WO2015110518A1

WO2015110518A1 - Fermentative process involving cell cultivation at a low dissolved-oxygen concentration

Info

Publication number: WO2015110518A1
Application number: PCT/EP2015/051226
Authority: WO
Inventors: Thomas Haas; Markus PÖTTER; Eva-Maria Eckl; Simon Beck; Anja Thiessenhusen; Martin DEMLER
Original assignee: Evonik Degussa Gmbh
Priority date: 2014-01-27
Filing date: 2015-01-22
Publication date: 2015-07-30
Also published as: AR100302A1; DE102014201384A1

Abstract

The invention relates to a process involving the cultivation of hydrogen-oxidizing bacteria at a low dissolved-oxygen concentration of 0.001 mg/l to 0.5 mg/l. Said process can also involve synthesis of target products selected among substances which are formed via the intermediate product acetyl coenzyme A or metabolites of the citric acid cycle, e.g. fatty acids, fatty acid esters, fatty alcohols, fatty aldehydes, butanol, butene, propene, acetone, 2-hydroxy-methylpropanoic acid, or 2-propanol.

Description

FERMENTATIVE METHOD COMPRISING A CULTURE OF CELLS UNDER

LOW SOLVENT OXYGEN CONCENTRATION

Field of the invention

The invention relates to a method comprising culturing cells under low dissolved oxygen concentration. State of the art

In the fermentative production of products, efforts are always made to avoid or minimize the formation of undesirable by-products. In general, one can thus increase the product yield and also facilitate the purification of the final product.

Cells that use inorganic carbon as a carbon source to synthesize organic molecules are known in particular in the form of oxyhydrogen bacteria. Organisms that produce polyhydroxyalkanoate use as precursor 3-hydroxyalkanyl-CoA. This metabolite can be considered excellent

Original substance serve, in particular by artificially inserted

Metabolic pathways to other high-quality organic substances to be metabolized.

In particular, the polyhydroxybutyrate-producing oxyhydrogen bacterium Cupriavidus necator has been extensively characterized and studied in recent years.

The object of the invention was to provide a method which the

By-product formation is reduced in fermentation and thus increases the yield of monomer units of the polyhydroxyalkanoate reduced in the biosynthesis. Description of the invention

Surprisingly, it has been found that the method described below is capable of solving the problem posed by the invention.

The subject of the present invention is therefore a method comprising a

Cultivation of oxyhydrogen bacteria under low dissolved oxygen concentration.

All percentages (%) are by mass unless otherwise specified. The advantage of the present invention is that the limited supply of oxygen prevents an excess metabolism of the microorganism, which leads to the formation of undesired by-products. Thus for the present

Invention to increase the carbon selectivity.

Another advantage of the present invention is that reduced by the

By-product formation simplifies the purification in process step C and thus becomes more cost-effective.

Another advantage of the present invention is that only a small proportion of oxygen in the H ₂ -containing substrate gas is necessary for this purpose. Thus, the risk of the formation of an explosive atmosphere inside the fermenter is minimized. Thus, the apparatus for the present method need not be designed for EX-protected, which can save significant costs in the construction of a production plant.

Subject matter is a process for the cultivation of methanogenic bacteria in a medium comprising a step, in which the medium is contacted with a gas containing H _2, C0 ₂ and 0 _2, and the medium is a

Dissolved oxygen concentration of 0.001 mg / L to 0.5 mg / L, preferably from 0.005 mg / L to 0.3 mg / L, more preferably from 0.01 mg / L to 0.2 mg / L.

The term "oxyhydrogen bacterium" is a bacterium to be understood which is capable of chemolithoautotrophically to grow and build up from H ₂ and C0 ₂ in the presence of oxygen carbon frameworks with more than one carbon atom, wherein the hydrogen oxidation and oxygen as a terminal According to the invention, it is possible to use both those bacteria which are inherent in nature

Bacteriophilic bacteria, or even bacteria which have been by genetic modification to Oxy-gas bacteria, such as an E. coli cell, which has been made by recombinant insertion of the necessary enzymes in a position capable of H ₂ and C0 ₂ in the presence of oxygen to build carbon skeletons with more than one carbon atom, the hydrogen oxidized and the oxygen as the terminal

Electron acceptor is used. Preferably, the im

According to the invention used explosive gas bacteria to those which already represent as wild-type explosive gas bacteria.

