EP2788494A1 - Biotechnological production of 3-hydroxyisobutyric acid - Google Patents

Abstract

The invention relates to a method having the steps of a) providing isobutyric acid, b) bringing the isobutyric acid into contact with the combination of isobutyrate kinase and phosphotransisobutyrylase and/or isobutyryl-coenzyme A-synthetase/ligase and/or isobutyrate-coenzyme A-transferase, c) bringing the product of step a) into contact with isobutyryl-coenzyme A-dehydrogenase, d) bringing the product of step b) into contact with methacrylyl-coenzyme A-hydratase, and e) hydrolyzing the product of step d) with the formation of 3-hydroxyisobutyric acid. At least one of the enzymes is used in the form of a cell which comprises an activity of 3-hydroxyisobutyric acid dehydrogenase or a variant thereof, said activity being reduced with respect to the wild type of the cell. The invention also relates to a cell which comprises at least one enzyme of the group comprising isobutyryl-coenzyme A-synthetase/ligase, isobutyrate-coenzyme A-transferase, isobutyrate-kinase, phosphotransisobutyrylase, isobutyryl-coenzyme A-dehydrogenase, methacrylyl-coenzyme A-hydratase, and 3-hydroxisobutyryl-coenzyme A-hydrolase and which has an activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof, said activity being reduced with respect to the wild type of the cell. Additionally, the cell preferably has a monooxygenase, even more preferably a monooxygenase of the AlkBGT type or a variant thereof. The invention also relates to the use of such a cell for producing 3-hydroxyisobutyric acid

Description

 Biotechnological production

 of 3-hydroxyisobutyric acid

The invention relates to a process comprising the steps of a) providing isobutyric acid, b) contacting isobutyric acid with the combination of isobutyrate kinase and phosphotransisobutyrylase and / or isobutyryl-coenzyme A synthetase / ligase and / or isobutyrate-coenzyme A transferase, c ) Contacting the product of step b) with isobutyryl-coenzyme A dehydrogenase, d) contacting the product of step c) with methacrylyl-coenzyme A hydrate, and e) hydrolysing the product of step d) to give 3-hydroxyisobutyric acid, wherein at least one of the enzymes is provided in the form of a cell comprising a reduced activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof, a cell comprising at least one enzyme selected from the group consisting of isobutyryl-coenzyme A synthetase / ligase , Isobutyrate-coenzyme A-transferase, isobutyrate kinase, phosphotransisobutyrylase, isobutyryl-coenzyme A dehydrogenase, methacryl yl coenzyme A hydratase and 3-Hydroxisobutyryl-coenzyme A hydrolase and a reduced compared to their wild-type activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof, wherein the cell preferably additionally a monooxygenase, more preferably an alkane hydroxylase of the AlkBGT type or a variant thereof, and the use of such a cell for the preparation of 3-hydroxyisobutyric acid.

Methacrylic acid is one of the most important industrially manufactured chemicals. In the form of its monomeric methyl ester, it is required for the production of polymethylmethacrylate as a polymerization educt, which is known to the public under the trade name Plexiglas and indispensable for a variety of applications. Examples of the use of polymethacrylate include dentistry, where it is used for prostheses, the automotive industry, in which it is used for turn signal and taillight glasses, the optics, especially as a material for contact lenses and spectacle lenses, the building industry, where it is polymer concrete as well is used as a two-component adhesive, the textile industry as Component of polyacrylic fibers and in the household as a material for items such as bowls and cutlery.

Traditionally, methacrylic acid is produced from fossil fuels such as oil. For example, isobutylene and tertiary butanol may be converted to methacrylein, which is subsequently further oxidized to methacrylate (William Bauer, Jr. "Methacrylic Acid and Derivatives" in Ullmann's Encyclopedia of Industrial Chemistry 2002, Wiley-VCH, Weinheim). Alternatively, amide sulfates of methacrylic acid produced from corresponding 2-hydroxynitriles can be hydrolyzed to form methacrylic acid. However, the industrial production of methacrylic acid using such methods not only depends on a continuous supply of fossil reactants, but is done by consuming significant amounts of aggressive, environmentally harmful chemicals. Thus, the production of 1 kg of methacrylic acid by hydrolysis of amide sulfates of methacrylic acid requires 1.6 kg of sulfuric acid.

In order to overcome the dependence on fossil fuels as energy sources and educts for industrial syntheses, a variety of efforts are currently being undertaken, which aim to produce industrially demanded fine chemicals biotechnologically based on renewable raw materials. In the case of methacrylic acid or of methyl methacrylate, a biotechnological synthesis route via 3-hydroxyisobutyric acid, which can be easily dehydrated chemically or enzymatically to form methacrylic acid (Wlliam Bauer, Jr., "Methacrylic Acid and Derivatives" in Ullmann's Encyclopedia of Industrial Chemistry, 2002) , Wley-VCH, Weinheim). The prior art teaches the preparation of 3-hydroxyisobutyric acid from isobutyric acid using the Wld type isolates of bacteria and yeasts (Hasegawa et al., 1981, Hasegawa et al., 1982, WO 2007/141208 A2 and WO 2008/19738 A1). , As a substrate for the production of 3-hydroxyisobutyric acid by suitable strains, for example by Candida rugosa, isobutyric acid is used exclusively. The above-mentioned patent applications, WO 2007/141208 A2 and WO 2008/119738 A1, describe genetically engineered cell and methods for the production of 3-hydroxyisobutyric acid from carbohydrates, glycerol, carbon dioxide, methanol, L-valine, L-glutamate, CO, synthesis gas , Methane, etc. and their further chemical conversion to methacrylic acid or methacrylic acid esters. However, the production of 3-hydroxyisobutyric acid via the biotechnological methods described above is currently not economical. A major obstacle here is the high raw material costs for isobutyric acid with at the same time partly low yield in the implementation or use of suitable microorganisms.

Against this background, the object of the present invention is to develop an improved process for the biotechnological production of 3-hydroxyisobutyric acid, which is superior to the processes described in the prior art with regard to yield, purity and resource requirements.

Furthermore, the object underlying the present invention is to develop a biotechnological process for the preparation of 3-hydroxyisobutyric acid starting from unsubstituted, in particular non-heteroatom-containing alkanes. Furthermore, the present invention is based on the object to develop a biotechnological process for the production of 3-hydroxyisobutyric acid starting from renewable raw materials and / or without the use of or with less use of harmful starting materials, intermediates, catalysts or by-products.

These and other objects are achieved by the subject matter of the present application and in particular also by the subject of the appended independent claims, wherein embodiments result from the subclaims.

According to the invention the object is achieved in a first aspect by a process comprising the following steps: a) providing isobutyric acid, b) contacting isobutyric acid with the combination of isobutyrate kinase and phosphotransisobutyrylase and / or Isobutyryl-coenzyme A synthetase / ligase and / or isobutyrate-coenzyme A-transferase, c) contacting the product from step b) with isobutyryl-coenzyme A dehydrogenase, d) contacting the product from step c) with methacrylyl-coenzyme A- Hydratase, and e) hydrolysis of the product of step d) to form 3-hydroxyisobutyric acid, wherein at least one of the enzymes used in steps b), c) and d) is selected from the group comprising isobutyrate kinase, phosphotransisobutyrylase, isobutyryl coenzyme A Synthetase / ligase and isobutyrate-coenzyme A transferase, preferably all, enzymes in the form of a cell is used, which has a wild-type compared to reduced activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof.

In a first embodiment of the first aspect, the isobutyric acid is formed by contacting isobutane with a monooxygenase, preferably an alkane hydroxylase, more preferably one of the AlkBGT type or a variant thereof.

In a second embodiment of the first aspect, which is also an embodiment of the first embodiment, the hydrolysis in step e) is achieved by contacting the product of step d) with a 3-hydroxysobutyryl-coenzyme A hydrolase.

In a third embodiment of the first aspect, which is also an embodiment of the first to second embodiments, the cell has both the isobutyryl-coenzyme A dehydrogenase in step c) and the methacrylyl coenzyme A hydratase in step d) as well Combination of isobutyrate kinase and phosphotransisobutyrylase and / or isobutyryl-coenzyme A synthetase / ligase and / or Isobutyrate coenzyme A transferase on.

In a fourth embodiment of the first aspect, which is also an embodiment of the first to third embodiments, the cell additionally comprises an alkane hydroxylase, preferably one of the AlkBGT type or a variant thereof.

In a fifth embodiment of the first aspect, which is also an embodiment of the first to fourth embodiments, the 3-hydroxyisobutyric acid dehydrogenase is XP_50491 1.1 or a variant thereof.

According to the invention, the object is achieved in a second aspect by a cell which comprises at least one enzyme from the group comprising isobutyryl-coenzyme A synthetase / ligase, isobutyrate-coenzyme A-transferase, isobutyrate kinase, phosphotransisobutyrylase, isobutyryl-coenzyme A dehydrogenase, Methacrylyl coenzyme A hydratase and 3-Hydroxisobutyryl- coenzyme A hydrolase and a reduced compared to their wild type activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof.

In a first embodiment of the second aspect, in addition to an isobutyryl-coenzyme A dehydrogenase and in addition to a methacrylyl coenzyme A hydratase, the cell comprises the combination of isobutyrate kinase and phosphotransisobutyrylase and / or isobutyryl-coenzyme A synthetase / ligase and / or isobutyrate-coenzyme A-transferase. preferably further comprises a 3-hydroxyisobutyryl-CoA-hydrolase. on. In a second embodiment of the second aspect, which is also an embodiment of the first embodiment, the cell further comprises an alkane hydroxylase, preferably one of the AlkBGT type or a variant thereof.

In a third embodiment of the second aspect, which is also an embodiment of the first embodiment, is the 3-hydroxyisobutyric acid dehydrogenase and YaliOF02607g (XP_504911) or a variant thereof. In a third aspect, the problem underlying the invention is solved by the use of the cell according to any one of claims 7 to 10 for the preparation of 3-hydroxyisobutyric acid.

In one embodiment of the third aspect, the 3-hydroxyisobutyric acid dehydrogenase is XP_50491 1.1 or a variant thereof.

In a further embodiment of the first, second or third aspect, the cell is a bacterial or lower eukaryotic cell. In a further embodiment of the first, second or third aspect, the cell is a yeast cell from the group of genera comprising Yarrowia, Candida, Saccharomyces, Schizosaccharomyces and Pichia, and is preferably Yarrowia lipolytica. In a fourth aspect, the problem underlying the invention is solved by a reaction mixture comprising the cell according to the second aspect as well as isobutane or isobutyric acid.

The inventors of the present invention have surprisingly found that inactivating a gene encoding an enzyme identified as 3-hydroxyisobutyric acid dehydrogenase in an microorganism results in an increased yield of 3-hydroxyisobutyric acid. The inventors have furthermore surprisingly found that it is possible to produce 3-hydroxyisobutyric acid biotechnologically starting from alkane educts, in particular isobutane. The execution of the method according to the invention first requires the preparation of isobutyric acid. First, there is the possibility to commercially available isobutyric acid abest the possibility to produce isobutyric acid using isolated enzymes or whole organisms with suitable catalytic ability starting from other starting materials, for example by culturing an organism which naturally produces isobutyric acid. In a preferred embodiment, the isobutyric acid is prepared by contacting isobutane or another suitable alkane precursor with a suitable monooxygenase, preferably alkane hydroxylase, which in a particularly preferred embodiment is an alkyd hydroxylase of the AlkBGT type or a variant thereof. In a preferred embodiment, the term "alkane hydroxylase" as used herein means an oxidoreductase capable of oxidizing saturated hydrocarbons, particularly isobutane or 3-hydroxyisobutane, to the carboxylic acid, preferably at a terminal carbon atom Technique describes a number of suitable microorganisms and enzymes: Patel et al (Journal of Applied Biochemistry, 1983, 5 (1-2), 107-120) describe 16 new strains of bacteria capable of producing gaseous alkanes Grant et al. (2011) describe the oxidation of alkanes to corresponding acids by means of the alkB-alkane hydroxylase (Grant, C, Woodley, J & Baganz, et al , F 2011, 'Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli', Enzyme and Mic Robial Technology, vol 48, no. 6-7, pp. 480-486).

