WO2012049172A1

WO2012049172A1 - Phage receptor proteins in fermentation processes

Info

Publication number: WO2012049172A1
Application number: PCT/EP2011/067722
Authority: WO
Inventors: Stefan Miller
Original assignee: Lysando Holding Ag
Priority date: 2010-10-11
Filing date: 2011-10-11
Publication date: 2012-04-19

Abstract

The present invention relates to a fermentation process for the production of an organic compound of interest, wherein the fermentation process comprises the following steps: incubation of components comprising polysaccharides with a phage receptor protein in an aqueous medium and fermentation of the mixture of the incubation step to the organic compound of interest, wherein the organic compound of interest consists of C, H and O atoms only.

Description

Phage receptor proteins in fermentation processes

Phage receptor proteins are proteins originally derived from bacteriophages being specific viruses that only infect bacteria. To infect and replicate within the bacterial cell, phage particles have to achieve close proximity to the bacterial surface for being able to inject their nucleic acid. The natural function of phage receptor proteins is the specific binding to receptors located on the bacteria's surface. For allowing or facilitating i.e. positioning the phage particle close to the bacterial surface for the infection of the bacteria with the bacteriophage these phage receptor proteins hydrolyse components of the bacteria's cell wall for decreasing the bacteria's integrity.

The receptors to which the phage receptor proteins specifically bind are for example components of the lipopolysaccharide (LPS) of gram negative bacteria, of teichoic or lipoteichoic acids in gram positive bacteria, of peptidoglycan, of capsular polysaccharides or of membrane associated proteins of bacteria. However, said receptors may also be on special bacterial protrusions like flagella, pili or fimbria or extracellular components of bacteria such as capsules or slime layers, carbohydrates, polysaccharide matrices, surface protein layers or cell wall associated proteins.

Currently, several phage receptor proteins have been identified and described in the art as e.g. the P22 tailspike as e.g. described by Steinbacher et al. (J Mol Biol. 1997 Apr 11;267(4):865- 80) or the Sf6 tailspike as e.g. described by Freiberg et al. (J Biol Chem. 2003 Jan 17;278(3): 1542-8. Epub 2002 Nov 6). Most of said phage receptor proteins exhibit an enzymatic activity, namely mostly a hydrolytic activity. However, some phage receptor proteins have been identified having no enzymatic activity. The present invention however relates only to those phage receptor proteins having an enzymatic activity, namely a hydrolytic activity. The phage receptor proteins may also show significant stability and activity in the presence of e.g. increasing concentrations of ethanol or butanol produced in the fermentation process.

Fermentation processes offer a wide range of possibilities of producing organic compounds of interest from renewable resources such as biomass. However, in fermentation processes the compounds to be fermented by bacteria, algae, yeast or fungi have often to be pretreated for generating suitable components being fermentable by said bacteria, algae, yeast or fungi. Said pretreatment can for example occur chemically, mechanically, enzymatically, thermically or by combinations thereof. Moreover, in view of the fact that the fermentation is carried out by living organisms such as bacteria, algae, yeast or fungi fermentation by-products and/or inhibiting products are generated by said living organisms as well.

Fermentation processes have been optimized in the past in several ways. An example for different kinds of optimization in regard to the fermentation of ethanol is demonstrated in Cardona et al. (Cardona et al., Fuel ethanol production: process design trends and integration opportunities, Bioresource Technology 98 (2007), 2415-2457). However, there is still a need for optimizing fermentation processes in view of efficiency, costs and environmental sustainability.

This object is solved by the subject matter defined in the claims. The following figures illustrate the present invention.

Fig. 1 shows a graphical representation of increasing reducing ends during a hydrolysis assay. For said hydrolysis assay samples of 40 μΐ containing 10 mg/ml reduced polysaccharide and purified P22 tailspike protein at a subunit concentration of 0.3 μΜ in 50 mM sodium phosphate, 1 mM EDTA, pH 7.0 were prepared. In addition, a sample of 40 μΐ containing 10 mg/ml reduced polysaccharide and purified P22 tailspike active site variants D392N (Baxa et al.) at a subunit concentration of 0.3 μΜ in 50 mM sodium phosphate, 1 mM EDTA, pH 7.0 was prepared. As a negative control, a respective sample containing BSA was used. Reactions were terminated by adding 40 μΐ 3,5-dinitrosalicylic acid (20 mg/ ml) in 0.7 M NaOH, boiling the samples for 5 min and subsequently cooling on ice. Finally, the samples were diluted by the addition of 520 μΐ ¾0. Absorbance at 535 nm were measured in cells of 1 cm path length against a reagent blank and the concentration of reducing ends was calculated using a calibration graph obtained by measurement of glucose.

Fig. 2 shows a graphical representation of ethanol yields obtainable by three different fermentation processes A, B and C for the production of ethanol.

For A yeast cultures of Saccharomyces cerevisiae are grown in YP medium (10 g/L yeast extract, 20 g/L Bacto peptone) containing 20 g L of glucose to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from YP media containing 20 g L of glucose are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 400 mL of YP medium containing 20 g/L of glucose at 30°C with initial OD600 of about 1 or 10, at an agitation speed of 200rpm under oxygen limited conditions. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. Said fermentation process is performed with and without addition of the phage receptor protein PHIV10 TF at a final concentration of lOOmg/ml.

For B yeast cultures of Saccharomyces cerevisiae are grown in YP medium (10 g/L yeast extract, 20 g/L Bacto peptone) containing 20 g/L of glucose to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from YP media containing 20 g/L of glucose are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 400 mL of YP medium containing 20 g/L of pretreated lignocelluloses at 30 °C with initial OD600 of about 1 or 10, at an agitation speed of 200rpm under oxygen limited conditions. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for 72 h resulting in a yield of 0.47 g ethanol per 1 g glucose. Said fermentation process is performed with and without addition of the phage receptor protein Det 7 tsp at a final concentration of lOOmg/ml.

For C cultures of Zymomonas mobilis are grown in ZM medium (50mM Phosphate, pH 5.5, 5 g/L yeast extract, 10 g/L Bacto peptone, 50 g/L of glucose, 30mg/ml Ampicillin) to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from ZM media containing are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 1000 mL of ZM medium at 30 °C with initial OD600 of about 1 or 10, at an agitation speed of 150rpm under oxygen limited conditions. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for up to 160 h resulting in a yield of 0.47 g ethanol per 1 g glucose. Said fermentation process is performed with and without addition of the phage receptor protein PHIVIO TF at a final concentration of lOOmg/ml.

Fig. 3 shows a graphical representation of the gamma-linolenic acid yield obtainable by a fermentation processes for the production of gamma-linolenic acid by Hansenula polymorpha. For said process yeast cultures of Hansenula polymorpha are grown in GBS medium (one liter of the GBS medium consisted of 26.7 ml 85% H₃P0₄, 0.93 g CaS0₄, 18.2 g K₂S0₄, 14.9 g MgS0₄-7H₂0, 4.13 g KOH, 40.0 g glucose, and 4.35 ml trace salts). The trace salts contained 6.0 g CuS0₄-5H₂0, 0.08 g KI, 3.0 g MnS0₄ H₂0, 0.2 g Na₂Mo0₄-2H₂0, 0.02 g H₃BO₃, 20.0 g ZnCl₂, 65 g FeS0₄-7H₂0, 0.5 g CoCl₂-6H₂0, 5.0 ml H₂S0₄, and 0.2 g biotin in 1 1 of distilled water) supplemented with adenine sulfate to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from GBS media are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 1000 mL of GBS medium (26.7 ml 85% H₃P0₄, 0.93 g CaSC¼, 18.2 g K₂S0₄, 14.9 g MgS0₄-7H₂0, 4.13 g KOH, 40.0 g glycerol, and 4.35ml trace salts) at 30 °C with initial OD600 of about 1 or 10, at an agitation speed of 200rpm under 1-41/h aeration with oxygen gas (to keep saturation at 20% 0₂) and pH 5.0. A 25% ammonium hydroxide solution is used to adjust the pH of the culture broth. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for 48 h and is performed with and without addition of the phage receptor protein PHIVIO TF at a final concentration of lOOmg/ml.