Bacteriostatic bacteria preferably used according to the invention are selected from the genera Achromobacter, Acidithiobacillus, Acidovorax, Alcaligenes, Anabena,

Ancyclobacter, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter, Herbaspirillum, Hydrogenobacter, Hydrogenobaculum,

Hydrogenophaga, Hydrogenophilus, Hydrogenothermus, Hydrogenovibrio, Ideonella sp. 01, Kyrpidia, Metallosphaera, Mycobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Pseudonocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Seliberia, Streptomyces, Thiocapsa, Variovorax, Xanthobacter, Wautersia, with Cupriavidus being particularly preferred.

especially from the species Cupriavidus necator (also known as Ralstonia eutropha, Wautersia eutropha, Alcaligenes eutrophus, Hydrogenomonas eutropha), Achromobacter ruhlandii, Acidithiobacillus ferrooxidans, Acidovorax facilis, Alcaligenes hydrogenophilus,

Alcaligenes latus, Anabena cylindrica, Anabena oscillaroides, Anabena sp., Anabena spiroides, Ancyclobacter vacuolatus, Aquifex aeolicus, Aquifex pyrophilus, Arthrobacter strain 11X, Bacillus schlegelii, Bradyrhizobium japonicum, Derxia gummosa, Escherichia coli, Heliobacter pylori, Herbaspirillum autotrophicum, Hydrogenobacter hydrogenophilus. , Hydrogenobacter thermophilus, Hydrogenobaculum acidophilum, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflav, Hydrogenophaga

taeniospirales, Hydrogeneophilus thermoluteolus, Hydrogenothermus marinus,

Hydrogenovibrio marinus, Ideonella sp. 0-1, Kyrpidia tusciae, Metallosphaera sedula, Myobacterium gordonae, Nocardia autotrophica, Oligotropha carboxidivorans,

Paracoccus denitrificans, Pelomonas saccharophila, Polaromonas hydrogenivorans, Pseudomonas hydrogenovora, Pseudomonas thermophila, Rhizobium japonicum, Rhodococcus opacus, Rhodopseudomonas palustris, Seliberia carboxydohydrogena, Thiocapsa roseopersicina, Variovorax paradoxus, Xanthobacter autrophicus,

Xanthobacter flavus, Cupriavidus necator being particularly preferred, in particular from the strains Cupriavidus necator H16, Cupriavidus necator H1 or Cupriavidus necator Z-1.

Particular preference is given to using the strain Cupriavidus necator H 16 PHB-4, DSM 541. Suitable methods for measuring the dissolved oxygen concentration are described, for example, in Suresh et al. (2009) Techniques for oxygen transfer measurement in bioreactors: a review, J Chem Technol Biotechnol, 84, pp. 1091-1103.

In particular, the dissolved oxygen concentration is used by measurement with an amperometric dissolved oxygen probe according to the Clark principle. This probe is calibrated under process conditions at the oxygen concentration used in the gas to 100% p0 ₂ and under nitrogen to 0% 0 ₂ . The measured pO ₂ values are directly proportional to the respective dissolved oxygen concentration in the medium. The measured pO ₂ values are converted into the respective dissolved oxygen concentration via the corresponding Henry constant. Henry's constants for dissolved gases can be found in Wilhelm et al. (1977) Low-pressure solubility of gases in liquid water (77), pp. 219-262.

The amount of dissolved oxygen in an aqueous medium is dependent on many factors, such as pressure, temperature, and oxygen concentration of the gas adjacent to the medium.

The skilled person can thus take direct influence on the dissolved oxygen concentration by targeted modification of these parameters.

In a fermenter, the dissolved oxygen concentration can be regulated, for example, by the fermenter geometry, the type of stirrer, the stirrer speed and the type of oxygen input.

The dissolved oxygen concentration can be monitored in real time by using, for example, the above-mentioned probes.

A method preferred according to the invention is characterized in that the oxyhydrogen bacterium has been genetically engineered such that it has a content comparable to its

Wild type has a reduced polyhydroxyalkanoate synthesis.

A "cell type" of a cell is preferably referred to as a cell whose genome is in a state naturally formed by evolution

Term is used for both the entire cell and for individual genes. The term "wild type" therefore does not include in particular cells or genes whose gene sequences are at least partially recombinant by humans

Procedures have been changed.

