Enhanced biocompatible metathesis

Abstract

The disclosed technology relates to a method of biocompatible metathesis employing a biocompatible metathesis catalyst (i.e., a catalyst that is not toxic to cells at less than 5 mM) and a bacterium, where interactions between the biocompatible metathesis catalyst and bacterial fimbriae influence catalysis.

Description

TITLEENHANCED BIOCOMPATIBLE METATHESIS BACKGROUND OF THE INVENTION

[0001] The disclosed technology relates to a method of biocompatible metathesis employing a biocompatible metathesis catalyst (i.e., a catalyst that is not toxic to cells at less than 5 mM) and a bacterium, where interactions between the biocompatible metathesis catalyst and bacterial fimbriae influence catalysis.

[0002] Sustainable olefin production is a challenge and may be accessed from renewable feedstocks by combining engineered microbes with abiotic transition metal catalysts. For example, alkene cross-metathesis represents an established organometallic route to various petrochemicals. Synthetic chemical methods represent productive routes to long chain alkenes, including lubricants, owing to several decades of catalyst development. Their reliance on fossil fuels, however, remains a significant challenge for the petrochemical industry.

[0003] Advances in synthetic biology have enabled novel routes to many of these chemicals from renewable feedstocks. Although Escherichia coli represents a genetically tractable chassis, appropriate biocatalysts are lacking for several chemical reactions, including alkene cross-metathesis. Biocompatible chemistry demonstrates that de novo pathways can be interfaced with abiotic catalysts, to catalyze biochemical transformations previously inaccessible through synthetic biology alone.

[0004] However, Chemistries completed using biological systems often require the destruction of the cell media to ensure extraction of the relevant raw materials, thereby minimizing flux (yield) of product.

[0005] The aim of the disclosed technology is to improve yields (flux) of product by utilizing biocompatible catalysis.SUMMARY OF THE INVENTION

[0006] It was found that cell surface bacterial fimbriae enhance yields of product when employing a biocompatible metathesis catalyst.

[0007] The disclosed technology therefore provides a method of biocompatible metathesis. The method includes introducing a starting material to a medium containing a bacterium and a biocompatible metathesis catalyst. The biocompatible metathesis catalyst is selected to be compatible with the bacterium and fimbriae, allowing the catalyst to interface with the bacterium.

[0008] Generally, the starting material for the method will be a saccharide and the product being sought can be an olefin, however any type of metathesis is envisioned.

[0009] The technology also includes a composition including a biocompatible metathesis catalyst and a bacterium having fimbriae compatible with the catalyst.DETAILED DESCRIPTION OF THE INVENTION

[0010] Various preferred features and embodiments will be described below by way of non-limiting illustration.

[0011] The technology has, as one embodiment, a method of biocompatible metathesis, or in other words, the use of living organisms in reactions in which two compounds exchange ions, typically with precipitation of an insoluble product.

[0012] In the instant technology, bacteria are used as the living organisms employed in the biocompatible metathesis method. Bacteria can include, for example, fimbriae containing gram-negative bacteria, such as; Klebsiella pneumoniae, Klebsiella variicola, Legionella pneumophila, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Bacillus subtilis, Ser- ratia marcescens, Helicobacter pylori, Bacillus amyloliquefaciens, Salmonella enteritidis, Salmonella typhi, Pseudomonas oleovorans, Cupriavidus necator Chlamydia trachomatiss,; Yersinia pestis, Pseudomonas nitroreducens, Acineto- bacter baumani; Shigella; and other Enterobacteriaceae; Pseudomonas; Morax- ella; Helicobacter; Stenotrophomonas; Bdellovibri.o; acetic acid bacteria; Legionella; cyanobacteria; spirochaetes; green sulfur; and green non-sulfur bacteria; Acinetobacter baumannii; cocci including Neisseria gonorrhoeae , Neisseria meningitidis, Moraxella catarrhalis, and Haemophilus influenzae ; as well as fimbriae gram-positive bacteria.

[0013] Fimbriae are long multimeric proteins that protrude from the membrane surrounding a bacterium. These thin tubes are made up of proteins, called pilin, which include structures corresponding to specific structures, generally glycoproteins or glycolipid receptors, of a cell host to which the fimbriae can adhere. These pilin structure may also adhere to the catalyst.