The term "fermentation" as used herein refers to any biotechnological process for the conversion of biologic material by bacteria, yeast, fungi, or algae. The term "fermentation" also comprises a process in which the biologic material is converted by a combination of bacteria, yeast, fungi or algae, as e.g. the combination of bacteria and yeast for the production of bioethanol or biofuels, which can be e.g. ethanol, butanol, fatty acid ethyl ester (Palmitate ethylester) or hydrogen. The term "fermentation" as used herein refers both to anaerobic and aerobic fermentation. The biologic material being converted during a fermentation process is preferably a biomass, preferably comprising starch or saccharose. Examples for such biologic materials are crops, corn, wheat, sugar cane, cottonwoods, paper, switchgrass, bagasse, wood, cornstover and corn fibers. However, the term "fermentation" as used herein refers to the fermentation of any material being fermentable by bacteria, yeast, fungi, algae or combinations thereof. The bacteria, yeast, fungi or algae used for the fermentation can be naturally occurring organisms or genetically modified organisms. In addition, the term "fermentation" as used herein refers to a process, in which the conversion of the biologic material is not carried out by a living organism but by an enzyme, a so called ferment. Moreover, the fermentation can be carried out by a combination of a living organism as listed above and an enzyme.

The term "organic compound of interest" as used herein refers to any organic compound consisting of carbon (C), hydrogen (H) and oxygen (O) atoms only. In particular, said term refers to an alcohol or a lipid.

The term "alcohol" as used herein refers to any linear, branched, and cyclic hydroxylated hydrocarbons of the formula R-OH, wherein R is an alkyl group. An alcohol can comprise one or more double or triple bonds. Examples of alcohols are methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol, pentanol, and hexanol.

The term "lipid" as used herein refers to a substance that is soluble in organic solvents and includes for example triacylglycerol, glycolipid, sterols, sterol esters, cartenoids, xanthophylls or fatty acids, in particular free fatty acids, unsaturated fatty acids, polyunsaturated fatty acids or esters of fatty acids.

The term "phage receptor protein" as used herein refers to bacteriophage proteins, prophage proteins and proteins of cryptic phages encoded by bacterial genomes and located on phage islands. Phage islands are clusters of genes encoding two or more proteins originated from a phage genome. Moreover, the term "phage receptor proteins" as used herein refers only to those phage receptor proteins which

(i) bind specifically to bacterial surface receptors, and

(ii) have a hydrolytic activity.

In particular, said hydrolytic activity is an activity being able to degrade polysaccharide structures. Preferably said polysaccharide structures are polysaccharide structures of bacteria such as bacterial polysaccharides as e.g. the O-antigens of lipopolysaccharides or the K- antigens of capsular polysaccharides. In addition, said hydrolytic activity can be an activity being able to degrade further bacterial cell components, in particular of the bacteria's cell wall, as e.g. components of teichoic or lipoteichoic acids in gram positive bacteria, peptidoglycan, or extracellular components like capsules or slime layers or polysaccharide matrices. In particular, the hydrolytic activity of the phage receptor proteins may be a hydrolytic activity selected from the group consisting of: endoglucanase, endorhamnosidase, endo-N-acetylglucosaminidase, endoglycosaminidase, hyaluronate lyases, endosialidase, polysaccharide lyase, cutinase and chitinase. Thus, phage receptor proteins may also degrade compounds not having a bacterial origin but comprising polysaccharide structures as e.g. carbohydrate structures or polymer structures such as cellulose, starch, glycan and polysaccharides. Phage receptor proteins include bacteriophage tail spikes, tail fibers, tail pins, baseplate proteins or shaft proteins and other tail proteins involved in binding to receptors on bacterial surfaces and the lysis of components on the cell surface via their enzymatic function. Said phage receptor proteins are usually located in the phage tail. However, they can also be located at the phage head or at the phage's base plate. Moreover, the term "phage receptor proteins" as used herein refers not only to phage receptor proteins as originally encoded by bacteriophages but also to variants and fragments thereof. The phage receptor proteins may also show significant stability and activity in the presence of e.g. increasing concentrations of ethanol or butanol produced in the fermentation process.

The term "variants" as used herein refers to phage receptor proteins exhibiting in comparison to phage receptor proteins naturally occurring in bacteriophages modifications in the form of one or more deletions, substitutions, additions, inversions and/or chemical modifications of amino acids or nucleotides. Said variants still exhibit the functions of (i) specifically binding to bacterial surface receptors, and (ii) having a hydrolytic activity, in particular of hydrolysing polysaccharide structures.

The term "fragments" as used herein refers to phage receptor proteins comprising only a part of the amino acid sequences coding for phage receptor proteins naturally occurring in bacteriophages as long as they exhibit the functions of (i) specifically binding to bacterial surface receptors, and (ii) having an hydrolytic activity, in particular of hydrolysing polysaccharide structures. Moreover, the term "fragments" as used herein refers to receptor proteins from which the binding region of the phage receptor protein to the phage has been removed. One example for such a fragment with removed binding region of a phage receptor protein is described in Danner et al. (Eur J Biochem. 1993 Aug 1 ; 215(3):653-61). The fragment described in Danner et al. lacks the N-terminal domain of P22 tailspike, wherein said N-terminal domain is the binding region of the phage receptor protein to the phage P22. Moreover, Danner et al. describes in said paper the identification of regions which can be removed from phage receptor proteins without having an influence on the enzymatic activity and the specific binding to bacterial surface receptors.

The term "bacterial surface receptors" as used herein refers to components to which phage receptor proteins can specifically bind. Example for such components are lipopolysaccharides (LPS), in particular the O-antigen or the LPS core of gram negative bacteria, capsular polysaccharides, in particular K-antigens being present on some gram negative bacteria, components of teichoic or lipoteichoic acids in gram positive bacteria, peptidoglycan, membrane associated proteins of bacteria, special bacterial protrusions like flagella, pili or fimbria or extracellular components like capsules or slime layers, carbohydrates, polysaccharide matrices, surface protein layers or cell wall associated proteins.

The term "O-antigen" as used herein refers to the glycan polymer contained within an LPS. The O-antigen is the most surface exposed part of the bacterial lipopolysaccharide being highly variable from strain to strain and is thus a target for specific recognition. It is composed of repetitive units of oligosaccharides of different lengths with usually 2 to 7 sugar moieties per unit.

The term "K-antigen" as used herein refers to different structures being part of a bacterial capsule. Said "capsular" antigens may be composed of proteinaceous organelles associated with colonization such as CFA antigens or are made of polysaccharides. Regardless of their chemistry, these capsules may be able to promote bacterial virulence by decreasing the ability of antibodies and/or complement to bind to the bacterial surface, and the ability of phagocytes to recognize and engulf the bacterial cells. An example for a "K-antigen" is K-l antigen of E.coli being composed of a polymer of N-acetyl neuraminic acid (sialic acid).