On the other hand, it is possible to prepare isobutyric acid by suitable strains of microorganisms naturally or genetically modified with metabolic pathways involving the production of isobutyric acid when fed with suitable carbon sources, for example glucose. Exemplary microorganisms include e.g. B. Yarrowia lipolytica, Candida rugosa, Hanseniaspora valbyensis, Hansenula anomala, Trichosporon aculeatum, Trichosporon fennicum, Endomyces reessii, Geotrichum loubieri, Micrococcus flavus, Micrococcus luteus, Micrococcus lysodeikticum, Candida parapsilosis, Pichia membranaefaciens, Torulopsis Candida, Coccidioides posadasii, Coccidioides immitis, Verticillium dahliae, zeae, Gibberella, Thielavia terrestris, Metarhizium acridum oryzae Magnaporthe, Sordaria macrospora, Metarhizium nisopliae, Ajellomyces dermatitidis, Chaetomium globosum, Paracoccidioides brasiliensis, Nectria haematococca, Neurospora tetrasperma, Chaetomium thermophilum and Neurospora crassa.

In a preferred embodiment, the isobutyric acid is provided by oxidation of isobutane by the oxidoreductase AlkB from the alkBGT system from Pseudomonas putida or a variant thereof. AlkB represents an oxidoreductase from the alkBGT system from Pseudomonas putida, which is known for its alkane hydroxylase activity. This is dependent on two other polypeptides, AlkG and AlkT. AlkT is characterized as FAD-dependent rubredoxin reductase, which transfers electrons from NADH to AlkG. AlkG is a rubredoxin, an iron-containing redox protein that acts as a direct electron donor for AlkB. In a preferred embodiment, the term "AlkBGT-type alkane hydrahydroylase" as used herein is understood to mean a membrane-bound alkane monooxidase In a further preferred embodiment, a polypeptide having a sequence homology of increasingly preferred is the same term "alkBGT-type alkane hydrohylase" at least 75, 80, 85, 90, 92, 94, 96, 98 or 99% to the sequence of the AlkB of Pseudomonas putida Gpo1 (database code: CAB54050.1) understood. In a further preferred embodiment, the term is understood to mean a cytochrome-independent monooxygenase. In a further preferred embodiment, the term "alkBGT-type alkanohydrohylase" is understood to mean a cytochrome-independent monooxygenase which uses at least one rubredoxin or homologue as electron donor In a particularly preferred embodiment, the term "membrane-active, cytochrome-independent alkanoneoxygenase" is used with more preferably at least 60, 70, 80, 80, 85, 90, 92, 94, 96, 98 or 99% to the sequence of the AlkB of Pseudomonas putida Gpo1, which as electron donor at least AlkG (CAB54052.1), but preferably the Combination of AlkG with the reductase AlkT (CAB54063.1), where alkG and / or alkT may also be a homologue of the respective polypeptide The term "sequence" as used herein may refer to the amino acid sequence of a polypeptide and / or the nucleic acid sequence coding therefor. In another preferred embodiment, it is an "alkB-type oxidoreductase" as herein used to produce a cytochrome-independent oxidoreductase, ie an oxidoreductase that does not contain cytochrome as a cofactor.

It is possible to contact for this purpose purified components of the AlkBGT system with isobutane, in particular AlkB. In a preferred embodiment, isobutane is contacted with a AlkBGT-containing whole-cell catalyst, expressed in a bevorzugtesten embodiment with a recombinant £ .co // ^'strain of heterologous AlkBGT. The teachings of the present invention may be made not only by using the exact amino acid or nucleic acid sequences of the biological macromolecules described herein, but also by using variants of such macromolecules obtained by deletion, addition or substitution of one or more amino acids or nucleic acids can. In a preferred embodiment, the term "variant" of a nucleic acid sequence or amino acid sequence, hereinafter synonymous and interchangeable with the term "homologue" as used herein, means another nucleic acid or amino acid sequence that is unique with respect to the corresponding original wild-type nucleic acid sequence. or amino acid sequence has a homology, here used as identity, of 70, 75, 80, 85, 90, 92, 94, 96, 98, 99% or more percent, preferably other than the catalytically active center forming amino acids or for the structure or folding essential amino acids are deleted or substituted or the latter are only conservatively substituted, for example, a glutamate instead of an aspartate or a leucine instead of a valine. The prior art describes algorithms that can be used to calculate the extent of homology of two sequences, e.g. B. Arthur Lesk (2008), Introduction to bioinformatics, 3 ^rd edition. In a further preferred embodiment of the present invention, the variant of an amino acid or nucleic acid sequence, preferably in addition to the sequence homology mentioned above, has essentially the same enzymatic activity of the wild-type molecule or the original molecule. For example, a variant of a polypeptide enzymatically active as protease has the same or substantially the same proteolytic activity as the polypeptide enzyme, ie the ability to catalyze the hydrolysis of a peptide bond. In a particular embodiment, the term "substantially the same enzymatic activity "means an activity with respect to the substrates of the wild-type polypeptide that is significantly above the background activity and / or that differs by less than 3, more preferably 2, more preferably an order of magnitude from the K _M and / or k _cat values In a further preferred embodiment, the term "variant" of a nucleic acid or amino acid sequence comprises at least one active part or fragment of the nucleic acid or amino acid sequence. In a further preferred embodiment, the term "active part" as used herein means an amino acid sequence or nucleic acid sequence which is less than full length of the amino acid sequence or less than full length of the amino acid sequence encoding the amino acid sequence or amino acid sequence the encoded amino acid sequence of lesser length than the Wldtype amino acid sequence has substantially the same enzymatic activity as the Wldtype polypeptide or a variant thereof, for example as alcohol dehydrogenase, monooxygenase or transaminase In a particular embodiment, the term "variant" of a nucleic acid comprises a nucleic acid whose complementary strand, preferably under stringent conditions, binds to the wild-type nucleic acid. The stringency of the hybridization reaction is readily determinable by those skilled in the art and generally depends on the length of the probe, the temperatures of washing and the salt concentration. In general, longer probes require higher temperatures for hybridization, whereas shorter probes manage at low temperatures. Whether hybridization takes place generally depends on the ability of denatured DNA to anneal to complementary strands present in their environment, below the melting temperature. The stringency of hybridization reaction and corresponding conditions are more fully described in Ausubel et al. 1995 described. In a preferred embodiment, the term "variant" of a nucleic acid as used herein includes any nucleic acid sequence encoding the same amino acid sequence as the original nucleic acid or a variant of that amino acid sequence in the context of degeneracy of the genetic code.

After providing isobutyric acid, this invention is contacted with an enzyme or enzyme system capable of converting it to isobutyryl-CoA. It is possible to contact isobutyric acid with the combination of isobutyrate kinase and phosphorotransisobutyrylase. By the term "isobutyrate kinase" as used herein is meant an enzyme capable of adding isobutyric acid with hydrolysis of ATP phosphorylate. In a particularly preferred embodiment, the term "phosphotransisobutyrylase" as used herein means an enzyme which catalyzes the reaction of the phosphorylated butyric acid with consumption of coenzyme A to form isobutyryl-CoA .. Suitable enzymes can be taken from the prior art, for example : NP_348286.1, YP_001311072.1, YP_001311673.1, YP_003845108.1, CCC57671.1, ZP_02993103.1, YP_001255907.1, YP_001788766.1, YP_001783065.1, ZP_02613551.1, ZP_05129586.1, NP_783068.1, ZP_02616584 .1, YP_001392779.1, ZP_02642313.1, YP_697036.1, NP_563263.1, YP_699607.1, ZP_05129585.1, ZP_05394270.1, ZP_04821992.1, YP_001086582.1, YP_001884532.1, YP_001919732.1, YP_001513941.1 , YP_001322041.1, NP_349675.1, YP_001307350.1, ZP_02074622.1, AAA75487.1, ZP_05979314.1, ZP_02950703.1, YP_003935519.1, YP_001126403.1, ZP_03147541.1, BAD11094.1, ZP_08532470.1, CBK83142 .1, ZP_02027123.1, ZP_02206646.1, ZP_03226899.1, ZP_05791023.1, YP_002950277.1, YP_003825 215.1, ZP_0409145, ZP_0363, 015, ZP_0403, 0351, ZP_06425397.1, ZP_06425397.1 ZP_04187800.1, ZP_04302360.1, ZP_04098303.1, ZP_00394509.1, YP_085496.1, NP_846616.1, ZP_04313571.1, YP_896508.1, NP_980529.1, ZP_04170515.1, YP_001646798.1, ZP_00240356.1, ZP_04269443. 1, ZP_04291055.1, ZP_04176201.1, ZP_01860365.1, ZP_03237173.1, YP_001471381.1, ZP_08211979.1, ZP_04208847.1, YP_004820818.1, ZP_04103879.1, YP_003666354.1, NP_833876.1, ZP_04285820.1, ZP_04086212.1, ZP_04147476.1, NP_623752.1, ZP_03231885.1, ZP_04066826.1, ZP_08211238.1, ZP_07709086.1, YP_002315321.1, YP_002447739.1, ZP_00741048.1, ZP_04073820.1, YP_003988617.1, YP_002368967. 1, YP_148233.1, YP_003251443.1, AEN87678.1, YP_003564885.1, YP_004660977.1, YP_004587379.1, NP_349675.1, AAA75487.1, YP_001788766.1, YP_001255907.1, ZP_02993103.1, YP_001783065.1, ZP_02613551.1, ZP_02616584.1, YP_001392779.1, ZP_05394270 .1, NP_783068.1, CCC57671.1, YP_697036.1, NP_563263.1, YP_699607.1, ZP_02642313.1, YP_001919732.1, ZP_07525976.1, YP_001884532.1, ZP_04821992.1, ZP_03292064.1, ZP_05129586.1 , YP_001307350.1, ZP_02950703.1, ZP_05129585.1, YP_001086582.1, YP_001311072.1, YP_001513941.1, YP_003845108.1, YP_001322041.1, ZP_06425397.1, YP_001311673.1, YP_003935519.1, NP_348286.1, ZP_02027123 .1, ZP_08532470.1, YP_001664465.1, YP_002950277.1, YP_004820818.1, ZP_07709086.1, ZP_08211979.1, ZP_02211576.1, ZP_05979314.1, ZP_02074622.1, YP_003677577.1, CBK83142.1, YP_003477715.1, Z P_02206646.1, BAD11094.1, ZP_01860365.1, ZP_03226899.1

NP_623752.1, YP_003988617.1, ZP_08005605.1, YP_003599607.1, YP_003831638.1

YP_003564885.1, YP_004587379.1, AEN87678.1, YP_001376086.1, YP_004819378.1

ZP_04152833.1, YP_001126403.1, ZP_03147541.1, ZP_04218902.1, ZP_08609344.1 ZP_00240356.1, YP_002368967.1, ZP_04147476.1, ZP_04176201.1, ZP_04269443.1

YP_003666354.1, ZP_04073820.1, ZP_04103879.1, NP_833876.1, ZP_04285820.1

ZP_04187800.1, ZP_04313571.1, ZP_03231885.1, ZP_03237173.1, ZP_04086212.1

ZP_04302360.1, ZP_08094177.1, NP_980529.1, ZP_04098303.1, YP_085496.1

ZP_04170515.1, YP_002447739.1, BAD11089.1, ZP_00394509.1, YP_001646798.1 YP_002315321.1, NP_846616.1, ZP_04066826.1, YP_896508.1, YP_003866727.1 ZP_00741048.1, YP_001487375.1, ZP_06875892.1, ZP_04291055.1

All database codes used in this application for indicating sequences of the prior art are from the database NCBI (National Center for Biotechnology Information, access date: 19.10.2011), more precisely using the available on 19.10.2011 online release.