By the term "reduced polyhydroxyalkanoate synthesis compared to wild-type" in the context of the present invention is meant that the cells under consideration, when cultured under the same conditions as the wild type, less

Polyhydroxyalkanoate per cell form as the wild-type. The oxyhydrogen bacteria preferably used according to the invention have, due to at least one genetic modification, a polyhydroxyalkanoate synthesis reduced compared to their wild-type. In this connection, it is particularly preferred that the oxyhydrogen bacteria used according to the invention do not synthesize detectable amounts of polyhydroxyalkanoate. Preferably, the polyhydroxyalkanoate whose synthesis is reduced, polyhydroxybutyrate.

The polyhydroxyalkanoate synthesis, which is reduced compared to the Wld type, can preferably be achieved by a genetic modification, such that the activity of at least one enzyme which is reduced compared to the cell type of the enzyme which converts 3-hydroxyalkanyl-coenzyme A to polyhydroxyalkanoate, preferably 3-hydroxybutyryl Coenzyme A catalyses to polyhydroxybutyrate.

Accordingly, by the term "reduced activity of an enzyme" used, it is preferred to use a factor of at least 0.5, more preferably at least 0, 1, more preferably at least 0.01, even more preferably at least 0.001, and Most preferably, the term "decreased activity" does not include any detectable activity ("zero activity")

Reduction of the activity of a particular enzyme can be carried out, for example, by targeted mutation or by other measures known to those skilled in the art for reducing the activity of a particular enzyme.

Methods for reducing enzymatic activities in microorganisms are known to the person skilled in the art, these include insertion of foreign DNA into the gene coding for the target enzyme, deletion of at least parts of the gene coding for the target enzyme, point mutations in the gene sequence coding for the target enzyme, RNA Interference (siRNA), antisense R A or modification (insertion, deletion or point mutations) of regulatory sequences such as promoters and terminators or of

Ribosome binding sites flanking the gene encoding the target enzyme. By foreign DNA is meant in this context any DNA sequence which is "foreign" to the gene (and not to the organism), i.e. endogenous DNA sequences may also function as "foreign DNA" in this context.

In the enzyme, which involves the reaction of 3-hydroxyalkanyl-coenzyme A too

Polyhydroxyalkanoate is preferably a polyhydroxyalkanoate synthase, more preferably a polyhydroxybutyrate synthase. In particular, this enzyme is preferably encoded by the genes phbC or phaC, with phaC being particularly preferred. In the inventive process, the medium is brought into contact with a gas containing H _2, C0 ₂ and 0. ₂

As a result, the explosive gas bacterium H ₂ , C0 ₂ and 0 ₂ containing gas as

Carbon source provided. The oxyhydrogen bacterium synthesized at least partially from this carbon source acetoacetyl-CoA, which to other organic

Compounds can be metabolized. Unlike in an acetogenic

Process, which takes place under strictly anaerobic conditions, the process step described here of the method according to the invention is carried out under aerobic conditions.

Preferably, the partial pressure of the hydrogen in the H ₂ , C0 ₂ and 0 ₂

containing gas 0, 1 to 100 bar, preferably 0.2 to 10 bar, more preferably 0.5 to 4 bar.

The partial pressure of carbon dioxide in the H _2, C0 ₂ and 0 ₂ -containing gas is preferably 0.03 to 100 bar, particularly preferably 0.05 to 1 bar, in particular 0.05 to 0.3 bar

The partial pressure of oxygen in the H _2, C0 ₂ and 0 ₂ -containing gas is preferably 0.04 to 100 bar, particularly preferably 0.04 to 1 bar, in particular 0.04 to 0.5 bar as the source of H _2, and C0 ₂ is particularly suitable synthesis gas, which is used mixed with oxygen; Therefore, it is preferred according to the invention that the gas containing H ₂ , C0 ₂ and 0 ₂ contains synthesis gas.

Syngas can, for. B. be provided from the by-product of the coal gasification. The oxyhydrogen bacterium thus converts a substance that is a waste product into a valuable raw material.

Alternatively, synthesis gas may be provided by the gasification of widely available, inexpensive agricultural raw materials for the process of the present invention. There are many examples of raw materials that can be converted into synthesis gas, since almost all forms of vegetation can be used for this purpose. Preferred raw materials are selected from the group comprising

perennial grasses such as miscanthus, grain residues, processing waste such as sawdust. In general, synthesis gas is recovered in a gasification apparatus from dried biomass, mainly by pyrolysis, partial oxidation and steam reforming, the primary products being CO, H ₂ and CO ₂ .