The term "bacterial cell components" as used herein refers to all components of a bacterial cell as e.g. components comprising bacterial carbohydrate structures such as bacterial polysaccharides as e.g. the O-antigens of lipopolysaccharides or the K-antigens of capsular polysaccharides, teichoic or lipoteichoic acids, peptidoglycan, membrane associated proteins of bacteria, special bacterial protrusions, in particular flagella, pili or fimbria, of extracellular components, in particular capsules or slime layers, polysaccharide matrices, lipopolysaccharides, surface protein layers or cell wall associated proteins or fragments of these components. The term„cell wall" as used herein refers to all components that form the outer cell enclosure of bacteria and thus guarantee their integrity. In particular, the term„cell wall" as used herein refers to peptidoglycan, the outer membrane of the gram negative bacteria with the lipopolysaccharide, the bacterial cell membrane, but also to additional layers deposited on the peptidoglycan as e.g. capsules, outer protein layers or slimes.

The term "polysaccharides" as used herein refers to polymeric carbohydrate structures, which are formed of repeating units joined together by glycosidic bonds. Said repeating units are preferably mono- or disaccharides. The term "polysaccharides" as used herein comprises linear polysaccharides, branched polysaccharides, homopolysaccharides and heteropolysaccharides. Examples of "polysaccharides" are storage polysaccharides such as starch and glycogen and structural polysaccharides such as cellulose and chitin. Moreover, the term "polysaccharides" as described herein refers not only to compounds consisting of polysaccharides only but also to compounds consisting of polysaccharides and further non- polysaccharidic molecules such as lipids. Examples for such compounds are lipopolysaccharides consisting of both polysaccharides and lipids.

The term "biofuel" as used herein refers to any kind of fuels being derived from biomass and thus from a renewable energy source of biological material from living or recently living organisms. Examples for biofuels are bioalcohols such as bioethanol and biobutanol, green diesel, biodiesel, vegetable oil, bioethers and biogas. Thereby, the term "biodiesel" refers in particular to methyl esters obtained from transesterification of e.g. rapeseed, soy bean, palm or jatropha oil.

The present invention relates to a fermentation process for the production of an organic compound of interest, wherein the fermentation process comprises the following steps:

(a) incubation of components comprising polysaccharides with a phage receptor protein in an aqueous medium, and

(b) fermentation of the mixture of step (a) to the organic compound of interest, wherein the organic compound of interest consists of C, H and O atoms only.

In a preferred embodiment of the present invention step (a) and step (b) of the fermentation process according to the present invention are performed at the same time. Thus, the phage receptor proteins is preferably be added in said fermentation process together with the inoculum.

The components comprising polysaccharides of step (a) of the fermentation process according to the present invention are preferably bacterial polysaccharides, biomass or pretreated biomass or a combination thereof. Said bacterial polysaccharides can be polysaccharide structures of the cell wall of living bacteria, of non-living bacteria, of bacterial cell components or combinations thereof. In particular, said bacterial polysaccharides can be O- antigens of lipopolysaccharides or K-antigens of capsular polysaccharides, components of teichoic or lipoteichoic acids, of peptidoglycan, of extracellular components, in particular capsules or slime layers, of carbohydrates, of polysaccharide matrices, of lipopolysaccharides, or fragments of these components. Alternatively or in addition, said polysaccharides can be biomass such as crops, corn, wheat, barley, sorghum, rye, sugar cane, potato, cottonwoods, paper, switchgrass, bagasse, wood, cornstover or corn fibers.

Before said biomass is incubated with phage receptor proteins in step a) of the fermentation process according to the present invention said biomass can be pretreated. The pretreatment can be mechanically, chemically, enzymatically, by heat or a combination of these different pretreatments. However, step (a) of the fermentation process according to the present invention can also be integrated into already known pre-treatments steps. For example, in case of an enzymatically pretreatment the phage receptor proteins can be added in said enzymatically pretreatment step as well. The pre-treatment can be carried under conditions known by a person skilled in the art and depends amongst other on the organic compound of interest as well as the microorganism used for the fermentation. For example, when the compound of interest is ethanol and the microorganism used for fermentation is yeast than the biomass may be liquefied and saccharified enzymatically as e.g. described in US 5,231,017. Further information regarding the pre-treatment in ethanol processes can e.g. be found in Lysons et al., 1995, The Alcohol Textbook: Ethanol Production by Fermentation and Distillation, Nottingham University Press. The enzymes used for the pre-treatment steps can be immobilized on suitable supporting structures. Said supporting structures may consist of e.g., polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, or silicium. In a preferred embodiment of the present invention step (a) of the fermentation process according to the present invention is integrated into an enzymatically pre-treatment step using immobilized enzymes. Thereby, the phage receptor protein is active in preventing contaminating bacteria and/or byproducts of these contaminating bacteria from adhesion to surfaces immobilized with catalyzing enzymes.

The components comprising polysaccharides in step (a) of the fermentation process according to the present invention can also be a by-product or an inhibitory product generated during the pre-treatment. Thus, the incubation of said by-products or inhibitory products in step (a) of the fermentation process according to the present invention may result in that the phage receptor proteins degrade said by-products or inhibitory product into compounds being fermentable in step (b) of the fermentation process according to the present invention. For example degradation of said by-products by the phage receptor proteins may result in polysaccharide fragments such as hexoses and pentoses which can further be fermented by the bacteria, yeast, fungi or algae used for the fermentation. Alternatively or in addition, said incubation may result in that the inhibitory products become non-inhibitory for the fermentation process according to the present invention.

Moreover, the components comprising polysaccharides in step (a) can also be a bacterial contamination. In many fermentation processes bacterial contamination is a problem. For example, in a process for producing biofuel or lipids by the aid of yeast or bacteria like E. coli, Zymomonas sp., Clostridium sp., Bacillus sp. or Klebsiell sp. bacterial contaminations occur. Said bacterial contamination is often a contamination of gram-positive bacteria such as gram-positive lactic acid bacteria in bioethanol production by yeast, wherein said gram- positive lactic acid bacteria are ethanol tolerant and growing faster than the yeast. However, also gram negative bacteria may contaminate such processes. Thus, the incubation with phage receptor proteins in a fermentation process according to the present invention results in that components of the cell wall of the contaminating bacteria are degraded. Said degradation results in a destabilization of the cell wall. Said destabilization enhances the effect of further additives for removing or reducing said bacterial contaminations such as detergents or antibiotics. Moreover, said degradation reduces the propagation rate of the contaminating bacteria.

The phage receptor protein used in step (a) of the fermentation process according to the present invention is able to bind specifically to bacterial surface receptors and has hydrolytic activity, in particular of degrading polysaccharide structures. The hydrolytic activity is preferably an endoglucanase, endorhamnosidase, Endo-N-acetylglucosaminidase, endoglycosaminidase, hyaluronate lyase, endosialidase, polysaccharide lyase, cutinase or chitinase activity. Thus, the phage receptor protein used in step (a) is able to degrade the components comprising polysaccharides. In particular, the phage receptor protein is able to degrade O-antigens of lipopoly saccharides or K-antigens of capsular polysaccharides, components of teichoic or lipoteichoic acids, of peptidoglycan, of extracellular components, in particular capsules or slime layers, of carbohydrates, of polysaccharide matrices, of lipopolysaccharides, or fragments of these components. Moreover, the phage receptor proteins are able to degrade other polysaccharide structures such as starch, glycogen, cellulose or chitin. The degradation carried out by the phage receptor proteins may result for example in polysaccharide fragments such as pentoses, hexoses or combinations thereof. Said polysaccharide fragments such as pentoses and hexoses can then further be converted in step b) of the fermentation process according to the present invention into the organic compound of interest.