Alternatively, isobutyryl-CoA can be obtained using isobutyryl-coenzyme A synthetase or ligase from isobutyric acid. In a preferred embodiment, the term "isobutyryl-coenzyme A ligase" as used herein refers to an enzyme which catalyzes the conversion of isobutyric acid to isobutyryl-CoA consuming coenzyme A and nucleotide triphosphate, in a particularly preferred embodiment For example, the term "isobutyryl-coenzyme A synthetase" as used herein means an isobutyryl-coenzyme A ligase wherein the NTP hydrolyzed in the course of the reaction is ATP. Suitable enzymes can be found by those skilled in the art, for example: NP_579516.1, NP_125992.1, YP_004423263.1, YP_004070968.1, YP_182878.1, YP_002306709.1, YP_002959654.1, YP_004762301.1, YP_002581616.1, YP_002994502. 1, YP_004623157.1, NP_143577.1, YP_002307387.1, NP_579566.1, YP_004623106.1, YP_004763660.1, YP_002583149.1, YP_002959108.1, YP_002993622.1, YP_001736558.1, YP_003649375.1, YP_004423314.1, ZP_04874839.1, YP_183356.1, ZP_04874991.1, YP_001041242.1, YP_003860266.1, NP_126044.1, NP_143628.1, YP_003669211.1, YP_002428622.1, YP_001013511.1, YP_004175933.1, YP_004174284.1, YP_001012369.1, YP_004781520.1, ΥΡ_004071372.1, ΝΡ_769799.1, ΥΡ_429257.1, CAJ70793.1, ΖΡ_08257475.1, ΥΡ_930006.1, ΝΡ_618478.1, ΖΡ_08667208.1, ΥΡ_001239140.1, ΥΡ_004438172.1, ΥΡ_003400029. 1, ΥΡ_001206872.1, ΝΡ_070039.1, ΥΡ_003727548.1, ΥΡ_004625200.1, ΖΡ_08422040.1, ΥΡ_460839.1, ΖΡ_08631067.1, ΖΡ_02883796.1, ΥΡ_002953381.1, ΖΡ_08110577.1, ΥΡ_003542795.1, ΖΡ_08905433.1, ΖΡ_06908198.1, ΥΡ_001581540.1, ΝΡ_632517.1, ΥΡ_004519194.1, ΥΡ_004515899.1, ΥΡ_004120624.1, ΖΡ_07293826.1, CAJ73927.1, ΖΡ_07331643.1, ΥΡ_001278815.1, ΖΡ_08840735.1, ΥΡ_002289530.1, ΥΡ_001637486. 1, ΥΡ_004342727.1, ΖΡ_07026137.1, ΖΡ_08112481.1, ΥΡ_003487360.1, ΥΡ_001540910.1, ΖΡ_07946223.1, ΥΡ_685524.1, ΥΡ_004812635.1, ΖΡ_08677213.1, ΖΡ_08803651.1, ADI05837.1, ΥΡ_874976.1, ΥΡ_002465105.1, ΥΡ_003355503.1, ΖΡ_07026765.1, ΥΡ_001430231.1, ΥΡ_004893707.1, ΥΡ_003766745.1, ΥΡ_004627663.1, ΥΡ_003649567.1, ΖΡ_073076 86.1, ΥΡ_001546514.1, ΥΡ_686303.1, ΥΡ_002992636.1, ΥΡ_004516207.1, ΥΡ_001154008.1, ΥΡ_004338243.1, ΥΡ_003357973.1

Finally, isobutyryl-CoA can be prepared from isobutyric acid by means of isobutyrate-coenzyme A transferase. In a preferred embodiment, the term "isobutyrate-coenzyme A transferase" as used herein is an enzyme that catalyzes the production of isobutyryl-CoA from isobutyric acid to transfer coenzyme A from a donor-acting acyl-CoA Suitable enzymes can be found by the person skilled in the art, for example: NP_149326.1, AAB53234.1, YP_001310904.1, AAD54947.1, AAP42564.1, CAQ57984.1, YP_001886322.1, NP_622378.1, ZP_08693244.1, ZP_07926619 .1, ZP_08555875.1, ZP_04390377.1, ZP_01867058.1, ZP_04573915.1, ZP_07913714.1, ZP_07923474.1, ZP_08691337.1, ZP_08600063.1, ZP_02692961.1, ZP_08582386.1, ZP_00144733.1, ZP_05815087.1 , ZP_06524353.1, ZP_07952599.1, YP_004254308.1, YP_003039857.1, YP_003828410.1, ZP_06175535.1, NP_602657.1, ZP_06748826.1, ZP_06749807.1, ZP_04970682.1, AD077683.1, AA018070.1, ZP_05887867 .1, AEJ99145.1, EGB63075.1, NP_931005.1, YP_003968227.1, ZP_08327315.1, ZP_06025832.1, YP_003260518.1, YP_003016746.1, YP_001452140.1, EGW68380.1, ZP_02424926.1, ZP_03828051.1, EGB72667.1, EFW53769.1, ZP_03001766.1, ZP_05402063.1, YP_049390.1, ZP_08690265.1, YP_003936148.1, YP_002382110.1, NP_416725. 1, YP_003941041.1, YP_001744415.1, YP_003296165.1, ZP_07151665.1, ZP_05275011.1, YP_541501.1, YP_002335226.1, YP_001089188.1, ZP_04054428.1 YP_001784143.1, YP_001306376.1, ZP_07820010.1, ZP_03065638.1, YP_004441693.1 NP_905281.1, YP_001321984.1, ZP_06636026.1, YP_003296060.1, ZP_07903700.1 YP_004509865.1, ZP_08626421.1, YP_002933615.1, YP_002771575.1, ZP_06715088.1 ZP_01548307.1, ZP_06982396 .1, ZP_08643079.1, YP_002497558.1, YP_001513889.1 NP_438933.1, YP_248482.1, YP_531878.1, YP_002746872.1, YP_002744076.1 ZP_01792337.1, YP_002940319.1, YP_001471175.1, ZP_01220381.1

YP_002123783.1, NP_149327.1, CAQ57985.1, YP_001886321.1, AAD54948.1 YP_001310905.1, AAP42565.1, ZP_05092257.1, NP_622379.1, ZP_08555876.1 ZP_02692960.1, YP_002335225.1, YP_001306375.1, ZP_08693245.1, YP_001513888.1 YP_001321983.1, ZP_07926620.1, ZP_02424916.1, YP_001409735.1, ZP_06983458.1 YP_003828409.1, YP_004707898.1, YP_003936149.1, ZP_07577382.1, YP_001089189.1 YP_001471174.1, ZP_05272742 .1, YP_002940318.1, ZP_05402064.1, YP_004254317.1 YP_003968226.1, CBK81879.1, ZP_07819217.1, YP_001740168.1, YP_049389.1 ZP_08709179.1, YP_001918401.1, YP_003016745.1, NP_905290.1, YP_004509856 . ZP_03828050.1, YP_426559.1, YP_001929287.1, YP_001568118.1, ZP_04390271.1 ZP_07913715.1, ZP_07820042.1, YP_003260519.1, ZP_08691338.1, ZP_01548308.1 ZP_08327314.1, ZP_04970681.1, ZP_04054413. 1, ZP_08690266.1, ZP_05887868.1 ZP_06748825.1, YP_001918068.1, ZP_08731713.1, NP_602656.1, ZP_06524354.1 ZP_01867057.1, ZP_04573916.1, ZP_05815086.1, ZP_06025831.1, ZP_00144734.1 ZP_08600062.1 , YP_001784144.1, EC ZP_01220382.1, YP_597732.1, YP_003945474.1, YP_004219013.1, YP_002562694.1 YP_001788168.1, YP_002285170.1, NP_268527.1, YP_001392125.1, CBZ04738.1 ZP_06921170, ZP_06175534.1, AD077682.1 .1, ZP_07461446.1, YP_531879.1, YP_002805353.1, YP_003452384.1 YP_001255346.1, YP_001385091.1, YP_878775.1, EGC06741.1, NP_663914.1 YP_059486.1, YP_002382109.1, YP_001884900.1, ZP_07307813 .1, ZP_02621896.1 ZP_07903699.1, ZP_08729700.1, YP_001309762.1, YP_001768507.1, YP_602949.1 EGL48602.1, XP_501388.1, XP_002841479.1, XP_002481641.1, XP_002147496.1 XP_001879757.1, XP_661332. 1, XP_003295652.1, XP_001936644.1, AAK40365.1 XP_001802255.1, XP_002383405.1, XP_001390976.1, XP_001212457.1, EGP83670.1 XP_001227675.1, XP_003176313.1, XP_001268294.1, XP_751117.1, EGF82407. 1 XP_001258382.1, EFW13413.1, XP_003065510.1, EGD93452.1, EEH22314.1 XP_001247983.1, EGE09225.1, XP_003022194.1, CBX98353.1, XP_003014185.1 XP_002564448.1, XP_003239146.1, EGX51025.1, EGO01287.1, XP_002792936.1 EFQ30503.1, XP_001838038.1, XP_002847737.1, EGO55093.1, XP_957979.1 XP_001547410.1, EGR48848.1, XP_003038014.1, EER44122.1, EFY99528.1, EGC42434.1 EEH09119.1, EFY92418.1, EGS22244.1, XP_002629107.1, XP_002548743.1 XP_001904655 .1, EFX03923.1, XP_716276.1, XP_002422356.1, XP_388121.1 XP_001586993.1, EGX96527.1, XP_368380.1, NP_595848.1, XP_003345762.1, XP_759662.1 XP_003001963.1, EGG06029.1, XP_002175297 .1, XP_001386078.2, XP_003040746.1 XP_459426.2, EDK36000.2, CBQ73919.1, EGU85384.1, XP_001525161.1, XP_001731221.1 XP_002490853.1, XP_001486721.1

The next step in the biotechnological synthesis of 3-hydroxyisobutyric acid according to the invention comprises contacting the product from step b), the isobutyryl-CoA, with an isobutyryl-coenzyme A dehydrogenase. In a preferred embodiment, the term "isobutyryl-coenzyme A dehydrogenase" as used herein means an enzyme which catalyzes the oxidation of isobutyryl coenzyme A to methacrylyl coenzyme A to release reducing equivalents Prior art known polypeptides with the database codes XP_501919.2, EDP50227.1, XP_001267173.1, XP_751977.1, EFW17827.1, XP_001241675.1, XP_003070631.1, XP_002376988.1, EGS22147.1, XP_001271742.1, XP_002794645. 1, XP_001214528.1, XP_959931.1, EG055678.1, EEH46977.1, XP_002543210.1, XP_001401697.1, EEH07104.1, BAE59223.1, EGU83504.1, XP_002627767.1, XP_002483661.1, EFX03379.1, XP_003048205.1, EGX54111.1, XP_002150528.1, EFQ35634.1, XP_001548029.1, XP_659303.1, XP_002841387.1, XP_001791413.1, XP_001904778.1, XP_390966.1, XP_003015640.1, EGD94205.1, XP_003233609. 1, XP_003018507.1, XP_002561081.1, XP_360875.1, EFY92504.1, XP_003344493.1, CBX95087.1, EGC41158.1, EGX 92432.1, XP_003169516.1, EFZ00003.1, EGR49157.1, EGP87134.1, XP_002849756.1, XP_003302688.1, XP_001937377.1, EEH 18077.1, XP_001227538.1, XP_001586947.1, AAK63186.1, XP_001538624.1, AAQ04622 .1, EGF84480.1, EER45206.1, XP_502873.1, EGS21840.1, XP_752854.1, XP_003170973.1, CBF88712.1, XP_001264273.1, XP_003232243.1, EGD97329.1, XP_002479178.1, XP_658428.1 , XP_002844533.1, EGE05996.1, XP_001223344.1, EGX95352.1, XP_001389698.2, CAK37343.1, EGR51332.1, XP_003068884.1, XP_002146883.1, XP_001243830.1, CAP65331.1, XP_001268770.1, XP_001818195.1, EFQ34732.1, XP_001210950.1, XP_962250.1, EEH50726.1, XP_001804465.1, XP_003023750.1, EG058886.1, CBX97678.1, XP_002561648. 1, EEH15844.1, XP_389837.1, XP_363106.2, EFY97392.1, EFY90016.1, EGU81915.1, XP_002621381.1, EEQ89840.1, EGX53945.1, XP_003295397.1, XP_003011911.1, EER38173.1, XP_002792725.1, XP_003045885.1, XP_001935173.1, EGP88451.1, XP_001584930.1, XP_001836878.1, EGG04035.1, XP_003005525.1, XP_001553916.1, XP_762332.1, CBQ71452.1, EFX05387.1, XP_568632. 1, XP_001884381.1, EGO01481.1, XP_003332014.1, XP_003197572.1, XP_001731213.1, XP_003026264.1, EGF80206.1, XP_003346492.1, XP_003324899.1, EEH09831.1, XP_002582344.1, EGT41105.1, XP_002640134.1, CBJ32167.1, XP_002607968.1, EFW43327.1, XP_003099748.1, CAF95757.1, NP_491859.1, XP_002471089.1, YP_001611803.1, XP_002904727.1, EGG17601.1, EFX71478.1, CBN81547. 1, XP_002640162.1, ADY46184.1, XP_003099703.1, EFA76871.1, AAH82665.1, 2JIF_A, XP_001658431.1, CAD38535. 2, AAH13756.1, NP_001124722.1, NP_001600.1, XP_003255101.1, XP_003283361.1, CAJ81939.1, XP_002640145.1, NP_491871.1, XP_001104844.1, AAH 54428.1