Normally, some of the product gas is treated to optimize product yields and prevent tar formation. Cracking the unwanted tar in Synthesis gas and CO can be carried out using lime and / or dolomite. These processes are described in detail in z. B. Reed, 1981 (Reed, TB, 1981, Biomass gasification: principles and technology, Noves Data Corporation, Park Ridge, NJ.) Mixtures of different sources for the generation of

Synthesis gas can be used.

In particular, it is preferred that the carbon contained in the synthesis gas at least 50 wt .-%, preferably at least 70 wt .-%, particularly preferably at least 90 wt .-% of the carbon of all carbon sources, the

Oxygen gas bacterium by the gas containing H ₂ , C0 ₂ and 0 ₂ are available makes up, wherein the weight percent of carbon refer to the carbon atoms. The remaining carbon sources, for example in the form of

Carbohydrates be contained in the aqueous medium or else represent C0 ₂ from a source other than synthesis gas.

Other C0 ₂ sources include exhaust gases such as flue gas, petroleum refinery emissions, gases produced by yeast fermentation or clostridial fermentation, exhaust gases from the

Gasification cellulosic materials or coal gasification.

These exhaust gases do not necessarily have as byproducts of others

Processes may have arisen, but may be extra for use in the

inventive method are produced.

In the inventive method also can be used in particular gas generator, so that the gas of H ₂ containing containing C0 ₂ and 0 ₂ gas generator

To achieve a high cell density in a short period of time it can

be advantageous according to the invention, if you first a larger amount of

Attracts oxyhydrogen bacteria with the help of a preculture. Thus, a preferred

Embodiment of the method characterized in that it comprises the method steps

A) Increase pop-up gas bacteria in a medium up to a cell density X cells / liter and

B) culturing the methanogenic bacteria in a medium, wherein the medium is contacted with a gas containing H _2, C0 ₂ and 0 _2, and the medium is a

In this case, process step B) corresponds to the method step explained in the introduction, in which the medium is contacted with a gas containing H _2, C0 ₂ and 0 _2, and the medium has a dissolved oxygen concentration of from 0.001 mg / L to 0.5 mg / L, preferably from 0.005 mg / L to 0.3 mg / L, more preferably from 0.01 mg / L to 0.2 mg / L.

In method step A), any form of carbon source can be used according to the invention, including, for example, sugar. Even the low

Dissolved oxygen concentration is not mandatory in process step A). According to the invention, it is even preferable not to limit the cells with respect to any nutrient.

The cell density X in method step A is preferably at least 1 × ^{10 5} , more preferably at least 1 × ¹⁰ ⁷ , in particular at least 1 × 10 ⁸ cells / ml of total culture. An inventively preferred method is characterized in that in

Process step B) the cell density does not exceed 2 × cells / ml, in particular 1.5 × cells / ml, particularly preferably 1.2 × cells / ml. Process step B) is advantageous and thus preferably at least one

Part-time area used for product production.

The target product to be produced by the process according to the invention is preferably selected from substances comprising three or four carbon atoms, in particular butanol, butene, propene, acetone, 2-hydroxyisobutyric acid and 2-propanol.

Alternatively preferred target products to be prepared by the method according to the invention are selected from substances whose formation takes place via the intermediate acetyl coenzyme A; such target products are, for example, fatty acids, fatty acid esters, fatty alcohols, fatty aldehydes and substituted derivatives of the abovementioned compounds, substitution radicals being especially amino, hydroxyl and keto groups, preference being given in this connection to an omega substitution, in particular an amino group.

Further alternatively preferred to be produced by the process according to the invention target products are selected from substances whose formation on metabolites of the citric acid cycle such. B. succinate occurs.

In a preferred embodiment of the process according to the invention, target products are synthesized in process step B) by the oxyhydrogen gas bacteria, whose formation via the intermediate 3-hydroxyalkanyl-CoA, in particular

3-hydroxybutyryl-coenzyme A, takes place.

The term "intermediate" in this context defines a chemical compound which can be converted to the desired product by the use of at least one enzyme enzymatically, using as a "chemical compound" those with or without coenzyme A thioester functionalization should be considered equivalent and thus the thioester forming or cleaving enzymes should not be counted. It may be advantageous if the oxyhydrogen bacteria were modified in accordance with the desired target product by genetic modification such that the

Target product reinforced by the oxyhydrogen bacteria is formed.