The phage receptor protein is preferably present in step (a) of the fermentation process according to the present invention in an amount, which enhances the productivity of the fermentation process. Preferably, the productivity of the fermentation process is increased by the phage receptor protein by at least about 2 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 % or even by at least about 70%. The quantity of phage receptor proteins in step (a) of a fermentation process according to the present invention depends on the enzymatic activity of the phage receptor protein. For example, the phage receptor protein is added in an amount of about 0.005 to about 5 mL of a 1% to 5 % phage receptor protein solution to 500 mL aqueous medium. It is also preferred that the phage receptor proteins are added at a final concentration of about 10 μg/ml, 100 μg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110 mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, 500 mg/ml or 1 g/ml.

The incubation conditions of step (a) of the fermentation process according to the present invention may range from seconds to several hours up to several days depending on the activity of the phage receptor proteins and the temperature during the assay. Preferably, the incubation of step (a) is carried out for about 1 second up to about 72 hours, in particular for about 5 minutes to about 48 h, at temperatures of about 10°C to about 45°C, in particular at temperatures of about 20°C to about 37 °C, most preferred of about 30 °C. The phage receptor protein in the fermentation process according to the present invention may not only be enzymatically active in step (a) of the fermentation process according to the present invention but also in step (b). For example, the phage receptor proteins may degrade by-products which are produced in step (b) by the bacteria, yeast, fungi or algae. The degradation of said by-products by the phage receptor proteins may result in polysaccharide fragments such as hexoses and pentoses which can be further fermented by the bacteria, yeast, fungi or algae used for the fermentation.

In another embodiment of the present invention the fermentation of step b) is performed by fermentation of organisms such as bacteria, yeast, algae or fungi, which are immobilized on suitable supporting structures. Said supporting structures may consist of e.g., polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, or silicium. In this embodiment the phage receptor protein is active in preventing contaminating bacteria from adhesion to surfaces with said immobilized fermentation organisms.

The organic compound of interest in the fermentation process according to the present invention is preferably an alcohol or a lipid. Said alcohol is preferably methanol, ethanol, butanol or pentanol. The lipid is preferably a triacylglycerol, glycolipid, sterols, sterol esters, cartenoids, xanthophylls or fatty acids, in particular free fatty acids, unsaturated fatty acids, polyunsaturated fatty acids or esters of fatty acids. In a preferred embodiment of the present invention the lipid is gamma-linolenic acid.

The organic compound of interest in the fermentation process according to the present invention may be recovered from the fermented material of step (b). Thus, the fermentation process of the present invention may additionally comprise a step (c) comprising the recovery of the organic compound of interest. Usually, the fermentation process of the present invention results in that the compound of interest is extracellularly available in the fermented material at the end of step (b). However, the fermentation process for the production of some organic compounds of interest as e.g. specific lipids may result in that the compound of interest remain intracellularly at the end of step (b). Thus, in case the compound of interest remains intracellularly at the end of step (b), step (c) may comprise the lysis of cells to release the intracellular compound of interest. After recovery of the organic compound of interest said compound of interest might be further processed for example into biofuel, as e.g. bioethanol, biobutanol or biodiesel. Said further processing might comprise known processing steps in the art as e.g. distillation or refinement. For example, further information regarding the recovery and processing in ethanol processes are described in e.g. Lysons et al., 1995, The Alcohol Textbook: Ethanol Production by Fermentation and Distillation, Nottingham University Press.

The fermentation in the fermentation process according to the present invention is preferably carried out by bacteria, yeast, algae or fungi. Said bacteria, yeast, algae or fungi can be naturally occurring microorganisms or they can be genetically modified microorganisms. In particular, they might be genetically modified for producing the organic compound of interest. However, they might comprise further genetically modifications enhancing their productivity of the compound of interest.

The suitable host for the production of an organic compound of interest depends on the organic compound of interest. For the production of ethanol and butanol for example bacteria such as E.coli, Zymomonas sp, Clostridium sp., Bacillus sp. and Klebsiella sp., fungi such as strains of Penicillium species, yeast such as Pichia or Saccharomyces or algae such as spirogyra, cladophora or oedogonium can be used. Further information regarding suitable hosts for the production of alcohols such as ethanol can e.g. be found in Lysons et al. and Cardona et al. (Lysons et al., 1995, The Alcohol Textbook: Ethanol Production by Fermentation and Distillation, Nottingham University Press; Cardona et al., Fuel ethanol production: process design trends and integration opportunities, Bioresource Technology 98 (2007), 2412-2457).

More information about the various different hosts which may be used for biofuel production in accordance with the present invention can be found in Gowen CM, Fong SS. (Exploring biodiversity for cellulosic biofuel production. Chem Biodivers. 2010 May;7(5): 1086-97). Based on the invention described herein the respective competing microflora that negatively influences the production yield will be reduced or eliminated by addition of phage receptor proteins.

In one preferred embodiment of the present invention sugars such as glucose, fructose, and sucrose are converted into cellular energy during ethanol fermentation. Thereby ethanol and carbon dioxide are produced as metabolic waste products. This conversion is performed anaerobic in yeasts. Growth of contaminating gram positive bacteria like lactic acid bacteria which are tolerant to pH, temperature and ethanol will result in a decrease of ethanol yield of about 2-6% or even up to 10% as these bacteria compete for nutrients and produce organic acids that may inhibit the growth of yeasts. The addition of appropriate amounts of phage receptor proteins ^g-mg-g/ml range depending on cell density) will reduce growth of lactic acid bacteria and will also prevent the adhesion of lactic acid bacteria to surfaces where enzymes or yeast have been immobilized. Thus the ethanol production will be increased for at least 2%. More preferably, the ethanol production will be increased for about 2%, 3%, 4%, 5%, 6 %, 7%, 8 %, 9% or 10% or even higher.

For the production of lipids for example bacteria such as E.coli, algae such as heterokonts or fungi such as labyrinthulomycota or sordariomycetes as e.g. Gliocladium roseum can be used. Further information regarding suitable hosts for the production of lipids can e.g. be found in Meng et al. and Schorken et al. (Meng et. al, Biodiesel production from oleaginous microorganisms, Renewable Energy, Volume 34, Issue 1, 2009, p. 1-5; Schorken et al., Lipid biotechnology: Industrially relevant production processes, Eur. J. Lipid Sci.Technol. 2009, 111, 627-645).

However, for the fermentation process according to the present invention also a combination of different microorganisms can be used as e.g. a combination of different species of bacteria, yeasts, algae or fungi. For example, for the production of ethanol a combination of the two yeast species Saccharomyces cerevisiae and Pichia stipitis can be used for fermentation. In addition, for the fermentation process according to the present invention also a combination of different microorganisms can be used as e.g. a combination of a specific bacteria species and a specific yeast species. For example, for the production of ethanol a combination of the yeast Saccharomyces cerevisiae and the bacterium Bacillus sp. can be used. Also combinations of a living organism and additional enzymes can be used in the fermentation process according to the present invention. Further combinations which can be used for a fermentation process of the production of ethanol can e.g. be found in Cardona et al., Fuel ethanol production: process design trends and integration opportunities, Bioresource Technology 98 (2007), 2412-2457.

