acad Sci U S A 109, 5942-5947.

Typas, A., Banzhaf, M., Gross, CA., Vollmer, W. (201 1). From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10(2): 123- 36.

Vagner, V., Dervyn, E., and Ehrlich, S.D. (1998). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology (Reading, England) 144 ( Pt 11), 3097-3104.

Zamboni, N., Mouncey, N., Hohmann, HP., Sauer, U. (2003). Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis. Metab Eng 5(1):49-55.

Zhu, T.F., and Szostak, J.W. (2009). Coupled growth and division of model protocell membranes. J Am Chem Soc 131, 5705-5713.

Claims

1. 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) 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;

(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.

2. The recombinant cell according to claim 1 , wherein the first nucleic acid molecule, the second nucleic acid molecule and/or the third nucleic acid molecule is part of an expression vector.

3. The recombinant cell according to any preceding claim, wherein the first nucleic acid molecule encodes a carboxyltransferase subunit of acetyl CoA carboxylase.

4. The recombinant cell according to any preceding claim, wherein the first nucleic acid molecule comprises an inducible promoter.

5. The recombinant cell according to any preceding claim, wherein the first nucleic acid molecule comprises a mutation that increases expression of the encoded

carboxyltransferase.

6. The recombinant cell according to claim 5, wherein the mutation is in the 5' untranslated region (UTR) of the nucleic acid molecule.

7. The recombinant cell according to claim 6, wherein 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 ID NC .

8. The recombinant cell according to claim 6 or 7, wherein the 5'UTR has at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:2.

9. The recombinant cell according to claim 8, wherein the 5'UTR comprises or consists of the nucleic acid sequence of SEQ ID NO: 2.

10. The recombinant cell according to any one of the preceding claims, wherein the second nucleic acid molecule encodes a polypeptide involved in peptidoglycan synthesis.

1 1. The recombinant cell according to claim 10, wherein the second nucleic acid molecule encodes a polypeptide selected from the group consisting of murF, dapF, racE, yrpC, murAA and murC.

12. The recombinant cell according to claim 11 , wherein the second nucleic acid molecule encodes a polypeptide selected from the group consisting of murAA and murC.

13. The recombinant cell according to any one of claims 1 to 9, wherein the second nucleic acid molecule encodes a polypeptide involved in wall teichoic acid (WTA) synthesis.

14. The recombinant cell according to claim 13, wherein the second nucleic acid molecule encodes a polypeptide selected from the group consisting of tagA, tagB, tagD, tagE, tagF, manA and tagO.

15. The recombinant cell according to claim 14, wherein the second nucleic acid molecule encodes tagO.

16. The recombinant cell according to any one of claims 1 to 9, wherein the second nucleic acid molecule encodes a polypeptide involved in the regulation of peptidoglycan and/or wall teichoic acid synthesis.

17. The recombinant cell according to claim 16, wherein the second nucleic acid molecule encodes a polypeptide selected from the group consisting of glmS, glmM, gcaD and MreB.

18. The recombinant cell according to claim 17, wherein the second nucleic acid molecule encodes MreB.

19. The recombinant cell according to any preceding claim, wherein the second nucleic acid molecule comprises a repressible promoter.

20. The recombinant cell according to any preceding claim, wherein the second nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.

21. The recombinant cell according to any preceding claim, wherein the third nucleic acid molecule comprises a mutation that modifies the activity of the encoded ribosomal S9 protein.

22. The recombinant cell according to claim 21 , 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.

23. The recombinant cell according to any preceding claim, wherein the cell further comprises at least one of:

24. The recombinant cell according to claim 23, wherein 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.

25. The recombinant cell according to claim 23 or 24, wherein the fourth nucleic acid molecule comprises a repressible promoter.

26. The recombinant cell according to any one of claims 23 to 25, wherein the fourth nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.

27. The recombinant cell according to any one of claims 23 to 26, wherein the fifth nucleic acid molecule encodes a polypeptide selected from the group consisting of ptsH, ptsl, fbaA, gapA, pgk, tpiA, pgm and eno.

28. The recombinant cell according to any one of claims 23 to 27, wherein the fifth nucleic acid molecule comprises a repressible promoter.

29. The recombinant cell according to any one of claims 23 to 28, wherein the fifth nucleic acid molecule comprises a mutation that inhibits expression of the encoded polypeptide.

30. The recombinant cell according to any preceding claim, wherein the cell is a bacterial cell.

31. The recombinant cell according to claim 30, wherein the bacterial cell is B.subtilis.

32. 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:

(c) modified activity of ribosomal protein S9.

33. The semi-synthetic or synthetic cell according to claim 32, wherein the cell additionally has at least one of:

(d) decreased expression of at least one polypeptide involved in the respiratory chain; and/or (e) decreased expression of at least one polypeptide involved in the glycolysis pathway.

34. The semi-synthetic or synthetic cell according to claim 32 or 33, wherein the

carboxyltransferase is a carboxyltransferase subunit of acetyl CoA carboxylase.

35. The semi-synthetic or synthetic cell according to any one of claims 32 to 34, wherein the at least one polypeptide involved in cell wall synthesis is involved in peptidoglycan synthesis.

36. The semi-synthetic or synthetic cell according to claims 35, wherein the polypeptide is selected from the group consisting of murB, murG, murE, mraY, dal, murF, dapF, racE, yrpC, murAA and murC.