In the context of the present invention, a preferred target product is 2-hydroxyisobutyric acid. For this target product, it is preferable if the im

Oxy-gas bacteria used according to the invention were engineered such that they have an increased activity of the enzyme Ei compared to their wild-type, which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A to 2-hydroxyisobutyryl-coenzyme A. The enzyme egg is

preferably a hydroxyl isobutyryl-CoA mutase, an isobutyryl-CoA mutase (EC 5.4.99.13) or a methylmalonyl-CoA mutase (EC 5.4.99.2), in each case preferably a coenzyme B12-dependent mutase.

The enzyme Ei is preferably those enzymes which consist of the microorganisms Aquincola tertiaricarbonis L108, DSM 18028, DSM18512, Methylibium petroleiphilum PM1, Methylibium sp. R8, Xanthobacter autotrophicus Py2, Rhodobacter sphaeroides (ATCC 17029), Nocardioides sp. JS614, Marinobacter algicola DG893,

Sinorhizobium medicae WSM419, Roseovarius sp. 217, Pyrococcus furiosus DSM 3638, particularly preferably the coenzyme B12-dependent mutase described in PCT / EP2007 / 052830, as well as enzymes which are present in at least one of their

Partial sequence a sequence identity to the amino acid sequence of the small or large subunit of the mutase described in PCT / EP2007 / 052830 (accession number

DQ436457.1 and DQ436456.1) of at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably at least 99% at the amino acid level, determined according to the blastp algorithm with an expect threshold of 10, a word size of 3, a blosum62 matrix with gap costs of existence: 1 1 and extension: 1 and a conditional compositional score matrix adjustment.

The term "increased activity of an enzyme" as used above in connection with the enzyme Ei and in the following statements in connection with the enzymes E ₂ , etc., is preferably to be understood as an increased intracellular activity. The following statements on increasing the enzyme activity in cells apply both to the increase in the activity of the enzyme Ei and to all the enzymes mentioned below, the activity of which may optionally be increased.

In principle, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences which code for the enzyme, using a strong promoter, changing the codon usage of the gene, in various ways the half-life of the mRNA or the enzyme is increased, the regulation of the expression of the gene modified or uses a gene or allele coding for a corresponding enzyme with an increased activity and

if necessary, combine these measures. In particular, thus, the gene coding for the enzyme E _x is overexpressed, which leads to an increased activity by overexpression. Genetically modified cells according to the invention are produced, for example, by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a promoter which enables the expression of the gene. In particular, heterologous expression is achieved by integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.

An overview of the possibilities for increasing the enzyme activity in cells using the example of pyruvate carboxylase is given in DE-A-100 31 999, which is hereby incorporated by reference and the disclosure of which relates to the possibilities for

Increasing enzyme activity in cells forms part of the disclosure of the present invention.

The expression of the above and all of the enzymes and genes mentioned below can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software. If the increase in enzyme activity is based solely on an increase in the expression of the corresponding gene, then the quantification of the increase in enzyme activity can be readily determined by comparing the 1- or 2-dimensional protein separations between Wld type and genetically engineered cell. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)

Protein concentration may also be assessed by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical evaluation with appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5: 32-39;

Lottspeich (1999), Angewandte Chemie 1 11: 2630-2647). The activity of DNA-binding proteins can be assessed by DNA band-shift assays (also known as

Gel retardation) (Wilson et al., (2001) Journal of

Bacteriology, 183: 2151-2155). The effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989). The intracellular enzymatic activities can after

various methods described (Donahue et al., (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284;

Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823). Unless specific methods for determining the activity of a particular enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity are preferably carried out by means of the methods described in Hermann et al., Electophoresis, 22: 1712-23 ( 2001), Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 1 11: 2630-2647 (1999) and Wlson et al., Journal of Bacteriology 183: 2151-2155 (2001).

Wrd the increase of enzyme activity by mutation of the endogenous gene

accomplished, such mutations can be generated either undirected by conventional methods, such as by UV irradiation or by

Mutagenic chemicals, or specifically by genetic engineering methods such as deletion (s), insertion (s) and / or nucleotide exchange (s). These mutations result in altered cells. Particularly preferred mutants of enzymes are, in particular, also those enzymes which are no longer or at least less feedback-inhibitable compared to the Wld-type enzyme.