The fermentation step (b) can be carried out until fermentation conditions known by a person skilled in the art and depends amongst other on the organic compound of interest as well as the microorganism used for the fermentation. For example, when the compound of interest is ethanol and the microorganism used for fermentation is a Saccharomyces species than the fermentation may typically be carried out for about 12 hours to about 96 hours at a temperature of about 25 °C to about 35 °C. Especially preferred is fermentation for about 24, 48 or 72 hours. The fermentation is most preferably carried out at about 30 °C. Further information regarding the fermentation conditions in ethanol processes can e.g. be found in Lysons et al. (Lysons et al., 1995, The Alcohol Textbook: Ethanol Production by Fermentation and Distillation, Nottingham University Press).

In one embodiment of the present invention the fermentation process is carried out by bacteria. Said bacteria may be called producer bacteria. In such a process the phage receptor protein may degrade components of the cell wall of said producer bacteria. Said degradation may occur in step (a), step (b) or in steps (a) and (b) of the fermentation process according to the present invention. In particular, the phage receptor proteins may degrade bacterial polysaccharides, in particular O-antigens of lipopoly saccharides or K-antigens of capsular polysaccharides, components of teichoic or lipoteichoic acids, of peptidoglycan, of extracellular components, in particular capsules or slime layers, of carbohydrates, of polysaccharide matrices, of lipopolysaccharides, or fragments of these components. The degradation of said bacterial components of the cell wall of said producer bacteria may enhance the productivity of the producer bacteria. In addition or in the alternative, the cleaving of said bacterial components of the cell wall of said producer bacteria may inhibit the production of by-products or inhibiting substances produced by the producer bacteria.

One example for such an embodiment of the present invention is the production of biofuel by bacteria, in particular the production of bioethanol or biobutanol. For the production of biofuel several kinds of biomass such as crops, corn, wheat, barley, sorghum, rye, sugar cane, potato, cottonwoods, paper, switchgrass, bagasse, wood, cornstover and corn fibers can be used as raw material. Said raw material is preferably pretreated for receiving carbohydrates being fermentable by bacteria. Examples for bacteria being suitable for such are fermentation are recombinant E.coli, Zymomonas sp., Clostridium sp., Bacillus sp. and Klebsiella sp.. During fermentation the bacteria convert the carbohydrates, preferably hexoses and pentoses, into biofuel, in particular ethanol or butanol. However, beside the conversion of carbohydrates into useful biofuel products said bacteria also produce by-products. For example, levan, fructooligosaccharides and sorbital are typical by-products of a conversion of carbohydrates into ethanol. The production of such by-products can be inhibited by phage receptor proteins degrading components of the cell wall of the producer bacteria.

Phage receptor proteins which can be used in step (a) of fermentation processes according to the present invention are preferably proteins originally deriving from bacteriophages. Said phage receptor protein may be either isolated phage receptor proteins directly deriving from bacteriophages or recombinantly produced phage receptor proteins. However, also variants or fragments of such phage receptor proteins may be used. Said phage receptor proteins, variants or fragments thereof exhibit the function of specifically binding to receptors located on the bacteria's surface. In addition, said proteins exhibit a hydrolytic activity, in particular of hydrolysing components of polysaccharides, in particular bacterial polysaccharides such as O-antigens of lipopolysaccharides or K-antigens of capsular polysaccharides, components of teichoic or lipoteichoic acids, of peptidoglycan, of extracellular components, in particular capsules or slime layers, of carbohydrates, of polysaccharide matrices, of lipopolysaccharides, or fragments of these components.

An assay for the determination whether a protein exhibits hydrolytic activity on polysaccharides is the quantification of reducing ends arising from hydrolysis of the poly- and oligosaccharides by the proteins using the chromogenic substrate 3,5-dinitrosalicylic acid as e.g. described in Danner et al. (Danner et al., 1993, Eur. J. Biochem. 215, 653-661) or as outlined in the Examples of the present invention.

A protein exhibits hydrolytic activity on polysaccharides if the absorbance at 535nm is increasing at least 10% above the background resulting from incubation with BSA which exhibits no hydrolytic activity. Preferably, a phage receptor protein which can be used in a fermentation process according to the present invention increases the absorbance at 535 nm at least about 20 %, 30 %, 40 %, 50 %, 60 %, 70 % , 80 %, 90 % or 100 % above the background resulting from incubation with BSA. The incubation time for said incubation should be sufficient to detect the hydrolytic activity of a phage receptor protein having hydrolytic activity (e.g. as e.g. one of the preferred phage receptor proteins as described in the Table 1) and depends on the incubation conditions such as protein concentration and buffer composition. The hydrolytic activity on lipopoly saccharides can be determined in a LPS hydrolysis assay. For such an assay lipopolysaccharides (LPS) have to be incubated with respective proteins. In case said proteins exhibit a hydrolytic activity on lipopolysaccharides the degradation of the LPS can be observed on silver stained SDS-gels where complete LPS molecules and fragments produced after hydrolysis of the LPS migrate with different mobility. The time span for hydrolysis ranges from seconds to several hours up to several days depending on the activity of the phage receptor protein, and the temperature during the assay. Preferred time spans for the assays are 1 s up to 48 h and temperatures in the range of room temperature up to 37 °C.

To determine whether a phage receptor protein specifically binds to bacterial surface receptors the specific binding test of phage receptor proteins to bacterial surface receptors as described in the Examples can be used. Therefore, the phage receptor proteins are coupled to a solid support, like magnetic beads. The phage receptor proteins being coupled to said solid support are incubated in excess with bacteria of interest. Preferably, for said incubation a panel of different defined cultures of bacteria or bacteria strains is used. The bacteria that bind to the phage receptor proteins will be removed from the sample and plated onto appropriate growth media containing agar plates, whereas bacteria that are not bound will not be removed. The tested phage receptor protein specifically binds to bacterial surface receptors of said tested bacteria if at least 20 % of the bacteria in at least one panel are removed from the sample. Preferably, the tested phage receptor protein specifically binds to at least 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 98 %, 99 % or 100 % of said tested bacteria in said at least one panel.

Alternatively, to determine whether a phage receptor protein specifically binds to bacterial surface receptors a biotinylated phage receptor protein can be incubated in excess with bacteria of interest. Preferably, for said incubation a panel of different defined cultures of bacteria, bacteria strains or bacteria serotypes is used. After incubation for 10 to 30 minutes at room temperature unbound protein has to be removed by washing. Subsequently, the bacterial cells have to be pelleted by centrifugation. Afterwards, the bacterial pellet is incubated with Streptavidin-Horseradish Peroxidase and a respective substrate as e.g. o-phenylenediamine dihydrochloride. In case the phage receptor protein to be tested binds to bacterial surface receptors a color formation can be detected in comparison to a negative control with bacteria but no phage receptor proteins. A phage receptor protein specifically binds to bacterial surface receptors if it shows a specific binding of at least 20 % to at least one of the tested bacteria, bacteria strains or bacteria serotypes in the tested panels. Preferably, said phage receptor protein specifically binds to at least 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 98 %, 99 % or 100 % of said tested bacteria of at least one panel.

Preferred are phage receptor proteins, variants or fragments thereof deriving from bacteriophages known in the art or variants or fragments thereof. Examples for such bacteriophage are: P22, ε15, Sf6, SP6, Kl, K5, HK620, Det7, Tl, T5, T7, phage 14, P2, phiVIO, APSE-1, Al, A18a, ST104, ST64T, SETP3, JK106 or Gifsy.