[0014] Bacterial strains will vary in the fimbriae associated with each strain, and the fimbriae of a specific strain may be genetically altered. While the method herein does not include the method of choosing a strain of bacteria such that the fimbriae of that bacteria are compatible with the chosen catalyst, or vice-versa, choosing a catalyst such that the catalyst is compatible with the fimbriae of the chosen bacterial strain.

[0015] The term “compatible” means that at least one group of fimbriae of the bacteria strain correspond to structures on the catalyst, such that the catalyst adheres, or remains locked in a sufficiently close relationship to the bacteria to maintain a catalytic reaction. In simpler terms, the fimbriae will include pilin structures that adhere to the biocompatible metathesis catalyst.

[0016] The biocompatible metathesis catalysts employed in the method will depend on the product desired. The term “biocompatible” is used to convey that for a selected bacterium, the catalyst will be non-toxic at up to 5mM concentration in a solution of the bacterium. Thus, the catalyst can be included at concentrations of 0.1 to 5 mM, or from 0.2 to 4 mM, or 0.3 to 3 mM, or even 0.4 to 2 mM, or even from 0.5 to 1 mM. Biocompatible metathesis catalysts can include, for example, alkene metathesis catalyst, and more particularly, abiotic metathesis catalyst, such as a transition metal catalyst, including ruthenium, titanium, cobalt, copper, and platinum.

[0017] Metathesis reactions can be employed to prepare, for example, olefins, salts, alkynes, alkanes, acyclic dienes, ethenolysis, enynes. Starting materials will of course depend on the product desired from the reaction, but in general include olefin, saccharides, including, for example, monosaccharides such as glucose, fructose and galactose as well as disaccharides such as sucrose, lactose and maltose.

[0018] The method includes introducing a starting material to a medium containing a bacterium interfaced with biocompatible metathesis catalyst. In this method, the biocompatible metathesis catalyst is selected to be compatible with fimbriae on the bacterium.

[0019] It was found, unexpectedly, that cell surface fimbriae on the bacteria can lead to increased yields of product when employing a biocompatible metathesis catalyst that is compatible with the bacterial fimbriae. The compatibility of the fimbriae and catalyst can be achieved through the selection of an appropriate bacterium or by selecting a catalyst compatible with the fimbriae on a selected bacterium. Alternatively, the fimbriae on a particular bacterium can be adjusted for compatibility with a particular catalyst, such as through controlling the mRNA abundance of fimA. It was also envisaged that controlling fimD mRNA intracellular abundance may also alter the morphology of the fimbriae.

[0020] Methods of improving the compatibility of fimbriae for an abiotic metal catalyst using strain-catalysed ‘click’ chemistry are known to those of ordinary skill in the art. Using known methods, those of ordinary skill can, for example,introduce Z-4-azido-phenylalanine on the fimbriae surface using genetic code expansion, in combination with a non-heterocyclic carbene ligand containing a strained alkyne.

[0021] Catalyst can be chosen to be compatible with a particular bacteria having a known fimbriae structure by running computational simulation methodology, such as that used in the known molecular docking computational technique or homology modeling.

[0022] The aim of the foregoing biocompatible metathesis method is to improve yields (flux) of product by utilizing biocompatible catalysis, that enables consumption of raw materials that a bacteria produces, and thereby stimulates the bacteria to produce more, thus improving efficiency.

[0023] The amount of each chemical or biological component described is presented exclusive of any solvent or diluent oil, which may be customarily present in the commercial material, that is, on an active basis, unless otherwise indicated. However, unless otherwise indicated, each chemical or composition referred to herein should be interpreted as being a commercial grade material which may contain the isomers, by-products, derivatives, and other such materials which are normally understood to be present in the commercial grade.EXAMPLES

[0024] Several ligands were investigated for their role in the metathesis of styrene using ruthenium catalysts in cell culture. This identified the role of TV-heterocyclic carbene ligands in conferring catalysts reactivity under biological conditions. Reactions were completed in sterile Hungate tubes sealed with a rubber septa, with 10 mM styrene in 10 mL cell culture of E. coli BL21 pET22b with 10 mol% cat. loading (a) also with the addition of 2.5% w / v TPGS 1000 (b). Reactions were incubated for 18 h at 37 °C with shaking at 220 rpm. The products were extracted with 3x 5 mL of ethyl acetate before the organic phase was concentrated under reduced pressure before 1H NMR analysis with used as 1,3,5-trimethoxybenzene as internal standard to arrive at the catalyst samples set forth below.