The next step of the process of the invention involves the hydration of methacrylyl coenzyme A to give 3-hydroxyisobutyryl CoA by means of a methacrylyl coenzyme A hydratase. In a preferred embodiment, the term "methacrylyl-coenzyme A hydratase" as used herein means an enzyme which catalyzes the attachment of a water molecule to methacrylyl coenzyme A to form 3-hydroxyisobutyric acid Known polypeptides with the database codes XP_502475.1, XP_003067220.1, XP_001239658.1, XP_002567879.1, XP_002145078.1, XP_001259415.1, CAK97202.1, XP_001211164.1, XP_001401252.2, XP_753374.1, XP_664448.1, CBF71576.1, XP_001274572.1, XP_002340305.1, XP_002845201.1, XP_001824127.1, XP_002795848.1, XP_003304302.1, EGC44435.1, EER38804.1, EEH09966.1, XP_001936195.1, EEH50797.1, EEH 15784.1 , EG061149.1, XP_003170409.1, XP_961123.1, EGS18121.1, XP_001554659.1, XP_003230996.1, EGE07573.1, XP_002621253.1, EGE81015.1, EEQ89969.1, XP_361538.2, XP_001224889.1, XP_002381216 .1, CBX96718.1, EFY95366.1, XP_001595164.1, XP_001911969.1, EGX51365.1, EFY87978.1, EFQ29854.1, EGU81865.1 , XP_003050469.1, XP_003352189.1, XP_002839588.1, XP_003002061.1, EGD96635.1, XP_003022976.1, XP_003010670.1, EFX03685.1, EGR47862.1, XP_002584325.1, XP_387195.1, EGX94938.1, XP_002616791.1, XP_462069.2, XP_001800531.1

Finally, to release the desired product 3-hydroxyisobutyric acid, the hydrolysis of the product from step d), the 3-hydroxyisobutyryl-CoA, is required. For this, it is initially possible to expose the product to extreme pH conditions by adding acid or base, which promote hydrolysis without the action of another enzyme. In a particularly preferred embodiment, however, the hydrolysis is carried out by contacting the product from step c) with a 3-hydroxyisobutyryl-coenzyme A hydrolase. In a preferred embodiment, the term "3-hydroxyisobutyryl-coenzyme A-hydrolase" as used herein means an enzyme which hydrolyzes 3-hydroxyisobutyryl-coenzyme A to 3-hydroxyisobutyric acid and coenzyme A. As examples, the following proteins may be mentioned are: XP_504911.1, XP_003066853.1, XP_001246264.1, XP_385460.1, EFY92465.1, XP_363761.1, XP_003346508.1, EFY97405.1, EEQ90955.1, XP_002623105.1, XP_001223375.1, XP_002794385.1, XP_003046454.1, EGO58901.1, EEH47368.1, EGS21819.1, EEH18429.1, AAK07843.1, XP_002540650.1, XP_002484211.1, XP_002150057.1, EFQ36202.1, EDP49675.1, XP_750988.1, XP_003235672. 1, EGR51338.1, XP_003013762.1, XP_003019423.1, EGE05569.1, XP_003169276.1, EGD98503.1, EEH06901.1, XP_001544047.1, EGC47721.1, XP_001821058.1, EGE82172.1, XP_002842837.1, XP_002835633.1, EGX53716.1, EFX05960.1, XP_001590627.1, XP_001552254.1, XP_962266.2, EGX95378.1, XP_001392906.1, XP_003302932.1, CBF89164.1, EGU77782.1, EGP84417.1, XP_001258 223.1, XP_001268201.1, XP_002569077.1, XP_001214240.1, EER38573.1, ADD19825.1, XP_001939164.1, XP_658197.1, XP_001248657.1, XP_315590.3, XP_003047713.1, EFR21351.1, EGP83804.1, EFX82586.1, EFN83706.1, XP_001799030.1, XP_002423077.1, XP_002073789.1, XP_001987737.1, XP_002050275.1, XP_002091863.1, XP_002005175.1, XP_001974736.1, EGF81169.1, EFW47689.1, XP_002149358. 1, XP_002149354.1, CBY01415.1, NP_611373.1, CAP65353.1, XP_002569208.1, AAL39202.2, XP_002543763.1, XP_001664110.1, XP_002034506.1, XP_002565243.1, XP_003049329.1, XP_001360407.1, XP_001865614.1 According to the invention, biologically active enzymes are used in step b) to d). It may, as long as at least one of the enzymes is used in the form of a cell having a reduced their wild type activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof, as in all the invention used enzymatically active polypeptides to cells comprising enzymatically active polypeptides or whose lysates or preparations of the polypeptides in all purification stages, from the intact cell or its crude lysate to the pure polypeptide, which have the respective biologically active enzyme in endogenous or recombinant form, are preferably overexpressed. Numerous methods are known to those skilled in the art for over-expressing and isolating enzymatically-active polypeptides in suitable cells. Thus, for the expression of the polypeptides all expression systems available to the person skilled in the art can be used, for example vectors of the type pET or pGEX. Purification can be carried out by chromatographic methods, for example the affinity chromatographic purification of a tagged recombinant protein using immobilized ligands, for example a nickel ion in the case of a histidine tag, of immobilized glutathione in the case of glutathione S-transferase fused to the target protein or immobilized maltose in the case of a tag comprising maltose-binding protein. It is further known to those skilled in the art how to work out, under routine experimentation, suitable reaction conditions under which the enzyme of interest exhibits activity, preferably optimal activity. These conditions include, for example, the selection of suitable buffers, the determination and adjustment of the optimum pH, a certain salt concentration, and a certain minimum protein concentration, see, for example, Cornish-Bowden, 1995. If purified enzymes and non-living living cells are used in the process of the invention Thus, the former can be used either in soluble form or immobilized. Suitable methods are known to the person skilled in the art with which polypeptides can be immobilized covalently or noncovalently on organic or inorganic solid phases, for example by sulfhydryl coupling chemistry (eg kits from Pierce).

However, since the method of the present invention employs several enzymes having different cofactors, which may be stoichiometrically added in the case of purified polypeptides, the enzymes required for the process become particularly unique preferred embodiment in the form of a single whole-cell catalyst, ie in the form of a viable, metabolically active cell. The enzymes can be presented on the surface of the whole-cell catalyst, as described in the prior art, for example in DE 60216245. Most preferably, however, the enzymes to be regenerated by cofactors, more preferably all enzymes, are located so that their active sites are in contact with the interior of the cell, so that the required cofactors and cosubstrates are obtained from the metabolism of the cell and be redelivered.

The production of mutants of a cell which has a certain enzymatic activity, with the aim of reducing this enzymatic activity in the mutant to be obtained compared to the wild type of the cell, is known to those skilled in the art using standard techniques in the field of molecular biology, genetics and microbiology (Sambrook et ai, 1989). For example, undirected mutagenesis is possible by treating Wld-type cells with radioactive radiation followed by a step of selecting appropriate mutants by determining the enzymatic activity of isolated colonies using appropriate assays described for numerous enzymes in the art (Cornish-Bowden , 1995). Further methods comprise the insertion of deactivating point mutations, for example into the promoter or into the active center of the enzymatically active polypeptide, a likewise established method for decades (Fersht and Winter, 2008).

In a preferred embodiment, the cell used is a prokaryotic, preferably a bacterial cell. In a further preferred embodiment, it is a mammalian cell. In a further preferred embodiment, it is a lower eukaryotic cell, preferably a yeast cell. Exemplary prokaryotic cells include Escherichia, especially Escherichia coli, and strains of the genus Pseudomonas and Corynebacterium. Exemplary lower eukaryotic cells include the genera Saccharomyces, Candida, Pichia, Yarrowia, Schizosaccharomyces, especially the strains Candida tropicalis, Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica and Saccharomyces cerivisiae. The most preferred embodiment is Yarrowia lipolytica. An aspect which is essential for the teaching according to the invention is that a cell is used whose 3-hydroxyisobutyric acid dehydrogenase activity or the activity of a variant thereof is reduced. In a preferred embodiment, the term "3-hydroxyisobutyric acid dehydrogenase activity" as used herein means the activity of an enzyme that oxidizes 3-hydroxyisobutyric acid to the aldehyde, In a preferred embodiment, the 3-hydroisobutyric acid dehydrogenase an enzyme from the group comprising the polypeptides known from the prior art XP_504911.1, XP_003066853.1, XP_001246264.1, XP_385460.1, EFY92465.1, XP_363761.1, XP_003346508.1, EFY97405.1, EEQ90955. 1, XP_002623105.1, XP_001223375.1, XP_002794385.1, XP_003046454.1, EGO58901.1, EEH47368.1, EGS21819.1, EEH 18429.1, AAK07843.1, XP_002540650.1, XP_002484211.1, XP_002150057.1, EFQ36202 .1, EDP49675.1, XP_750988.1, XP_003235672.1, EGR51338.1, XP_003013762.1, XP_003019423.1, EGE05569.1, XP_003169276.1, EGD98503.1, EEH06901.1, XP_001544047.1, EGC47721.1 , XP_001821058.1, EGE82172.1, XP_002842837.1, XP_002835633.1, EFX05960.1, XP_001590627.1, XP_001552254.1, XP_ 962266.2, XP_001392906.1, XP_003302932.1, CBF89164.1, EGP84417.1, XP_001258223.1, XP_001268201.1, XP_002569077.1, XP_001214240.1, EER38573.1, XP_001939164.1, XP_658197.1, XP_001248657.1, XP_003047713.1, EGP83804.1, XP_001799030.1, EGF81169.1, XP_002149358.1, XP_002149354.1, CBY01415.1, CAP65353.1, XP_002569208.1, XP_002543763.1, XP_002565243.1, XP_003049329.1, XP_001933034. 1, XP_002551090.1, XP_001209304.1, XP_001796105.1, XP_003305815.1, CBY01417.1, XP_002144318.1, XP_003035025.1, XP_003047232.1, EDK39415.2, XP_003070936.1, XP_001878640.1, XP_001833463.1, XP_001484132.1, XP_003051032.1, XP_719244.1, XP_001796108.1, XP_456589.2, XP_001384410.2, XP_002421055.1, XP_719127.1, XP_001524095.1, XP_003336106.1, EGN96107.1, XP_003043164.1, XP_001903154. 1, XP_758336.1, XP_003051445.1, CBX93804.1, XP_002614534.1, EFW22385.1, XP_003047715.1, XP_003009661.1, XP_001903159.1, XP_002144320.1, XP_754672.2, EGG05569.1, even more preferably an enzyme from the group, the XP_50491 1.1, XP_003066853.1, XP_001246264.1, XP_385460.1, EFY92465.1, XP_363761.1, XP_003346508.1, EFY97405.1, EEQ90955.1, XP_002623105.1, XP_001223375.1, XP_002794385.1, XP_003046454.1, EGO58901.1, EEH47368.1, EGS21819.1, EEH 18429.1, AAK07843.1, XP_002540650.1, XP_002484211.1, XP_002150057.1, EFQ36202.1, EDP49675.1, XP_750988.1, XP_003235672.1, EGR51338.1 , XP_003013762.1, XP_003019423.1, EGE05569.1, XP_003169276.1, EGD98503.1, EEH06901.1, XP_001544047.1, EGC47721.1, XP_001821058.1, EGE82172.1, XP_002842837.1, XP_002835633.1, EFX05960.1, XP_001590627. 1, XP_001552254.1, XP_962266.2, XP_001392906.1, XP_003302932.1, CBF89164.1, EGP84417.1, XP_001258223.1, XP_001268201.1, XP_002569077.1, XP_001214240.1, EER38573.1, XP_001939164.1, XP_658197.1, XP_001248657.1, XP_003047713.1, XP_002551090.1, EDK39415.2, XP_001484132.1, XP_719244.1, XP_456589.2, XP_001384410.2, XP_002421055.1, XP_719127.1, XP_001524095.1, XP_002614534. 1, XP_504911.1, more preferably XP_504911.1 XP_002551090.1, EDK39415.2, XP_001484132.1, XP_719244.1, XP_456589.2, XP_001384410.2, XP_002421055.1, XP_719127.1, XP_001524095.1, XP_002614534.1 , and most preferably XP_504911.1.