Especially preferred phage receptor proteins are phage receptor proteins as listed in the following table (Table 1) as well as variants and fragments thereof:

Table 1 : Preferred phage receptor proteins

Phage Reference Enzymatic activity Additional receptor comment protein

P22 TSP J Mol Biol. 1997 Apr 11;267(4):865- Endorhamnosidase P22 TSP

(SEQ ID 80. Phage P22 tailspike protein: crystal hydrolyses the O- NO: 1) structure of the head-binding domain antigen of

at 2.3 A, fully refined structure of the Salmonella endorhamnosidase at 1.56 A

resolution, and the molecular basis of

O-antigen recognition and cleavage. :

Steinbacher S, Miller S, Baxa U,

Budisa N, Weintraub A, Seckler R,

Huber R.

Sf6 TSP J Biol Chem. 2003 Jan Endorhamnosidase Sf6 TSP

(SEQ ID 17;278(3): 1542-8. Epub 2002 Nov 6. hydrolyses the O- NO: 2) The tailspike protein of Shigella phage antigen of

Sf6. A structural homolog of Shigella

Salmonella phage P22 tailspike protein

without sequence similarity in the

beta-helix domain. Freiberg A,

Morona R, Van den Bosch L, Jung C,

Behlke J, Carlin N, Seckler R, Baxa U.

HK620 Mol Microbiol. 2008 Jul;69(2):303-16. Endo- HK620 TSP TSP Crystal structure of Escherichia coli Nacetylglucos- hydrolyses the O-

(SEQ ID phage HK620 tailspike: podo viral aminidase antigen of E.coli NO: 3) tailspike endoglycosidase modules are

evolutionarily related. Barbirz S,

Miiller JJ, Uetrecht C, Clark AJ,

Heinemann U, Seckler R. Det7 Tsp J Virol. 2008 Mar;82(5):2265-73. Endorhamnosidase Like P22 TSP (SEQ ID Epub 2007 Dec 12. Structure of the hydrolyses the O- NO: 4) receptor-binding protein of antigen of

bacteriophage det7: a podo viral tail Salmonella spike in a my o virus. Walter M, Fiedler

C, Grassl R, Biebl M, Rachel R,

Hermo-Parrado XL, Llamas-Saiz AL,

Seckler R, Miller S, van Raaij MJ

ε15 Nature. 2006 Feb 2;439(7076):612-6. unknown ε15 tailspike tailspike Structure of epsilonl5 bacteriophage hydrolyses the O- (SEQ ID reveals genome organization and DNA antigen of NO: 5) packaging/injection apparatus. Jiang Salmonella

W, Chang J, Jakana J, Weigele P, King

J, Chiu W.

K5 lyase J Biol Chem. 2010 Jul Polysaccharide KflA cleaves K5 A (KflA) 30;285(31):23963-9. Epub 2010 Jun 2. lyase capsular

(SEQ ID The K5 lyase KflA combines a viral polysaccharide, NO: 6) tail spike structure with a bacterial which is a

polysaccharide lyase mechanism. glycosaminoglyca Thompson JE, Pourhossein M, n

Waterhouse A, Hudson T, Goldrick M,

Derrick JP, Roberts IS.

HylP2 FEBS J. 2009 Jun;276(12):3392-402. Hyaluronate lyases HylP2 digests the (SEQ ID Epub 2009 May 8. Polysaccharide (class of hyaluronic acid NO: 7) binding sites in hyaluronate lyase— endoglycos- capsule during crystal structures of native phage- aminidase) phage invasion of encoded hyaluronate lyase and its the S. pyogenes complexes with ascorbic acid and host lactose. Mishra P, Prem Kumar R,

Ethayathulla AS, Singh N, Sharma S,

Perbandt M, Betzel C, Kaur P,

Srinivasan A, Bhakuni V, Singh TP.

K1F TSP Nat Struct Mol Biol. 2005 Endosialidases K1F TSP cleaves (SEQ ID Jan;12(l):90-6. Epub 2004 Dec 19. polysialic acid NO: 8) Crystal structure of the polysialic acid- degrading endosialidase of

bacteriophage K1F. Stummeyer K,

Dickmanns A, Muhlenhoff M,

Gerardy-Schahn R, Ficner R.

PhiVIO www.ncbi.nlm.nih.gov/protein/AAZ95 unknown PhiVIO TF TF (SEQ 917 cleaves o- antigen ID NO: 9) of E.coli 0157 H7

ACCESSION AAZ95917

DBSOURCE accession DQ126339.2

Perry, L.L. and Applegate,B.M. Sr.:

Complete nucleotide sequence of

Escherichia coli 0157:H7

bacteriophage PhiVIO In addition, phage receptor proteins, variants or fragments thereof comprising an additional marker moiety such as biotin or Streptavidin or a tag such as a HA-tag, His₆-tag, Strep-tag, Avi-tag, Myc-tag, GST-tag, JS-tag or Cystein-tag may be used in step (a) of the fermentation process according to the present invention. Said marker moiety or tag is preferably coupled to the C-terminus or the N-terminus of a phage receptor protein.

The choice of a specific phage receptor protein for use in the fermentation process according to the present invention depends on the bacterial targets to be degraded in the fermentation process according to the present invention by the phage receptor proteins. Since each phage receptor protein binds specifically only to a limited number of bacterial surface receptors, it is not efficient to use any phage receptor protein in any fermentation process. To determine which phage receptor protein can be used in a fermentation process in accordance with the present invention, the bacterial targets of said fermentation process have to be identified. Based on the identified bacterial target a phage receptor protein can be chosen, which is able to bind specifically to the identified bacterial target. The bacterial target of several phage receptor proteins is known in the art. As e.g. outlined in the Table above Salmonella is a suitable bacterial target for P22 TSP, ε15 tailspike or Det7 Tsp, Shigella, is a suitable bacterial target for Sf6 TSP, E.coli is a suitable bacterial target for HK620 TSP, KIF tsp or K5 lyase A, S. pyogenes is a suitable bacterial target of KflA and S. pyogenes is a suitable bacterial target of HylP2. Moreover, further suitable bacterial targets for any phage receptor protein can be identified by the specific binding test of phage receptor proteins to bacterial surface receptors as described herein and shown in the Examples.

The following examples explain the present invention but are not considered to be limiting. Unless indicated differently, molecular biological standard methods were used, as e.g., described by Sambrock et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Example 1: Detection of hydrolytic activity of P22 tailspike

1. Purification of Salmonella typhimurium polysaccharide:

Salmonella typhimurium polysaccharide was purified as described in Seckler et al. (Seckler, R., Fuchs, A., King, J. & Jaenicke, R. (1989) /. Biol. Chem. 264, 11 750-11 753).

Accordingly, 2.5 g Acetone-dried S. typhinurium LT2 cells were extracted twice with 50 ml of 0.1 M acetic acid for 1.5 h at 98 °C. Solubilized polysaccharide was purified by repeated fractional precipitation with 45 and 86% ethanol, dialyzed against water, and lyophilized. In order to minimize the background in subsequent assays, the polysaccharide was reduced with NaBH₄. Thus, 10 mg polysaccharide were dissolved in 2 ml 0.1 M boric acid, and 20 mg NaBH₄ in 1 ml 0.1 M NaOH was added. After 5 h incubation at room temperature, excess NaBH₄ was destroyed by adding dropwise 1 M acetic acid (until hydrogen emanation ended). Then the reduced polysaccharide was dialyzed against distilled water and lyophilized.