For example, by increasing the enzyme activity by increasing the synthesis of an enzyme, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or the ribosome binding site located upstream of the structural gene. Work in the same way

Expression cassettes that are installed upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time. Furthermore, the enzyme gene can be used as regulatory

Sequences but also so-called "Enhancer" be assigned, which has an improved Interaction between RNA polymerase and DNA also increased

Effect gene expression. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, a further

Overexpression of the genes in question can be achieved by changing the composition of the medium and culture. For instructions on this, the skilled person will find, inter alia, in Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-0 472 869, in US 4,601,893, in Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991), in Reinscheid et al. Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of

Bacteriology 175, 1001-1007 (1993)), WO-A-96/15246, Malumbres et al. (Gene 134, 15-24 (1993), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), and in known textbooks of genetics and molecular biology lead as well as the mutations to genetically modified cells.

To increase the expression of the respective genes, episomal plasmids are used, for example. In principle, all embodiments which are available to the person skilled in the art for this purpose are suitable as plasmids or vectors. Such plasmids and vectors may, for. B. the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL be removed. Further preferred plasmids and vectors can be found in: Glover, D.M. (1985), DNA cloning: a practical approach, Vol. I-Ill, IRL Press Ltd. , Oxford; Rodriguez, R.L. and Denhardt, D.T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D.V. (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J .; Fritsch, E.F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.

The plasmid vector containing the gene to be amplified is then converted by conjugation or transformation into the desired strain. The method of conjugation is described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination by means of a cross-over event, the resulting strain contains at least two copies of the gene of interest.

Under the formulation used above and in the following, "is an increased activity of an enzyme E _x " compared to its wild-type

preferably always by a factor of at least 2, more preferably of at least 10, more preferably of at least 100, more preferably still greater than at least 1,000, and most preferably at least 10,000 increased activity of the respective enzyme E _x to understand. Furthermore, the cell according to the invention comprises "an activity of an enzyme E _x which is increased compared with its wild type, in particular also a cell whose Wld type has no or at least no detectable activity of this enzyme E _x and which only after increasing the enzyme activity, for example by overexpression shows a detectable activity of this enzyme E _x In this context, the term "overexpression" or the phrase "expression increase" used in the following also encompasses the case that an initial cell, for example a Wld type cell, has no or at least none has detectable expression and only by recombinant methods a detectable synthesis of the enzyme E _{x is} induced.

It may furthermore be advantageous if the oxyhydrogen bacteria used in the process according to the invention have been genetically engineered in such a way that they have an increased activity of an enzyme E ₂ which, compared to their Wld type, increases the

Reaction of acetoacetyl-coenzyme A catalyzed to 3-hydroxybutyryl-coenzyme A have.

The enzyme E ₂ is preferably an enzyme selected from the group comprising:

a 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35),

an acetoacetyl-coenzyme A reductase (EC 1.1.1.36),

a long-chain 3-hydroxyacyl-CoA dehydrogenase ((EC 1.1.1.21 1) and

a 3-hydroxybutyryl-coenzyme A dehydrogenase (EC 1.1.1.157).

This enzyme is preferably encoded by the genes selected from the group consisting of phaB, phbB, fabG, phbN1, phbB2 or, with phaB, phbB being particularly preferred. The nucleotide sequence of these genes can be found, for example, in the "Kyoto Encyclopedia of Genes and Genomes" (KEGG database), the databases of the

National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) or the nucleotide sequence database of European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK, respectively). The process according to the invention preferably has a process step C)

Purification of the target product.

In the examples given below, the present invention is described by way of example, without the invention, whose scope of application is apparent from the entire description and the claims, referred to in the examples

Embodiments should be limited.

EXAMPLES Example 1 Autotrophic cultivation of C. necator at different stirrer speeds in a stirred tank reactor

The following example shows the genetically modified strain

C. necator H16 PHB-4 pBBR1 MCS-2 :: hcmA-hcmB (lac) (hereinafter referred to as RITA), which is used to produce 2-hydroxyisobutyric acid (2HIB), cultured autotrophically.

It has been used a defined medium consisting of 9 g / l Na ₂ HP0 ₄ x 12 H ₂ 0, 1, 5 g / l KH ₂ P0 _4, 1 g / l NH ₄ Cl, 0.2 g / l MgS0 ₄ , 0.2 g / l CaCl ₂ , 6 mg / l FeCl ₃ , 0.1 mg / l ZnSO ₄ × 7 H ₂ O, 0.03 mg / l MnCl ₂ × 4 H ₂ O, 0.3 mg / 1 H ₃ B0 ₃ , 0.2 mg / L CoCl ₂ × 6 H ₂ O, 0.01 mg / L CuCl ₂ × 2 H ₂ O, 0.02 mg / L NiCl ₂ × 6 H ₂ O and 0.03 mg / l Na ₂ M04 x 2 H ₂ 0.