In a preferred embodiment, the 3-hydroxyisobutyric acid dehydrogenase is a 3-hydroxyisobutyric acid dehydrogenase from the group comprising the polypeptides known from the prior art BAC82381.1, NP_746775.1, YP_004703920.1, YP_001670886.1, ADR61938.1, YP_001269834.1, YP_001747642.1, YP_606441.1, BAJ07617.1, YP_257885.1, EGH62730.1, ZP_06461142.1, ZP_05642104.1, YP_004351842.1, EGH86869.1, YP_002874705.1, EGH24275. 1, ZP_07777517.1, ZP_07003462.1, ZP_07264531.1, EGH72043.1, YP_233798.1, EGH66664.1, EGH33025.1, EGH77479.1, EGH54475.1, EFW86611.1, EGH13085.1, ZP_03399204.1, NP_790630.1, YP_346429.1, ZP_04590292.1, EGH46911.1, YP_004681354.1, YP_840711.1, YP_002007768.1, YP_298456.1, ZP_06495709.1, YP_001777122.1, YP_837675.1, YP_624179.1, YP_001117046. 1, YP_555519.1, YP_004360017.1, ZP_07673810.1, YP_004714189.1, YP_003607711.1, YP_557465.1, YP_001810630.1, YP_003749513.1, ZP_08140457.1, AEA83842.1, YP_001861262.1, YP_001892508.1, ZP_03264187. 1, ADY83615.1, YP_001844789.1, YP_002233766.1, ZP_02893156.1, ZP_06693498.1, YP_001705868.1, YP_001715485.1, YP_775329.1, ADX90568.1, ZP_06059137.1, ADX01770.1, ZP_05825497.1, ZP_06069662.1, YP_372791.1, YP_002254778.1, AEG71628.1, YP_001584514.1, ZP_02358230.1, ZP_03569839.1, YP_439994.1, ZP_02365286.1, YP_554218.1, YP_004473140.1, YP_001889364.1, ZP_03585594. 1, YP_110641.1, YP_003734018.1, ZP_06839562.1, YP_001583635.1, ZP_00944342.1, ZP_03573494.1, YP_001115788.1, ZP_03582471.1, YP_776268.1, YP_623275.1, ZP_04522920.1, ZP_04947734.1, ZP_02910304.1, YP_371475.1, YP_001811564.1, YP_002798276.1, YP_003910785.1, YP_004230293.1, ΖΡ_02881368.1, ΖΡ_03822695.1, ΖΡ_02891780.1, ΖΡ_06726280.1, CBJ40252.1, ΖΡ_02376988.1, ΖΡ_05359923.1, ΥΡ_002908300.1, ΥΡ_004348870.1, ΥΡ_001172440.1, ΥΡ_003747923. 1, ΥΡ_046276.1, ΝΡ_522210.1, ΥΡ_001156335.1, ΖΡ_03268101.1, ΖΡ_07236476.1, ΥΡ_001861075.1, ΖΡ_05136769.1, ΥΡ_293155.1, ΥΡ_001083192.1, ΥΡ_001970187.1, ΥΡ_002026611.1, ΥΡ_003776462.1, ΖΡ_02381313.1, ADP96674.1, EGF41785.1, ΖΡ_08410855.1, ΖΡ_05911432.1, AEL06416.1, ΖΡ_01989307.1, ΖΡ_06178628.1, ΝΡ_636638.1, ΖΡ_01260019.1, ΖΡ_04929809.1, ΖΡ_08460617.1, ΥΡ_004068587. 1, ΥΡ_002553266.1, ΖΡ_06877402.1, ΥΡ_002439068.1, ΝΡ_800628.1, ΖΡ_08100296.1, ΝΡ_252259.1, ΥΡ_001279202.1, ΖΡ_01367000.1, ΥΡ_001346959.1, ΖΡ_01133887.1, EGM 15746.1, ΥΡ_339977.1, ΖΡ_04921053 .1, ΖΡ_04764368.1, ΝΡ_762450.2, ΥΡ_986257.1, ΥΡ_789596.1, ΥΡ_004191179.1, ΥΡ_004416219.1, ΝΡ_937094.1, ΥΡ_002794225.1, ΖΡ_02244049.1, ΖΡ_05943914.1, ΥΡ_450771.1, ΥΡ_200485.1, ΖΡ_08178115.1, ΝΡ_717293.1, ΖΡ_06730192.1, ΖΡ_04958952.1, ΖΡ_03698449.1, ΖΡ_04922804.1, ΥΡ_003288698.1, ΥΡ_001141739.1, ΖΡ_08188278.1, ΥΡ_786279. 1, ΖΡ_05127790.1, ΥΡ_363098.1, ΖΡ_01104144.1, ΖΡ_05886382.1, ΖΡ_05620602.1, ΖΡ_01614260.1, ΝΡ_641651.1, ΖΡ_06705559.1, ΖΡ_01260775.1, ΖΡ_06182137.1, ΖΡ_05879122.1, ΥΡ_001631119.1, ΖΡ_08552251.1, ΖΡ_08570660.1, ΖΡ_01738211.1, ΖΡ_01893199.1, ΖΡ_08520681.1, ΖΡ_08182597.1, ΥΡ_264191.1, ΥΡ_001414164.1, ΖΡ_08638362.1, ΥΡ_422950.1, EGF41343.1, ΖΡ_00056040.1, ΖΡ_08700476. 1, ΝΡ_800135.1, ΖΡ_08272093.1, ΖΡ_08181910.1, ΖΡ_02194300.1, ΖΡ_03698989.1, ΥΡ_004235082.1, preferably to an enzyme from the group comprising the enzymes BAC82381.1, NP_746775.1, YP_004703920.1, YP_001670886.1, ADR61938.1, YP_001269834.1, YP_001747642.1, YP_606441.1, BAJ07617.1, YP_257885.1, EGH62730.1, ZP_06461142.1, ZP_05642104.1, YP_004351842.1 , EGH86869.1, YP_002874705.1, EGH24275.1, ZP_07777517.1, ZP_07003462.1, ZP_07264531.1, EGH72043.1, YP_233798.1, EGH66664.1, EGH33025.1, EGH77479.1, EGH54475.1, EFW86611 .1, EGH13085.1, ZP_03399204.1, NP_790630.1, YP_346429.1, ZP_04590292.1, EGH46911.1, ZP_06495709.1, YP_004714189.1, ZP_08140457.1, AEA83842.1, ADY83615.1, YP_001844789.1 , ZP_06693498.1, YP_001705868.1, YP_001715485.1, ADX90568.1, ZP_06059137.1, ADX01770.1, ZP_05825497.1, ZP_06069662.1, YP_004473140.1, YP_003734018.1, YP_002798276.1, ZP_03822695.1, ZP_06726280 .1, ZP_05359923.1, YP_001172440.1, YP_046276.1, ZP_07236476.1, YP_001083192.1, ZP_04929809.1, ZP_08460617.1, ZP_06877402.1, YP_002439068.1, NP_252259.1, YP_001279202.1, ZP_01367000.1, YP_001346959.1, EGM 15746.1, YP_789596.1, ZP_05620602.1, YP_264191.1, YP_580776.1, YP_792542.1, YP_004379211.1, ZP_06880384.1, YP_001 186868.1, ZP_04932430.1, ZP_01364121.1, YP_002442182.1, NP_249434.1, YP_0013501 19.1, EGH98403.1, 30BB_A, 3Q3C_A. In a preferred embodiment, the phrase "a cell which has a reduced activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof compared to its wild type" means that it is a cell genetically engineered with respect to its wild-type, that the Activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant in this cell is reduced compared to the activity of the same 3-hydroyxisobutyric acid dehydrogenase or the corresponding variant in the Wld type of the cell, preferably by at least 10, 20, 30, 50, 75, 90, 95 or 99% In a particularly preferred embodiment, the activity of the enzyme is no longer detectable in the genetically engineered cell In a further particularly preferred embodiment, the genetic engineering effecting the activity reduction relates solely to the activity of a specific 3-hydroxy-butyric acid dehydrogenase of the cell, in contrast to the possibility that nonspecifically several enzymatic activities of the cell are reduced, for example by a defect in the folding machinery of the cell, which could cause numerous enzymatic activities of the cell are defective. A comparison of the enzymatic activities of the genetically modified cell and its wild type is carried out under the same conditions and with the use of standard assays for the determination of a dehydrogenase activity. Thus, for example, the dehydrogenase activity can be monitored in a continuous spectrophotometric assay when the enzyme, either pure or in the form of a cell lysate, is incubated with substrate, ie 3-hydroxyisobutyric acid and redox factor, and the reaction progress is monitored by the extinction of the redox factor.

Various conditions may be used to carry out the process according to the invention. The temperature may be more than 20 ° C, 30 ° C, 40, 50 with the proviso that in the case of using a living cell or a preparation of suitable enzymes, the selected cell or enzymes are viable or show activity , 60, 70 ° C or more than 80 ° C, preferably to 100 ° C. The person skilled in the art knows which organisms are viable at which temperatures, for example from textbooks such as Fuchs / Schlegel, 2007. In the case of a living yeast cell, the temperature may be 5 to 45 ° C, more preferably 15 to 42 ° C, even more preferably 20 to 30 ° C. In the case of a Gram-negative bacterium, preferably a bacterium of the family Enterobacteriaceae, most preferably E. coli, the temperature may be 5 to 45 ° C, more preferably 15 to 42 ° C, even more preferably 20 to 30 ° C, most preferably 35 to 40 ° C.

For cultivation of the cell according to the invention numerous culture media are suitable, in the case of using a yeast cell, for example YPD, YPN and YNB, with amino acids, for example with 0.01 g / L tryptophan, or with glucose, for example in a concentration of 1% ( w / v) can be supplemented. In the case of using a bacterium from the family Enterobacteriaceae, preferably E. coli, come to grow full media such as LB medium or high cell density medium (HZD medium) consisting of NH ₄ S0 ₄ 1, 76 g, K ₂ HP0 ₄ 19.08 g, KH ₂ PO 12.5 g, yeast extract 6.66 g, Na ₃ citrate 1.96 g, NH Fe citrate (1%) 17 ml, trace element solution US3 5 ml, feed solution (glucose 50% w / v, MgS0 [x 7 H ₂ 0 0.5% w / v, NH ₄ Cl 2.2% w / v) 30 ml per liter in question.

In a preferred embodiment, cells used in the method according to the invention are used in a medium other than that used for steps a) to d) of the method. In a particularly preferred embodiment, the medium used for the cultivation is a complete medium and the medium used for the steps a) to d) is a minimal medium. The method according to the invention, if it is carried out using viable cells, is preferably carried out after transformation of the cells in transformation buffer per liter of (NH) H ₂ PO ₄ 8 g, NaCl 0.5 g, MgS 0 × 7 H ₂ O 0.48 1 ml of the trace element solution US 3 was composed of HCl 37% 36.5 g, MnCl ₂ × 4H ₂ 0 1, 91 g, ZnSO ₄ × 7H ₂ 0 1, 87 g, Na EDTA x 2H ₂ 0 0.8 g, H ₃ B0 ₃ 0.3 g, Na ₂ Mo0 ₄ x 2H ₂ 0 0.25 g, CaCl ₂ x 2H ₂ 0 4.7 g, FeS0 ₄ x 7 H ₂ 0 17 , 8 g, CuCl ₂ x 2H ₂ 0 0.15 g, and its pH is adjusted to 5.4.

The concentration of isobutyric acid, if provided as a ready-to-use starting material at the beginning of the process, is from 0.01 to 2, more preferably from 0.05 to 1, most preferably from 0.1 to 0.2% (w / v) at the beginning of the reaction. , When 3-hydroxyisobutyric acid is prepared using glucose as a carbon source which is suitable for use via a competent strain Precursors is metabolized, their concentration is initially in the medium preferably 1% (w / v).