37. The semi-synthetic or synthetic cell according to claim 36, wherein the polypeptide selected from the group consisting of murB, murG, murE, mraY, dal, murAA and murC.

38. The semi-synthetic or synthetic cell according to any one of claims 32 to 34, wherein the at least one polypeptide involved in cell wall synthesis is involved in wall teichoic acid (WTA) synthesis.

39. The semi-synthetic or synthetic cell according to claim 38, wherein the polypeptide is selected from the group consisting of tagA, tagB, tagD, tagE, tagF, manA and tagO.

40. The semi-synthetic or synthetic cell according to claim 39, wherein the polypeptide is tagO.

41. The semi-synthetic or synthetic cell according to any one of claims 32 to 34, wherein the at least one polypeptide involved in cell wall synthesis is involved in the regulation of peptidoglycan and/or wall teichoic acid synthesis.

42. The semi-synthetic or synthetic cell according to claim 41 , wherein the polypeptide is selected from the group consisting of glmS, glmM, gcaD and mreB.

43. The semi-synthetic or synthetic cell according to claim 42, wherein the polypeptide is MreB.

44. The semi-synthetic or synthetic cell according to any one of claims 33 to 43, wherein 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.

45. The semi-synthetic or synthetic cell according to any one of claims 33 to 44, wherein 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.

46. 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:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or (c) an agent that modifies ribosomal protein S9 activity.

47. The method according to claim 46, wherein the agent comprises a nucleic acid molecule.

48. The method according to claim 46 or 47, wherein the nucleic acid molecule does not comprise the nucleic acid sequence of SEQ ID NO:3 .

49. The method according to any one of claims 46 to 48, wherein 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.

50. The method according to any one of claims 46 to 49, wherein 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.

51. The method according to any one of claims 46 to 50, wherein 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.

52. The method according to any one of claims 46 to 51 , wherein 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.

53. The method according to any one of claims 46 to 53, wherein the method further comprises providing to the cell at least one of:

(d) an agent that decreases respiratory chain activity; and/or (e) an agent that decreases the activity of the glycolysis pathway.

54. The method according to claim 53, wherein 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.

55. The method according to claim 53 or 54, wherein 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.

56. The method according to claim any one of claims 46 to 55, wherein the providing step(s) generate(s) a cell according to any one of claims 1 to 45.

57. The method according to any one of claims 46 to 56, further comprising culturing the cell under conditions that support the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.

58. The method according to any one of claims 46 to 57, further comprising recovering the produced fatty acids, fatty acid derivatives and/or membrane associated molecules.

59. Use of a cell according to any one of claims 1 to 45 in the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.

60. Use of a cell according to any one of claims 1 to 45 in drug or vaccine delivery.

61. Use of at least one of:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or (c) an agent that modifies protein S9 activity; in the production of fatty acids, fatty acid derivatives and/or membrane associated molecules.

62. A reaction vessel containing a cell according to any one of claims 1 to 45 and medium sufficient to support growth of the cell.

63. The reaction vessel according to claim 62, wherein the reaction vessel is a bioreactor or a fermenter.

64. The method according to any one of claims 46 to 58, wherein the method is performed in the reaction vessel according to claim 62 or 63.

65. A method of inducing L-form growth in a cell, comprising providing to a cell at least one of:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or

66. The method according to claim 65, wherein the agent that removes the cell wall and/or prevents cell wall synthesis is a lysozyme.

67. The method according to any one of claims 65 or 66, wherein the method further comprises providing to the cell at least one of: (d) an agent that decreases respiratory chain activity; and/or

(e) an agent that decreases the activity of the glycolysis pathway.

68. The method according to any one of claims 65 to 67, wherein the cell is cultured under conditions that support L-form growth.

69. A method of preparing a therapeutic composition comprising: (i) providing to a cell at least one of:

(a) an agent that increases carboxyltransferase activity;

(b) an agent that decreases cell wall synthesis; and/or (c) an agent that modifies ribosomal protein S9 activity;

(iii) formulating the cell as a therapeutic agent.

70. A method of identifying a DNA mutation that supports L-form growth in a cell comprising:

(ii) culturing the protoplast under conditions that support L-form growth;

(iii) identifying a cell capable of L-form growth; and

71. The method according to claim 70, wherein the cell has previously been provided with at least one of:

(a) an agent that decreases respiratory chain activity; and/or

(b) an agent that decreases the activity of the glycolysis pathway.

72. The method according to claim 71 , wherein 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.

73. The method according to claim 71 or 72, wherein 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.

74. The method according to any one of claims 70 to 73, wherein step (iii) further comprises culturing the identified cell under conditions that support cell wall regeneration and identifying a cell with a regenerated cell wall.

75. A recombinant cell substantially as described herein with reference to the accompanying drawings.

76. A semi-synthetic or synthetic cell substantially as described herein with reference to the accompanying drawings.

77. A method of producing fatty acids, fatty acid derivatives and/or membrane associated molecules substantially as described herein with reference to the accompanying drawings.

78. A reaction vessel substantially as described herein with reference to the accompanying drawings.

79. A method of inducing L-form growth substantially as described herein with reference to the accompanying drawings.

80. A method of preparing a therapeutic composition substantially as described herein with reference to the accompanying drawings.

81. 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.