Two identical autotrophic cultivations were carried out in Biostat B 2L stirred tank reactors of Sartorius filled with 1 l of medium, once at 1000 min ^-1 and once without stirring, The gas was introduced into the medium through a gassing frit having a pore size of 20 μm, which was centrally under Cultivations were carried out at 28 ° C. without pH control.

The reactors were fumigated with a premixed, non-explosive gas mixture of composition H ₂ 90%, C0 ₂ 6%, 0 ₂ 4% at atmospheric pressure with a gassing rate of 0.25 vvm.

At the start of the experiment, the reactor was inoculated with 2 colonies from an agar plate (300 mg / L kanamycin). On sampling, 10 ml of each sample was taken to determine OD ₆ oo, dry biomass and product spectrum. The determination the product concentration was determined by quantitative ¹ H NMR spectroscopy. The internal quantification standard was trimethylpropionic acid.

It was found that in cultivation without stirring the speed of the

Growth was significantly reduced as compared to the cultivation at 1000 min ^-1 . In comparison, however, no formation of the by-products acetate, pyruvate, 3-methyl-butyrate and isobutyrate was found.

Experiment 1 (without product preparation

NMR analysis

3

OD methyl

Dissolved O ₂ , (600 BTM, 3HB, 2HIB, acetate, pyruvate, butyrate, iso-butyrate,

Time, h mg / L pH nm) g / L mg / L mg / L mg / L mg / L mg / L mg / L

0,0 1,56 6,92 0,00 0,00 0 0 0 0 0 0

46.4 0.006 6.90 0.35 0.28 0 0.87 0 0 0 0

54.8 0.006 6.87 0.46 0 2.51 0 0 0 0

70.3 0.006 6.83 0.92 0.50 0 11.33 0 0 0 0

78.6 0.006 6.79 0.95 0 18.07 0 0 0 0

95.6 0.006 6.74 1.29 0.67 0 32.16 0 0 0 0

102.8 0.006 6.71 1.45 0 33.82 0 0 0 0

166.7 0.006 6.51 2.41 1.15 5.02 57.94 0 0 0 0

197.7 0.006 6.43 2.79 0 69.85 0 0 0 0

219.5 0.006 6.40 3.35 1.63 0 60.01 0 0 0 0

242.6 0.006 6.32 3.79 0 55.54 0 0 0 0

268.5 0.006 6.23 4.22 2.02 0 49.51 0 0 0 0

295.0 0.006 6.17 4.41 3.37 59.15 0 0 0 0

316.8 0.006 6.14 4.51 2.13 6.82 63.91 0 0 0 0

335.5 0.006 6.13 4.44 5.71 68.96 0 0 0 0

357.8 0.006 6.13 4.60 2.22 0 69.55 0 0 0 0 Experiment 2 (stirred at 1000 rpm) product formation

NMR analysis

3

OD methyl

Time, h mg / L pH nm) g / L mg / L mg / L mg / L mg / L mg / L mg / L

0,0 1,56 6,88 0,00 0,00 0 0 0 0 0 0

46.4 1.42 6.83 0.73 0.40 0 1.06 0 0 0 0

50.8 1.33 6.77 1.19 0 2.4 0 0 0 0

54.8 1.09 6.67 2.21 1.03 0 3.92 0 0 0 0

70.3 0.94 5.90 5.06 2.10 10.76 15.32 15.55 44.19 12.37 10.35

74.9 0.85 5.86 4.90 14.27 20.71 27.82 30.29 17.56 17.37

78.6 0.83 5.89 4.86 2.08 23.29 21.91 24.72 9.97 26.57 17.58

95.6 0.73 5.98 4.51 1.93 44.55 37.11 0 5.85 41.42 25.27

102.8 0.74 5.99 4.14 1.95 30.19 36.05 0 4.52 31.87 14.11

Claims

claims

Anspruch [en] A process for cultivating oxyhydrogen bacteria in a medium comprising

Method step, in which the medium is brought into contact with a gas containing H ₂ , C0 ₂ and O ₂ , and the medium has a dissolved oxygen concentration of 0.001 mg / L to 0.5 mg / L, preferably from 0.005 mg / L to 0, 3 mg / L, more preferably from 0.01 mg / L to 0.2 mg / L.