The steps a) to d) of the process according to the invention are preferably carried out at atmospheric pressure. In the case of production of isobutyric acid from isobutane, it may be advantageous for the alkane hydroxylase to comprise the reaction in the presence of higher pressures in the presence of a gas mixture, preferably predominantly comprising isobutane. In a preferred embodiment, the pressure is more than 1, 5, 2, 3 or 4 bar. In a further embodiment, the pressure is 0.5 to 4, preferably 1 to 3, most preferably 1 to 1, 5 bar.

In a most preferred embodiment of the present invention, the object underlying the invention is achieved in that a cell of the genus Yarrowia, preferably Yarrowia lipolytica, in which the activity of the hydroxyisobutyric acid dehydrogenase YaliOF02607g or a variant thereof by deletion from the activity of the corresponding wild-type cell is reduced, is contacted in aqueous solution with isobutyric acid

The present invention will be further illustrated by the following figures and non-limiting examples to which further features, embodiments, aspects and advantages of the present invention may be derived.

1 shows a particularly preferred sequence according to the invention of enzymatically catalyzed reactions comprising the conversion of isobutyric acid to isobutyryl-CoA by isobutyrate kinase and phosphotransisobutyrylase or isobutyryl-coenzyme A synthetase / ligase or isobutyrate-coenzyme A transferase in step b) , the oxidation of isobutyryl-coenzyme A to methacrylyl-coenzyme A by isobutyryl-coenzyme A-dehydrogenase in step c), the addition of water to methacrylyl-coenzyme A to give 3-hydroisobutyryl-coenzyme A in step d) and its hydrolysis to 3-hydroxyisobutyric acid in step e). Examples

example 1

 Preparation of a recombinant Yarrowia lipolytica cell with attenuated 3-hydroxyisobutyric acid dehydrogenase activity

1. Construction of the gene disruption cassette GDC-YI02607PT for the deletion of the 3-hydroxybutyric acid dehydrogenase gene Ya // QF02607q Accession Number: XP_50491 1.1)

 To construct a Va // 0F02607g / (noc / roi / f mutant in Y. lipolytica, the promoter and terminator region of the Va // 0F02607g gene was cloned Chromosomal DNA from Y. lipolytica H222 (MATa) served as the template for the PCR The gene knockout was carried out in the following strains: V. lipolytica H222-41 (MATa ura3-41) and Y. lipolytica H222-SW4-2 (MATa ura3-302 SUC2 ku70A-1572 trp1A-1199). and terminator region of the gene coding for the 3-hydroxyisobutyric acid dehydrogenase (Va // 0F02607g) was determined using the oligonucleotides 3HIBDH-Pfw (SEQ ID NO: 01), 3HIBDH-Prv (SEQ ID NO: 02) (Promoter region), 3HIBDH-Tfw (SEQ ID NO: 03) and 3HIBDH-Trv (SEQ ID NO: 04) (terminator region) were amplified from the chromosomal DNA of Y. lipolytica H222 in a PCR were used for the PCR: promoter region, 1 x: initial denaturation, 98 ° C, 3 min; 35 x: denaturation, 98 ° C, 0:10 min, annealing, 59.5 ° C, 0:45 min; elongation, 72 ° C, 0:35 min; 1 x: terminal elongation, 72 ° C, 5 min. Terminator area, 1 x: initial denaturation, 98 ° C, 3 min; 35x: denaturation, 98 ° C, 0:10 min, annealing, 59.5 ° C, 0:45 min; Elongation, 72 ° C, 0:35 min; 1 x: terminal elongation, 72 ° C, 5 min. For amplification, the Phusion ™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) was used according to the manufacturer's recommendations. In this way, the promoter fragment at the 3 'end was provided with an I-Scel interface and the terminator fragment at the 5' end with an I-Scel interface. The following oligonucleotides were used for this:

3HIBDH-Pfw:

 5'-CAC ACA TCC AGA GCT CTA TG -3 '(SEQ ID NO: 01) 3HIBDH-Prv:

5'-TAT ATA CTA TAT TAC CCT GTT ATC CCT AGC GTA ACT ACA AAT ACA AGT TTT AAG CTG -3 '(SEQ ID NO: 02, containing an I-Scel recognition sequence at the 5' end) 3HIBDH-Tfw:

 5'-TAT ATA AGT TAC GCT AGG GAT AAC AGG GTA ATA TAG GCT GTG TAT GTG TTA GGG TG - 3 '(SEQ ID NO: 03, containing a / -Sce / recognition sequence at the 5' end) 3HIBDH -Trv:

 5'-GGT GAC CTT CAG GTG CAC CA -3 '(SEQ ID NO: 04)

2. Fusion of the promoter and terminator fragments

 The PCR products of the promoter and terminator region (1060 or 970 base pairs) were purified by means of the "QIAquick PCR Purification Kit" (Qiagen, Hilden) according to the manufacturer's instructions In a subsequent crossover PCR, the two PCR products used as a template and amplification was carried out with the primers 3HIBDH-Pfw (SEQ ID No. 01) and 3HIBDH-Trv (SEQ ID No. 04) The following parameters were used for the PCR: 1 ×: initial Denaturation, 98 ° C, 3 min; 35x: denaturation, 98 ° C, 0:10 min, annealing, 59.5 ° C, 0:45 min; elongation, 72 ° C, 1: 00 min; 1 x: terminal elongation, 72 ° C, 5 min. Complementarity of the / -Sce / restriction site resulted in a 2.071 kilobase pair PCR product (SEQ ID NO: 05). Cut the DNA out of the gel with a scalpel and clean with the "Quick Gel Extraction Kit" (Qiagen, Hilden). The procedure was carried out according to the manufacturer's instructions. In the next step, the PCR product was ligated into the vector pCR-Blunt II-Topo (Zero Blunt TOPO PCR Cloning Kit with One Shot TOP10 Chemically Competent E. coli, Invitrogen, Karlsruhe). The resulting plasmid pCRBIuntllTopo :: P_T_3HIBDH_YI (SEQ ID NO: 06) is 5.59 kilobase pairs. The ligation and the transformation of chemically competent E. coli were carried out according to the manufacturer. The authenticity of the plasmid was checked by restriction analysis with EcoRI, Xho \ and Psti.

3. Construction of the / cnoc / couf mutants Y. lipolytica H222-SW- -2 Δ 3HIBDH and Y. lipolytica H222-41 A3HIBDH

For the construction of the / (noc / "/ i mutant, the integration of a marker gene is necessary, for which the 1.3 kilobase pair" loxP-URA3-loxR "DNA fragment containing the URA3 gene was amplified by means of a - -Sce / Restriction out of the vector pJMP113 (Fickers et al., 2003) The "loxP-l / fi / A3 / ox / '' fragment was prepared using the Quick Gel Extraction Kit (Qiagen, Hilden) according to The resulting fragment was ligated into the I-Scel cut vector pCRBIuntllTopo :: P_T_3HIBDH_YI (SEQ ID NO: 06) resulting plasmid pCRBIuntllTopo :: P_T_3HIBDH_YI_ura (SEQ ID NO: 07) is 6.899 kilobase pairs in size. The ligation and the transformation of chemically competent E. coli DH5a cells (New England Biolabs, Frankfurt) were carried out in a manner known to those skilled in the art. The authenticity of the plasmid was checked by restriction with Xmal, Seal and Sad.

In order to obtain the gene disruption cassette GDC-YI02607PT necessary for the gene knockout to delete ORF Va // 0F02607g, the plasmid pCRBIuntllTopo :: P_T_3HIBDH_YI_ura (SEQ ID NO: 07) was used as a template for the PCR with the following oligonucleotides and Parameters employed: 3HIBDH-Pfw (SEQ ID NO: 01) and 3HIBDH-TRV (SEQ ID NO: 04); 1 x: initial denaturation, 98 ° C, 3 min; 35x: denaturation, 98 ° C, 0:10 min, annealing, 65 ° C, 0:45 min; Elongation, 72 ° C, 1:45 min; 1 x: terminal elongation, 72 ° C, 5 min. The desired 3.38 kilobase pair fragment (SEQ.ID.NO.:8) was purified using the "Quick Gel Extraction Kit" (Qiagen, Hilden) according to the manufacturer's instructions on an agarose gel, checked by restriction with Xmal and XmnI and for the integrative transformation of Y. lipolytica H222-SW4-2 and Y. lipolytica H222-41.

The transformation was carried out by the lithium acetate method (Barth G and Gaillard C (1996) Yarrowia lipolytica.) In: Wolf, K. (eds) Nonconventional yeasts in biotechnology Springer, Berlin Heidelberg New York, pp 313-388). The obtained uracil-prototrophic transformants were checked by colony PCR. The following parameters were used for the PCR: 1 ×: initial denaturation, 98 ° C., 3 min; 35x: denaturation, 98 ° C, 0:10 min, annealing, 60 ° C, 0:45 min; Elongation, 72 ° C, 1:30 min; 1 x: terminal elongation, 72 ° C, 5 min. The following oligonucleotides were used: fw-3HIBDH-ah:

 5'-GAG TCG CAG ATT CAG GAA AT -3 '(SEQ ID NO: 09) rv-3HIBDH-ah:

 5'-TCA CCT TCT GAT CAC GGT GT -3 '(SEQ ID NO: 10)

In the event of a successful disruption of the Ya // 0F02607g gene encoding the 3-hydroxyisobutyric acid dehydrogenase, a 1.005 kilobase pair fragment should be amplified. In fact, corresponding clones could be identified, which were further processed in the following. 4. Restoration of uracil auxotrophy

 Neither the production of uracil auxotrophy in the uracil prototrophic transformants was carried out according to Fickers et al., 2003. First, competent cells were prepared. To do this, transformants were added in 5 mL of YPD pH 4 (10 g / L yeast extract, 10 g / L peptone, 10.5 g / L citric acid, 2% (w / v) glucose, and 0.5 M sodium citrate for pH adjustment ) for 8 h at 30 ° C and 190 rpm in 100 mL baffled flasks. After 8 h, this preculture was used to inoculate 10 mL of YPD medium pH 4 in 250 mL baffled flasks with an optical density (OD600) of 0.05. All flasks were incubated at 30 ° C and 190 rpm. The next day, when a cell count between 9-107 and 1, 5-107 per ml was counted (counted using the Neubauer chamber), these precultures were harvested (500 g, 5 min, RT), twice in 10 mL TE buffer (10 mM Tris-HCl, 1 mM Na-EDTA, pH 8), then diluted in 30 mL of 0.1% (w / v) lithium acetate and incubated for 1 h at 28 ° C and 60 rpm. The cells were then recentrifuged (500 g, 5 min, RT), resuspended in 3 mL of 0.1% (w / v) lithium acetate, aliquoted into 100 and used immediately. All further steps were done on ice. For the transformation of the plasmid pUB4Cre (Fickers et al, 2003), 100 μί competent cells were mixed with 200-800 ng of plasmid DNA and 2.5 boiled salmon sperm DNA (10 mg / ml) (Invitrogen, Karlsruhe). After addition of 0.7 ml of 40% PEG4000 (w / v, dissolved in 0.1 M lithium acetate, pH 6), the cells were incubated for 1 h at 28 ° C. with shaking. The heat shock at 39 ° C followed for 10 min in a water bath. 1, 2 mL of 0.1% (w / v) lithium acetate was added to the cells and twice 250 applied very carefully to YPD plates with 500 μg / mL hygromycin.

After 3 to 10 days of incubation at 30 ° C, the resulting clones were phenotypically tested on agar plates with YNB medium (6.7 g / L Difco ™ Yeast Nitrogen Base w / o amino acids) and glucose with or without uracil. In order to remove the plasmid pUB4Cre from the cell, the tranformants were incubated several times in 10 mL YPD medium in 100 mL baffled flasks at 30 ° C. and 190 rpm for 24 h and spread on YPD plates. The resulting clones were monitored by PCR (as described in point 3) and phenotypically on agar plates.

In this way, the following strains were constructed:

- H222-SW4-2 Ä3HIBDH

- H222-41 Ä3HIBDH Example 2

 Production of 3-hydroxyisobutyric acid with glucose as the sole carbon source by means of genetically engineered Y. Hpolytica cells in which the activity of 3-hydroxyiosbutyric acid dehydrogenase was attenuated

The V. // o / yfca strain H222-41 Ä3HIBDH constructed in Example 1 was compared to the corresponding wild type H222-41 in 10 mL YNB medium (6.7 g / L Difco ™ Yeast Nitrogen Base w / o Amino Acids ) were cultured with 0.2 g / L uracil, 0.01 g / L tryptophan and 5% (w / v) glucose overnight at 28 ° C. and 190 rpm. The next morning, these precultures were used to mix 20 mL of YNB medium with 0.2 g / L uracil, 0.01 g / L tryptophan and 1% (w / v) glucose in 100 mL baffled flasks with an optical density ( OD600) of 0.5 inoculate. All flasks were incubated at 28 ° C and 190 rpm.