2. Hydrolysis assay

For the hydrolysis assay samples of 40 μΐ containing 10 mg/ml reduced polysaccharide and purified P22 tailspike protein at a subunit concentration of 0.3 μΜ in 50 mM sodium phosphate, 1 mM EDTA, pH 7.0 were prepared. In addition, a sample of 40 μΐ containing 10 mg/ml reduced polysaccharide and purified P22 tailspike active site variant D392N (Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide U. Baxa, S. Steinbacher, S. Miller, A. Weintraub, R. Huber, R. Seckler Biophysical Journal - 1 October 1996 (Vol. 71, Issue 4, pp. 2040-2048)) at a subunit concentration of 0.3 μΜ in 50 mM sodium phosphate, 1 mM EDTA, pH 7.0 was prepared. Reactions were terminated by adding 40 μΐ 3,5-dinitrosalicylic acid (20 mg/ ml) in 0.7 M NaOH, boiling the samples for 5 min and subsequently cooling on ice. Finally, the samples were diluted by the addition of 520 μΐ ¾0. Absorbance at 535 nm was measured in cells of 1 cm path length against a reagent blank. During hydrolysis with P22 tailspike the concentration of reduced polysaccharide fragments is increasing, which is resulting in a significant increased absorbance at 535nm. Performing the experiment using the P22 tailspike active site variants (either D392N, D395N or E359Q) as a control there is only a slight increase visible. The concentration of reducing ends is determined in comparison with calibration curve measured with glucose. To rule out unspecific binding effects a control curve incubating polysaccharide with BSA as negative control is measured.

Thus, the assay shows that P22 tailspike exhibits a hydrolytic activity of degrading bacterial polysaccharides in contrast to the P22 tailspike active site variants (either D392N, D395N or E359Q) that show no significant hydrolytic activity.

Using the same test as described above, it was confirmed that (i) Det7 Tsp has a hydrolytic activity of degrading Salmonella typhimurium polysaccharides

(ii) PHIV10 TF has a hydrolytic activity of degrading E.coli 0157 polysaccharides

(iii) SF6 Tailspike has a hydrolytic activity of degrading Shigella polysaccharides, and

(iv) ε15 tailspike has a hydrolytic activity of degrading Salmonella

polysaccharides.

Example 2: LPS hydrolysis assay

The LPS hydrolysis assay is another test for hydrolytic activity of phage receptor proteins. LPS containing the O-antigen was isolated from gram negative bacterial cells. Therefore, bacterial cells (50 ml) from a culture growing in logarithmic phase were centrifuged (20 min, 4000 rpm, 4 °C), and the cell pellet was resuspended in 10 ml buffer (10 mM Tris, 50 mM EDTA, pH 8). The suspension was incubated at room temperature for 10 min and vortexed several times. Afterwards, the cells were centrifuged again (20 min, 4000 rpm, 4 °C), and the supernatant containing extracted LPS was transferred into a fresh tube. Acetone was added up to a volume of 50 ml, and the tube was incubated at - 20 °C for 30 min. Subsequently, the precipitate containing the extracted LPS was centrifuged (4000 rpm, 4 °C for 60 min). The resulting precipitate was solubilized in 10 ml water in an ultrasonic bath. The precipitation was repeated once with acetone. Finally, the precipitate containing the extracted LPS was desiccated in a vacuum exsiccator for 2 h and the LPS -pellet was resolved in 1 ml water.

For the LPS hydrolysis assay LPS was incubated with phage receptor proteins (0.2 mg protein per sample) in hydrolysis buffer (50 mM sodium phosphate, pH 7.5) at room temperature for 0 hours, 4 hours and 30 hours. After the respective incubation times, the enzymatic reaction was stopped by adding SDS-sample buffer and boiling the samples. Subsequently, the samples were applied on 15% SDS-PAGE. The gel was run in Tris-Tricin buffer and stained by a silver staining procedure. It was observed that the samples containing LPS only result in a smear of bands beginning from high molecular weight fractions down to low molecular weight fractions since EDTA extracted LPS contains molecules of different molecular weight. In contrast, hydrolyzed LPS contains clearly less high molecular weight fractions but several bands of low molecular weight since the phage receptor protein hydrolyzes the O-antigen at specific saccharide bonds of the repeating units. Moreover, it was observed that the sample after addition of Det 7 Tsp was already hydrolyzed after a few seconds.

Example 3: Specific binding test of phage receptor proteins to bacterial surface receptors

The test strains were grown over night in pre-cultures at 37 °C. The pre-cultures were diluted 1:5 in mTSB-medium (modified TSB medium, Oxoid) and grown to an OD₆oo of 1. Subsequently, cells were diluted to a bacterial cell concentration between 10⁸ and 10⁴ cfu/ml. The bacteria cell capture was performed at a bacteria concentration of 10⁴ cfu/ml in PBST buffer (2.25 mM NaH₂P0₄, 7.75 mM Na₂HP0₄, 150 mM NaCl, 0.05 % Tween, pH 6.7) in a sample volume of 500 μΐ. Therefore, 0.3 μg of the phage receptor protein PHIVIO TF were immobilized to 10 μΐ MCB45 magnetic beads (Hyglos GmbH, beads precoated with streptavidin, concentration 10 mg/ml) for 15 min, added to the bacterial sample, and incubated at room temperature in a rolling incubator. After 20 min incubation, the complexes of bacteria, PHIVIO TF and magnetic beads were collected in a magnetic separator, and washed with PBST-buffer twice. Subsequently, 100 μΐ sample including the complexes of bacteria, PHIVIO TF and magnetic beads were plated to Caso-agar plates, and incubated at 37 °C over night. As a control for unspecific binding samples were used with streptavidin coated beads without the phage receptor protein PHIVIO TF. The fraction of specifically bound bacteria was calculated in relation to the bacterial cell number used in the test, which was also plated on Caso-agar plates directly after bacterial dilution, for comparison.

The following Table shows the results of the specific binding test with PHIVIO TF:

Table 2: Results of binding test with PHIVIO TF

Bacteria genus Species % specific

E.coli 0157:H7 100

Citrobacter freundii 0157 100

Staphylococcus aureus 0

Pseudomonas aeruginosa 0 As shown in the Table the phase receptor protein PHIV10 TF specifically binds to bacterial surface receptors of E.coli 0157:H7 and Citrobacter freundii 0157 but does not specifically binds to bacterial surface receptors of Staphylococcus aureus and Pseudomonas aeruginosa.

In view of the specific binding of PHIV10 TF to at least 0157:H7, the examples prove that the phage receptor protein PHIV10 TF binds specifically to bacterial surface receptors.

Example 4: Production of ethanol by Saccharomyces cerevisiae - with and without the bacteriophage receptor proteins PHIV10 TF.

Yeast cultures of Saccharomyces cerevisiae are grown in YP medium (10 g/L yeast extract, 20 g/L Bacto peptone) containing 20 g L of glucose to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from YP media containing 20 g L of glucose are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 400 mL of YP medium containing 20 g L of glucose at 30°C with initial OD600 of about 1 or 10, at an agitation speed of 200rpm under oxygen limited conditions. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for 72 h. A respective fermentation results in a yield of 0.42 to 0.44 g ethanol per 1 g glucose.

In addition, the bioreactor fermentation is performed in the presence of PHIV10 TF. PHIV10 TF is added at a final concentration of lOOmg/ml together with the inoculum. The fermentation is run for 72 h resulting. A respective fermentation results in a yield of 0.44 to 0.46 g ethanol per 1 g glucose.

Example 5: Production of ethanol by Saccharomyces cerevisiae - with and without the phage receptor proteins Det 7 tsp.