[0025] The stability of the C9 sample under prolonged biological conditions was subsequently investigated, to ensure that this catalyst system was compatible with the microbial environment. To test stability, reactions were completed at 1.5 mM styrene with 10 mol% of the C9 sample catalyst. Reactions were completed in 10 mL E. coli BL21 pET22b cell culture. A further 1.5 mM of styrene was added at t=48 h. Products were extracted with 3x 5 mL ethyl acetate, concentrated under reduced pressure before 1H NMR analysis with used as 1,3,5-trimethoxybenzene as internal standard. This identified that the C9 system remained active after 48 h in cell culture.Substrate Time Added Incubation Avg. Yield S.D.Loading (h) (h) (%)(mM)10 0 18 34 7.710 0 24 59 9.410 0 36 35 9.310 0 65 30 2.220 0 and 48 65 70 8.0

[0026] A number of strategies were employed to optimize stilbene synthesis without success, which lead to a review of the relationship between several E. coli bacterium strains and ruthenium catalysts. Reactions were completed with 10 mL E. coli culture at an Optical Density of 0.6 - 0.8, 10 mM styrene with 10 and 2.5 mol% cat. loading of C9. Reactions were completed for 18 h at 37 °C with 220 rpm. Products were extracted with 3x 5 mL ethyl acetate, concentrated under reduced pressure before 1H NMR analysis with 1,3,5-trimethoxybenzene used as internal standard. This review indicated the concentration of catalyst contributed to yields of product along with the role of phosphine ligands in the decomposition pathway.E. coli Strain Cat. Loading Avg. Yield S.D.(mol. %) (%)BL21 2.5 34 7.7BL21 10 73 10.01TOP10 2.5 38 2.88TOP10 10 41 3.39BW25113 2.5 19 5.1BW25113 10 35 9.3MG1655 2.5 43 12.19MG1655 10- 63 4.02MC4100 2.5 36 2.5MC4100 10 36 1.29JM109A 2.5 70 8.0JM109A 10 27 12

[0027] The inventors were aware that exopolysaccharides are secreted by bacteria in response to environmental stress, which were hypothesized as potential influencers of catalyst reactivity. As such, an assessment of an E. coli strain (JM109A) that cannot produce colanic acid was made. The results indicated that colanic acid was unlikely to influence catalyst reactivity. The low observed reactivity of the catalyst in the exopolysaccharide producing strain (MC4100) indicated some other influence of cell morphology on catalyst reactivity.

[0028] It was initially envisaged that microbial toxicity conferred by the metathesis catalyst (C9) may have influenced these results at higher catalyst loading. Bacterial toxicity of C9 was investigated for E. coli BL21 pET22b and E. coli MC4100 pET22b. Growth of E. coli BL21 and MC4100 pET22b in the presence of 0, 3 and 5 mM C9. A stock solution of C9 was prepared in CH2CI2 and kept on ice. Volumes of this stock solution were transferred to the 96-well plate, where the solvent was allowed to evaporate for 24 hours in a ventilated fume hood. Liquid E. coli culture was prepared in Lysogeny Broth before the ODeoo was adjusted to 0.1 with sterile M9-Glucose. 5 uL of this inoculum was added to 95 uL of sterile M9-Glucose. Positive controls included 95 uL of sterile M9-Glucose and 5 uL of E. coli inoculum, positioned in wells which did not contain any catalyst residue. Negative controls included no inoculum and were completed for each tested concentration. Blanks contained 100 uL of Milli-Q H2O. The ODeoo of each well was measured on the BMG Labtech POLARstar Omega every hour for 24hours, with shaking at 180 rpm for 10 seconds before each measurement. All tested catalyst concentrations, their corresponding negative controls and positive controls were completed in triplicate. Abs. datapoints were averaged and normalised using Graphpad.