After a period of 70 hours, 1 g / L of ammonium sulfate was added and after 95 hours, the concentrations of 3-hydroxyisobutyric acid in the batches were analyzed by IC. For the chromatographic separation in the ICS-2000 RFIC (Dionex Corporation, Synnyvale, USA) was the RFICTM TonPac column (2 ^χ 250 mm, column temperature 30 ° C, + precolumn AG15 4 ^χ 50 mm, flow rate 0.38 mlJmin) used ,

The strains reached an OD600 of about 30. While the control strain Y. lipolytica H222-41 did not produce any 3-hydroxyisobutyric acid, in the case of the Y. lipolytica H222-41 Ä3HIBDH derived therefrom, the formation of 4.5 mg / L 3-hydroxyisobutyric acid be detected.

Example 3

 Production of 3-hydroxyisobutyric acid from isobutyric acid with genetically engineered Y. Hpolytica cells, in which the activity of 3-hydroxyisobutyric acid dehydrogenase was attenuated

The Y. // po / yfca strains H222-SW-4-2 Ä3HIBDH and H222-41 Ä3HIBDH constructed in Example 1 were compared to an unmodified control strain (H222-SW-4-2) in 10 mL YNB- Medium (6.7 g / L Difco ™ Yeast Nitrogen Base w / o Amino Acids) with 0.01 g / L tryptophan and 1% (w / v) glucose cultured overnight at 30 ° C and 190 rpm. The next morning, these precultures were used to mix 20 mL of YNB medium with 0.2 g / L uracil, 0.01 g / L tryptophan, 1% (w / v) glucose, and 0.2% (w / v). Isobutyric acid (titrated with NaOH, pH = 5.1, 0.2% isobutyric acid added after 24 h) is inoculated into 100 mL baffled flasks with an optical density (OD600) of 0.5. All flasks were incubated at 30 ° C and 190 rpm. The H222-SW4-2 strains achieved an OD600 of 2-3 while the H222-41 strains grew to an OD600 of about 10.

After a period of 24 h and 48 h, the concentrations of 3-hydroxyisobutyric acid in all batches were analyzed by IC (see FIG. 1). The culture supernatant was diluted 1:10 with ddH20 so that the readings were within the calibration range. For the chromatographic separation in the ICS 2000 RFIC (Dionex Corporation, Synnyvale, USA) was the RFICTM TonPac column (2 ^χ 250 mm, column temperature 30 ° C, + precolumn AG154 ^χ 50 mm, flow rate 0.38 mlJmin) was used.

After 24 h all Y. // po / yfca strains reached the stationary growth phase. The pH of the H222-41 strains decreased to between 3.3 and 3.7 during growth, and to pH values of 2 in the H222-SW-4-2 strains. After 24 h and 48 h, the modified Y. // po / yfca strains H222-SW-4-2 Ä3HIBDH and H222-41 Ä3HIBDH Ä3HIBDH and H222-41 Ä3HIBDH achieved a higher yield than the respective unmodified strains H222-SW1 -2 and H222-41. Y. lipolytica H222-41A3HIBDH converts over 90% isobutyric acid to 3-hydroxyisobutyric acid after 24 h and over 80% after 48 h. By the deletion of Ya // 0F02607g in Y. lipolyica H222-41 or H222-SW ^ l-2, the yield of 3-hydroxyisobutyric acid production from isobutyric acid could be significantly increased (see Fig. 1). Example 4

 Production of 3-hydroxybutyric acid (3-HIB) from glucose or isobutyric acid (IBA) as sole carbon source by Y. Iipolytica H222 (wild type, WT) and genetically engineered Y. Iipolytica H222 Ä3HIBDH (ura) -8 with attenuated 3-hydroxyisobutyric acid dehydrogenase activity (Δ). a) Biomass production on a shake flask scale

Cryocultures of Y. Iipolytica H222 λ3HIBDH (ura) -8 (hereinafter referred to as Δ) and Y. Iipolytica H222 (hereinafter referred to as wild type (WT)) were prepared on YPD agar plates per liter from yeast extract 10g, peptone 20g, agar -Agar 12g autoclaved and complemented with separately autoclaved glucose 10g, streaked and incubated for 24h at 28 ° C.

2 1000 ml shake flasks with chicanes per 100 ml of YPD medium (above medium without agar-agar, each with 3 drops of Delamite antifoam) were filled in each strain, inoculated with 2 full inoculation loops from the agar plates and for 20 h at 30 ° C. incubated at 180rpm (amplitude 2.5cm) (residual glucose Og / I, OD> 20).

Table 1

The cultures were then sterile-filled in 50 ml Falcons and spun down at 5000 rpm. The pellets were washed 4x with 0.9% saline. Thereafter, 2 pellets of each strain were resuspended in 50 ml of transformation buffer and pooled. The transformation buffer was composed per sterile-filtered liter of (NH ₄ ) H ₂ P0 ₄ 8 g,

NaCl 0.5 g, MgSO ₄ × 7 H ₂ O 0.48 g, trace element solution US3 15 ml. 1 liter of the trace element solution US 3 was composed of HCl 37% 36.5 g, MnCl ₂ × 4H ₂ O 1.91 g, ZnS0 ₄ x 7H ₂ 0 1, 87g, Na-EDTA x 2H ₂ 0 0.8g, H ₃ B0 ₃ 0.3g, Na ₂ Mo0 ₄ x 2H ₂ 0 0.25g, CaCl ₂ x 2H ₂ 0 4.7g, FeS0 x 7 H ₂ O 17,8g, CuCl ₂ x 2H ₂ 0 0,15g. This solution was separately sterile filtered and added to the buffer under sterile conditions. The pH of the buffer was adjusted to 5.4. biotransformation

In 4 1000 ml shake flasks with baffles, 50 ml each of the above-mentioned transformation buffer pH 5.4 with 3 drops of Delamex antifoam were added sterile. Per strain, sterile were placed in a shake flask for a final volume of 100 ml of 0.2% (w / v) isobutyric acid, in a second shake flask 1% (w / v) glucose. One shake flask each with IBA and one with glucose was inoculated with 50 ml resuspended Pelltet of Y. lipolytica H222 from a). Similarly, Y. lipolytica H222 Ä3HIBDH (ura) -8 was used. The starting OD was about 14. The shake flasks were incubated at 180 rpm at 30 ° C. Samples were taken after 0, 6 and 24 hours. Microscopic controls did not show lysis cells in any approach throughout. The measurement of the glucose was carried out with a YSI measuring instrument Kreienbaum, the measurement of isobutyric acid and 3-hydroxyisobutyric acid was carried out by HPLC over an Aminex column, the measurement of OD with a spectrophotometer at 600nm.

Results:

Tab. 2

OD

The glucose was partially metabolized to the biomass structure. With IBA as the sole source of C, no biomass production took place.

Tab. 3

 Concentration substrates IBA / glucose [mg / l]

 Strain WT IBA WT glucose Δ IBA Δ glucose

 t [h] IBA Glucose IBA Glucose IBA Glucose IBA Glucose

 0 1914 20 0 8450 1808 10 0 8520

 6 676 0 0 3240 1267 0 0 1770

24 0 0 0 0 422 0 0 0 Both substrates were metabolized by both Y. Iipolytica H222 and Y. Iipolytica H222 Ä3HIBDH (ura) -8. Table 4

 Concentration 3-HIB [mg / l]

In Y. Iipolytica H222, 3-HIB could only be detected as a metabolite for a short time with IBA as the substrate.

With Y. Iipolytica H222 Ä3HIBDH (ura) -8, small amounts of free 3-HIB could be detected with glucose as the substrate. The IBA used consumed 15.7 mmol / l. From this theory, 18.6 mmol / l of 3-HIB can be formed. Measured were 19.6 mmol / l. The spent IBA is thus fully converted to 3-HIB.

Example 5

 Production of 3-hydroxyisobutyric acid from ketoisovalerate with genetically engineered Y. Hpolytica cells, in which the activity of 3-hydroxyisobutyric acid dehydrogenase was attenuated

Y. // po / yfca strain H222-41 Ä3HIBDH constructed in Example 1 was compared to the corresponding wild-type strain Y. lipolytica H222-41 in 10 ml of YNB medium (6.7 g / L Difco ™ Yeast Nitrogen Base w / o amino Acids) with 0.2 g / L uracil, 0.01 g / L tryptophan and 1% (w / v) glucose overnight at 30 ° C and 190 rpm cultivated. The next day, these precultures were used to mix 25 mL of YNB medium with 0.2 g / L uracil, 0.01 g / L tryptophan, 6% (w / v) glucose, and 0.65% (w / v). Ketoisovalerate (after 25.5 h, 0.5% (w / v) of ketoisovalerate and, after 49.5 h, 0.3% (w / v) of ketoisovalerate were again added to the culture) in 100 ml baffled flasks with an optical density ( OD600) of 0.5 inoculate. All flasks were incubated at 30 ° C and 190 rpm. Over time, the optical density (OD600) was determined and the 3-hydroxyisobutyric acid content was analyzed by IC in all batches. During the culture run, sufficient glucose and ketoisovalerate were present in the medium at all times. After about 24 h all Y. lipolytica strains reached the stationary growth phase. The pH dropped to about 3 during growth.

The deletion of Ya // 0F02607g in Y. lipolyica H222-41 increased the 3-hydroxyisobutyric acid production from ketoisovalerate from 2 g / L 3-hydroxyisobutyric acid to more than 5 g / L 3-hydroxyisobutyric acid (see Fig. 2).

Example 6

Production of 3-hydroxyisobutyric acid with a 2-step process starting from iso-butane using genetically modified E. co // ^'W3110 monooxygenase (alkBGT) from P. putida GP01 to isobutyric acid, the genetically modified Y. Iipolytica H222 (ura) -8 in which the activity of 3-hydroxyisobutyric acid dehydrogenase has been attenuated, is further reacted with IBA as the sole carbon source.

step 1

 Production of isobutyric acid from iso-butane by E. coli W3110 with the monooxygenase (alkBGT) from P. putida GP01. a) Production of biomass on 101 scale

 Preseed culture: 1 liter of LB medium containing 50μΙ kanamycin was prepared from a solution of yeast extract 5g, peptone 10g, NaCl 0.5g, and 50μΙ kanamycin. The pH was adjusted to 7.4 with 5% NH40H. The solution is autoclaved at 121 ° C for 20 minutes.

From this solution, 5 × 25 ml each in 100 ml shake flasks were filled with baffles and each inoculated with 200μΙ of a glycerol cryoculture of E. coli W3110 pBT10 (DE10200710060705). These cultures were incubated for 18 hours at 37 ° C and 180rpm (amplitude 2.5cm).

Seed culture: 1 liter of high cell density medium (HZD medium) consisting of NH ₄ SO ₄ 1, 76 g, K ₂ HP0 ₄ 19.08 g, KH ₂ PO ₄ 12.5 g, yeast extract 6.66 g, Na ₃ citrate 1, 96 g, NH ₄ Fe citrate (1%) 17ml, trace element solution US3 5ml, feed solution (glucose 50% w / v, MgS0 ₄ [x 7 H ₂ 0 0.5% w / v, NH CI 2.2% w / v) 30ml , and 50 ug kanamycin was prepared with deionized water. 1 liter of

Trace element solution US 3 is composed of HCl 37% 36.5 g, MnCl ₂ x 4H ₂ O 1.91 g, ZnSO ₄ x 7H ₂ O 1, 87 g, Na EDTA x 2H ₂ O 0.8 g, H ₃ BO ₃ 0 , 3g, Na ₂ Mo0 ₄ x 2H ₂ 0 0.25g, CaCl ₂ x 2H ₂ 04.7g, FeS0 x 7H ₂ 0 17.8g, CuCl ₂ x 2H ₂ 0 0.15g. 948 ml of solution with NH ₄ S0 to Na ₃ citrate are autoclaved, the remainder was separately sterile filtered and then added sterile. The pH was 6.8.