2. The method according to claim 1, characterized in that the bang specimen

is selected from the genera Achromobacter, Acidithiobacillus, Acidovorax,

Alcaligenes, Anabena, Ancyclobacter, Aquifex, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Cupriavidus, Derxia, Helicobacter, Herbaspirillum, Hydrogenobacter, Hydrogenobaculum, Hydrogenophaga,, Hydrogenophilus, Hydrogenothermus,

Hydrogenovibrio, Ideonella sp. 01, Kyrpidia, Metallosphaera, Mycobacterium, Nocardia, Oligotropha, Paracoccus, Pelomonas, Polaromonas, Pseudomonas, Pseudonocardia, Rhizobium, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Seliberia, Streptomyces, Thiocapsa, Variovorax, Xanthobacter, Wautersia, with Cupriavidus being particularly preferred.

3. The method according to any one of the preceding claims, characterized

in that the gas H _2, C0 ₂ and 0 ₂ contains containing synthesis gas.

4. The method according to claim 3, characterized in that in the synthesis gas

contained carbon at least 50 wt .-%, preferably at least 70 wt .-%, particularly preferably at least 90 wt .-% of the carbon of all carbon sources, which are the explosive gas bacterium by the gas containing H ₂ , C0 ₂ and 0 ₂ , makes up ,

5. The method according to any one of claims 1 -4, characterized in that the C0 ₂ at least 50 wt .-%, preferably at least 70 wt .-%, particularly preferably at least 90 wt .-% of all carbon sources, the explosive gas bacterium for

Be available.

6. The method according to any one of the preceding claims, characterized

characterized in that the gas containing H ₂ , C0 ₂ and 0 ₂ contains generator gas.

7. The method according to any one of the preceding claims, characterized

characterized in that it comprises the method steps

A) Increase pop-up gas bacteria in a medium up to a cell density X.

Cells / liter and

B) culturing the oxyhydrogen bacteria in a medium, wherein the medium is brought into contact with a gas containing H ₂ , C0 ₂ and O ₂ , and the medium has a dissolved oxygen concentration of 0.001 mg / L to 0.5 mg / L, preferably 0.005 mg / L to 0.3 mg / L, more preferably from 0.01 mg / L to 0.2 mg / L.

8. The method according to claim 7, characterized in that in method step B) the cell density does not exceed 2 X cells / ml.

9. The method according to claim 7 or 8, characterized in that the

Bacteriostatic bacteria in process step B) synthesize target product.

0. The method according to claim 9, characterized in that the target product is selected from substances whose formation takes place via the intermediate acetyl-coenzyme A; Such target products are, for example, fatty acids, fatty acid esters, fatty alcohols, fatty aldehydes and substituted derivatives of the abovementioned compounds

1. The method according to claim 9, characterized in that the target product is selected from substances whose formation on metabolites of the citric acid cycle, such as. B. succinate occurs.

2. The method according to claim 9, characterized in that the target product is selected from substances comprising three or four carbon atoms, in particular butanol, butene, propene, acetone, 2-hydroxyisobutyric acid and 2-propanol.

13. The method according to claim 9, characterized in that the oxyhydrogen bacteria in step B) synthesize target product whose formation via the intermediate 3-hydroxyalkanyl-CoA, in particular 3-hydroxybutyryl-coenzyme A, takes place. 14. The method according to at least one of claims 7 to 9, characterized in that the oxyhydrogen bacteria in step B) synthesize 2-hydroxyisobutyric acid and the oxyhydrogen bacteria were genetically engineered such that they have a in

Compared to their wild-type have increased activity of the enzyme egg, which catalyzes the conversion of 3-hydroxybutyryl-coenzyme A to 2-hydroxyisobutyryl-coenzyme A.

15. The method according to claim 14, characterized in that the oxyhydrogen bacteria have been genetically engineered such that they have a compared to their wild type increased activity of an enzyme E ₂ , which catalyzes the conversion of acetoacetyl-coenzyme A to 3-hydroxybutyryl-coenzyme A. ,

16. The method according to any one of claims 7 to 15, characterized in that it comprises the method step

C) Purification of the target product

includes.