[0029] The exopolysaccharide producing E. coli strain employed included several mutations in the promoter and coding regions for a FimB recombinase. FimB is critical to the expression of the fim operon, with the particular A. coli strain unable to assemble fimbriae. The effect of fimbriae influencing catalyst reactivity in vivo was confirmed through screening single-gene E. coli mutants. Reactions completed with 10 mL E. coli culture at an Optical Density of 0.6 - 0.8, 1.5 mM styrene with 10 mol% catalyst loading. Reactions completed for 18 h at 37 °C with 220 rpm. Products were extracted with 3x 5 mL ethyl acetate, concentrated under reduced pressure before 1H NMR analysis with 1,3, 5 -trimethoxybenzene used as internal standard.E. coli Strain Avg. Yield S.D.(%)WT 28 4.1AfimF 25 1.41AfimG 20 3.75AfimZ 27 2.86AfimD 9 9.3AfimA 7 1.71

[0030] Computational modelling of C9 with the bacterial fimbriae was completed in order to understand the binding interactions. This suggested that the residuespresent in the FimH adhesin binding site may participate in non-covalent hydro- phobic interactions with the C9 ligand.

[0031] It is expected that similar functionality would be exhibited by other bacteria systems, given that the functionality of the system tested related to the fimbriae and not to complex chemical interactions between the strain of bacteria and catalyst. Thus, it is expected that the basic method of employing bacteria having fimbriae compatible with a particular catalyst would be successful over a broad spectrum of bacteria and catalyst to the extent the fimbriae and the catalyst are compatible.

[0032] Each of the documents referred to above is incorporated herein by reference. The mention of any document is not an admission that such document qualifies as prior art or constitutes the general knowledge of the skilled person in any jurisdiction. Except in the Examples, or where otherwise explicitly indicated, all numerical quantities in this description specifying amounts of materials, reaction conditions, molecular weights, number of carbon atoms, and the like, are to be understood as modified by the word "about." It is to be understood that the upper and lower amount, range, and ratio limits set forth herein may be independently combined. Similarly, the ranges and amounts for each element of the invention can be used together with ranges or amounts for any of the other elements. As used herein, the expression "consisting essentially of" permits the inclusion of substances that do not materially affect the basic and novel characteristics of the composition under consideration.

Claims

What is claimed is:

1. method of biocompatible metathesis comprising introducing a starting material to a medium containing a bacterium interfaced with a biocompatible metathesis catalyst, wherein said biocompatible metathesis catalyst is selected to be compatible with said bacterial fimbriae.

2. The method of claim 1, wherein the biologic starting material comprises a saccharide.

3. The method of claim 1, wherein said biocompatible metathesis catalyst comprises a biocompatible alkene metathesis catalyst.

4. The method of claim 1, wherein said biocompatible alkene metathesis catalyst comprises abiotic metathesis catalyst.

5. The method of claim 1, wherein said biocompatible alkene metathesis catalyst comprises transition metal catalyst.

6. The method of claim 1, wherein said biocompatible alkene metathesis catalyst comprises ruthenium catalyst.

7. The method of claim 1, wherein said bacterium comprises E. coli.

8. The method of claim 1, wherein said fimbriae comprise pilin structures that adhere to said biocompatible metathesis catalyst.

9. A composition comprising a biocompatible metathesis catalyst and a bacterium, wherein said bacterium comprises fimbriae compatible with said biocompatible metathesis catalyst.

10. The composition of claim 9, wherein said biocompatible metathesis catalyst comprises a biocompatible alkene metathesis catalyst.

11. The composition of claim 9, wherein said biocompatible alkene metathesis catalyst comprises abiotic metathesis catalyst.

12. The composition of claim 9, wherein said biocompatible alkene metathesis catalyst comprises transition metal catalyst.

13. The composition of claim 9, wherein said biocompatible alkene metathesis catalyst comprises ruthenium catalyst.

14. The composition of claim 9, wherein said bacterium comprises E.coli.

15. The composition of claim 9, wherein said fimbriae comprise pilin structures that adhere to said biocompatible metathesis catalyst.