5 × 75 ml of the HZD medium in 1000 ml shake flasks with baffles were inoculated with 25 ml preseed culture each and cultured for 30 h at 37 ° C. and 180 rpm (amplitude 2.5 cm). Induction culture: 25 ml each of the culture broth was poured into 75 ml of modified M9 medium (sterile filtered) with the following composition per liter: 15 g glucose, 6.79 g Na ₂ PO ₄ , 3 g KH ₂ PO ₄ , 0.5 g NaCl, 2 g, NH ₄ Cl, 15 g yeast extract, 0.49 g MgS0 ₄ * 7H ₂ 0, 1 ml of trace element solution (as in seed culture) and 50 μg kanamycin in 1000 ml shake flasks. The cultures are incubated for 7h at 35 ° C and 180rpm (amplitude 2.5cm).

A sterile 10 L fermentor was charged with 7 L of a sterile medium having the composition (per liter) (NH ₄ ) ₂ S0 ₄ 1, 75 g, K ₂ HP0 ₄ x 3 H ₂ 0 19 g, KH ₂ P0 ₄ 12, 5 g, yeast extract 6.6 g, Na ₃ citrate x 2H ₂ O 2.24 g, glucose 15 g, MgS0 ₄ × 7 H ₂ 0 0.49 g, NH ₄ Fe citrate (1% w / v) 16 , 6ml, trace element solution (as in seed culture) 15ml and Kanamycin 50μg and 2ml defoamer Delamex. The feed was an autoclave solution of glucose (50% w / v) with MgS0 ₄ x 7H ₂ 0 10g / l, for pH correction 0.5M H ₂ S0 ₄ and 25% NH ₄ OH.

The cultures from the shake flasks were combined sterile and inoculated via a transfer bottle into the fermenter. The fermentation conditions were set p0 ₂ 30%, Airflow 6nlpm, stirrer 400 - 1200rpm temperature 37 ° C, pH7, feed start 8 h, feed rate 150-250 g / h. After 19 h, the temperature was lowered to 30 ° C, the feed stopped and induced with 0.4 mM DCPK. After 23 hours, the OD in the fermenter is about 100, the culture broth was removed under sterile conditions and centrifuged off at 8000 rpm with 500 ml in 1000 ml centrifuge beakers. The supernatant was discarded, the pellets were aliquoted into sterile falcons so that a resuspended pellet in 180 ml of Trafopuffer gave an OD of about 20. The pellets could then be used immediately in the biotransformation, or frozen at -80 ° C for later use. b) Biotransformation iso-butane to isobutyric acid

The pellets from 200 ml of culture were resuspended in 10 ml of conversion buffer. The conversion buffer consisted of 70 mM (NH ₄ ) H ₂ PO ₄ buffer, pH 7 per liter with 8 g (NH ₄ ) H ₂ PO ₄ , 0.5 g NaCl, 0.49 g MgSO ₄ .7H ₂ O, 1 ml TE and 50 μg kanamycin. The pH was adjusted here with 5% NH ₄ OH.

150 ml of buffer with about 3 drops of autoclaved antifoam (Delamex) were placed in a 300 ml fermenter. The fermenter was filled with a gas mixture from a gas bottle of 5 bar initial pressure of 25% iso-butane and 75% synthetic air via a Metallsinterperlator with a pore size of 0.2 μηι gassed at a flow rate of 12.5Nl / h. The fermenter was heated to 35 ° C. in a water bath and stirred at 900 rpm using a magnetic stirrer. The exhaust air was discharged through a wash bottle filled with 150 ml of water.

The fermenter was inoculated via a sampling tube with 30 ml of a pellet prepared in a) and resuspended in buffer. The reaction was started with the start of a Glucosefeedes (or iso also glucose batch or template + feed ....) of 1, 5 g / lh. After 4.5 hours, a concentration of isobutyric acid of> 350 mg / l was achieved (3.97 mmol / kg).

The pH was adjusted to 5.4 with 1 MH ₂ S0 ₄ and the gassing was changed to compressed air at a flow rate of 12.5 NI / h, the temperature lowered to 30 degrees, (alternatively):

 • 50 ml transfer buffer buffers with cannula and syringe were taken sterile from the fermenters to resuspend the vaccines / cultures (see step 2a)

• The culture was harvested sterile and the biomass separated by centrifugation at 8000rpm with 500ml in 1000ml centrifuge beakers, and the supernatant stored at -20 ° C until transformation with the Ya / row / ^' cultures.

Level 2

Production of 3-hydroxyisobutyric acid from isobutyric acid from stage 1 by genetically engineered Y. lipolvtica H222 A3HIBDH (ura) -8 compared to the Wldtype Y. lipolytica H222. a) Production of biomass on shake flask scale (or optionally also in the fermenter) Cryocultures of Y. lipolytica H222 Ä3HIBDH (ura) -8 and Y. lipolytica H222 were prepared on YPD agar plates per liter from yeast extract 10g, peptone 20g, agar-agar 12g autoclaved and complemented with separately autoclaved glucose 10 g, streaked and incubated for 24 h at 28 ° C. 2 1000 ml shake flasks filled with baffles were each inoculated from the plates with 100 l of YPD medium (above medium without agar-agar, with 3 drops of Delamex antifoam) each with 2 full inoculation loops and incubated at 30 ° C. for 20 h 180rpm (amplitude 2.5cm) incubated (residual glucose Og / I, OD> 20). The cultures were then sterile-filled in 50 ml Falcons and spun down at 5000 rpm. The pellets were washed 4x with 0.9% saline. Then 2 pellets each of a strain were resuspended in 50 ml of Trafopuffer (from stage 1b) fresh or thawed and combined. b) Biotransformation of isobutyric acid to 3-hydroxyisobutyric acid

Once 50 ml of the resuspended pellets of Y. lipolytica H222 Ä3HIBDH (ura) -8 and Y. lipolytica H222 were inoculated in a 300 ml fermenter with the Trafopuffer from step 1b) and for 24 h at pH 5.4, 30 ° C and 900rpm stirred and fumigated with 1, 2vvm compressed air.

After 24 hours, no isobutyric acid and no 3-hydroxybutyric acid were detected in the supernatant in the wild-type Y. lipolytica H222. In Y. lipolytica H222 Ä3HIBDH (ura) -8 no more isobutyric acid could be detected in the supernatant after 24 h. 3-Hydroxyisobutyric acid corresponding to the isobutyric acid values of> 3.97 mmol measured at the end of step 1b) had concentrations of> 413 mg / l.

References:

A. Cornish-Bowden (1995), Fundamentals of Enzyme Kinetics, Portland Press Limited, 1995 DE 60216245 (2007): FUNCTIONAL DISPLAY OF POLYPEPTIDES

DE10200710060705 (2007): ω-aminocarboxylic acids or their lactams, producing, recombinant cells Sambrook / Fritsch / Maniatis (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 ^nd edition

William Bauer, Jr. "Methacrylic Acid and Derivatives" in Ullmann's Encyclopedia of Industrial Chemistry 2002, Wley-VCH, Weinheim

Hasegawa et al. (1981) J. Ferment. Technol. 59, pp 203-208

Hasegawa (1981b), Agric. Biol. Chem. 45, pp 2899-2901. Hasegawa et al. (1982) J. Ferment. Technol. 60, pp 501-508

WO 2007/141208: MICROBIOLOGICAL PRODUCTION OF 3-HYDROXYISOBUTYRIC ACID

WO 2008/119738 ENZYME FOR THE PRODUCTION OF METHYLMALONYL COENZYME A OR ETHYLMALONYL COENZYME A, AND USE THEREOF

Patel RN et al. (1983), Journal of Applied Biochemistry 5 (1-2), 107-120)

Barth G and Gaillard C (1996) Yarrowia lipolytica. In: Wolf, K. (eds) Nonconventional yeasts in biotechnology. Springer, Berlin Heidelberg New York, pp 313-388

Van Beilen et al. (2002): Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria ", Journal of Bacteriology, 2002, 184 (6), pp. 1733-1742.) Fersht AR and Winter G (2008): Redesigning Enzymes by Site-Directed Mutagenesis, Ciba Foundation Symposium 1 11 - Enzymes in Organic Synthesis

Fuchs / Schlegel (2007) General Microbiology, 2008, Georg Thieme Verlag William Bauer, Jr. "Methacrylic Acid and Derivatives" in Ullmann's Encyclopedia of Industrial Chemistry 2002, Wley-VCH, Weinheim

Claims

claims

A process comprising the following steps: a) providing isobutyric acid, b) contacting isobutyric acid with the combination of isobutyrate kinase and phosphotransisobutyrylase and / or isobutyryl-coenzyme A synthetase / ligase and / or isobutyrate-coenzyme A transferase, c) contacting the product from step b) with isobutyryl-coenzyme A dehydrogenase, d) contacting the product from step c) with methacrylyl-coenzyme A hydrate, and e) hydrolysing the product from step d) to give 3-hydroxyisobutyric acid, wherein at least one of the enzymes used in steps b), c) and d) from the group comprising isobutyrate kinase, phosphotransisobutyrylase, isobutyryl-coenzyme A synthetase / ligase and isobutyrate-coenzyme A transferase, preferably all, is provided in the form of a cell having a reduced activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof compared to its wild-type.

The process of claim 1 wherein the isobutyric acid is formed by contacting isobutane with a monooxygenase, preferably an alkane hydroxylase, more preferably one of the AlkBGT type or a variant thereof.

3. The method according to any one of claims 1 to 2, wherein the hydrolysis in step d) by contacting the product of step d) with a 3-Hydroxisobutyryl-coenzyme A hydrolase is achieved.

4. The method according to any one of claims 1 to 3, wherein the cell both the isobutyryl-coenzyme A dehydrogenase in step c) and the MethacrylyI coenzyme A hydrate in step d) and the combination of isobutyrate kinase and phosphotransisobutyrylase and /or

Isobutyryl-coenzyme A synthetase / ligase and / or isobutyrate-coenzyme A-transferase.

5. The method according to any one of claims 1 to 4, wherein the cell additionally comprises an alkane hydroxylase, preferably one of the AlkBGT type or a variant thereof.

6. The method according to any one of claims 1 to 5, wherein it is 3-hydroxyisobutyric acid dehydrogenase to XP_504911.1 or a variant thereof.

7. cell comprising at least one enzyme from the group comprising isobutyryl-coenzyme A synthetase / ligase, isobutyrate-coenzyme A transferase, isobutyrate kinase,

Phosphotransisobutyrylase, isobutyryl-coenzyme A dehydrogenase, and MethacrylyI coenzyme A hydratase and a reduced compared to their wild type activity of a 3-hydroxyisobutyric acid dehydrogenase or a variant thereof. The cell of claim 7, wherein the cell comprises, in addition to an isobutyryl-coenzyme A dehydrogenase and in addition to a methacrylyl coenzyme A hydratase, the combination of isobutyrate kinase and phosphotransisobutyrylase and / or Isobutyryl-coenzyme A synthetase / ligase and / or isobutyrate-coenzyme A-transferase, preferably furthermore a 3-hydroxysobutyryl-coenzyme A-hydrolase.

A cell according to any one of claims 7 to 8, further comprising an alkane hydroxylase, preferably one of the AlkBGT type or a variant thereof.

10. A cell according to any one of claims 6 to 8, wherein it is in the 3-hydroxyisobutyric acid dehydrogenase to XP_50491 1.1 or a variant thereof.

1 1. Use of the cell according to any one of claims 7 to 10 for the preparation of 3-hydroxyisobutyric acid.

12. Use according to claim 1 1, wherein it is in the 3-hydroxyisobutyric acid dehydrogenase to XP_50491 1.1 or a variant thereof. 13. The method according to any one of claims 1 to 6, cell according to any one of claims 7 to 10 or use according to any one of claims 11 to 12, wherein the cell is a bacterial or lower eukaryotic cell.

14. The method according to any one of claims 1 to 5 or 13, cell according to any one of claims 7 to 10 or 13 or use according to one of claims 11 to 13, which is a yeast cell from the group of genera, the Yarrowia, Candida , Saccharomyces, Schizosaccharomyces and Pichia, and is preferably Yarrowia lipolytica. 15. A reaction mixture comprising the cell according to any one of claims 7 to 10 or 13 to 14 and isobutane or isobutyric acid.