Using lignocelluose as fermentation feedstock, lignocelluose is pretreated with cellulase. This enzymatic hydrolysis pretreatment produces amongst other components glucose, lignin and unhydrolysed cellulose. Said pretreatment is performed once with the addition of the phage receptor protein Det 7 tsp at a final concentration of lOOmg/ml and once without the addition of any phage receptor protein. Yeast cultures of Saccharomyces cerevisiae are grown in YP medium (10 g/L yeast extract, 20 g/L Bacto peptone) containing 20 g L of glucose to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from YP media containing 20 g L of glucose are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 400 mL of YP medium containing 20 g L of pretreated lignocellulose at 30 °C with initial OD600 of about 1 or 10, at an agitation speed of 200rpm under oxygen limited conditions. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for 72 h.

The fermentation from the fermentation process in which the phage receptor protein Det 7 tsp has been added to the pretreatment step results in a yield of 0.48 to 0.50 g ethanol per 1 g glucose whereas the fermentation process without the use of any phage receptor protein results in a yield of 0.46 to 0.48 g ethanol per 1 g glucose.

Example 6: Production of gamma-linolenic acid by Hansenula polymorpha - with and without the bacteriophage receptor proteins PHIV10 TF.

To produce gamma-linolenic acid, yeast cultures of Hansenula polymorpha are grown in GBS medium (one liter of the GBS medium consisted of 26.7 ml 85% H₃P0₄, 0.93 g CaS0₄, 18.2 g K₂S0₄, 14.9 g MgS0₄-7H₂0, 4.13 g KOH, 40.0 g glucose, and 4.35 ml trace salts). The trace salts contained 6.0 g CuS0₄-5H₂0, 0.08 g KI, 3.0 g MnS0₄ H₂0, 0.2 g Na₂Mo0₄-2H₂0, 0.02 g H₃BO₃, 20.0 g ZnCl₂, 65 g FeS0₄-7H₂0, 0.5 g CoCl₂-6H₂0, 5.0 ml H₂S0₄, and 0.2 g biotin in 1 1 of distilled water) supplemented with adenine sulfate to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from GBS media are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 1000 mL of GBS medium (26.7 ml 85% H₃P0₄, 0.93 g CaS0₄, 18.2 g K₂S0₄, 14.9 g MgS0₄-7H₂0, 4.13 g KOH, 40.0 g glycerol, and 4.35ml trace salts) at 30 °C with initial OD600 of about 1 or 10, at an agitation speed of 200rpm under 1-41/h aeration with oxygen gas (to keep saturation at 20% 0₂) and pH 5.0. A 25% ammonium hydroxide solution is used to adjust the pH of the culture broth. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for 48 h resulting in a yield of about 14% of the total fatty acids being gamma-linolenic acid. In addition, the bioreactor fermentation is performed in the presence of PHIV10 TF. PhiVIO TF is added at a final concentration of lOOmg/ml together with the inoculum. The fermentation is run for 48 h resulting in a yield of about 18% of the total fatty acids being gamma- linolenic acid, wherein the amount of total fatty acids is increased up to 5% in comparison the fermentation not performed in the presence of PHIVIO.

Example 7: Production of ethanol in by Zymomonas mobilis - with and without the bacteriophage receptor proteins PHIVIO TF.

Cultures of Zymomonas mobilis are grown in ZM medium (50mM Phosphate, pH 5.5, 5 g/L yeast extract, 10 g/L Bacto peptone, 50 g L of glucose, 30mg/ml Ampicillin) to prepare inoculums for fermentation experiments. Cells at mid-exponential phase from ZM media containing are harvested and inoculated after washing twice by sterilized water. Bioreactor fermentation experiments are performed using 1000 mL of ZM medium at 30 °C with initial OD600 of about 1 or 10, at an agitation speed of 150rpm under oxygen limited conditions. Initial cell densities are adjusted to OD600 ¼ to about 1 or 10. The fermentation is run for up to 160 h. Said fermentation results in a yield of 0.46 to 0.48 g ethanol per 1 g glucose.

In addition, the bioreactor fermentation is performed in the presence of PHIVIO TF. PHIVIO TF is added at a final concentration of lOOmg/ml together with the inoculum. The fermentation is run for up to 160 h. Said fermentation results in a yield of 0.49 to 0.51 g ethanol per 1 g glucose.

Claims

1. A fermentation process for the production of an organic compound of interest, wherein the fermentation process comprises the following steps:

2. The fermentation process according to any one of the preceding claims, wherein the components comprising polysaccharides of step (a) are bacterial polysaccharides, biomass, pretreated biomass or a combination thereof.

3. The fermentation process according to claim 2, wherein the bacterial polysaccharides are O-antigens of lipopolysaccharides or K-antigens of capsular polysaccharides, components of teichoic or lipoteichoic acids, of peptidoglycan, of extracellular components, in particular capsules or slime layers, of carbohydrates, of polysaccharide matrices, of lipopolysaccharides, or fragments of these components.

4. The fermentation process according to claim 2, wherein the biomass is crops, corn, wheat, barley, sorghum, rye, sugar cane, potato, cottonwoods, paper, switchgrass, bagasse, wood, cornstover or corn fibers.

5. The fermentation process according to claim 2, wherein the pretreated biomass has been pretreated mechanically, chemically, enzymatically, by heat or by a combination thereof.

6. The fermentation process according any one of the preceding claims, wherein the organic compound of interest is an alcohol or a lipid.

7. The fermentation process according to any one of the preceding claims, wherein the fermentation is carried out by bacteria, yeast, algae, fungi or a combination thereof.

8. The fermentation process according to any one of the preceding claims, wherein the amount of phage receptor proteins in step a) is present in an amount which enhances the productivity of the fermentation process.

9. The fermentation process according to any one of the preceding claims, wherein the phage receptor protein is derived from a bacteriophage selected of the list consisting of: P22, ε15, Sf6, SP6, Kl, K5, HK620, Det7, Tl, T5, T7, phage 14, P2, phiVIO, APSE-1, Al, A18a, ST104, ST64T, SETP3, JK106 and Gifsy.

10. The fermentation process according to any one of the preceding claims, wherein the phage receptor protein is selected from the group consisting of P22 TSP according to SEQ ID NO: 1, Sf6 TSP according to SEQ ID NO: 2, HK620 TSP according to SEQ ID NO: 3, Det7 Tsp according to SEQ ID NO: 4, ε15 tailspike according to SEQ ID NO: 5, K5 lyase A (KflA) according to SEQ ID NO: 6, HylP2 according to SEQ ID NO: 7, K1F TSP according to SEQ ID NO: 8 and PhiVIO TF according to SEQ ID NO: 9.

11. The fermentation process according to any one of claims 6 to 10, wherein the alcohol is ethanol or butanol, or wherein the lipid is a triacylglycerol, glycolipid, sterol, sterol ester, cartenoid, xanthophyll or fatty acid, in particular a free fatty acid, unsaturated fatty acid, polyunsaturated fatty acid or ester of a fatty acid.

12. The fermentation process according to any one of the preceding claims, wherein step a) and step b) are performed simultaneously.

13. The fermentation process according to any one of the preceding claims, wherein the fermentation process comprises additionally the following step:

(c) recovering of the compound of interest from the fermented material.

14. The fermentation process according to claim 13, wherein the compound of interest is further processed into a biofuel.

15. The fermentation process according to claim 14, wherein the biofuel is bioethanol, biobutanol or biodiesel.