WO2011157573A2

WO2011157573A2 - An enzyme for the preparation of methylmalonate semialdehyde

Info

Publication number: WO2011157573A2
Application number: PCT/EP2011/059178
Authority: WO
Inventors: Markus PÖTTER; Achim Marx; Steffen Schaffer; Liv Reinecke; Thomas Haas
Original assignee: Evonik Röhm Gmbh
Priority date: 2010-06-14
Filing date: 2011-06-03
Publication date: 2011-12-22
Also published as: WO2011157573A3; TW201211253A

Abstract

The subject matter of the invention is an enzyme for the preparation of methylmalonate semialdehyde or malonate semialdehyde, nucleic acids comprising coding sequences for this enzyme, cells which have been transformed with these nucleic acids, and the use of this enzyme.

Description

An enzyme for the production of methylmalonate semialdehyde

Field of the invention:

The invention relates to an enzyme for the production of methylmalonate semialdehyde or malonate semialdehyde, nucleic acids containing coding sequences for this enzyme, cells which have been transformed with these nucleic acids, as well as the use of this enzyme.

State of the art:

DE102006025821 describes an enzyme from Sulfolobus tokodaii which is capable of producing S- or R-methylmalonyl-coenzyme A and malonyl-coenzyme A to the corresponding ones

To implement semialdehydes. This enzyme is used in processes for the preparation of (S) - or (R) - methylmalonate semialdehyde, (S) - or (R) -3-hydroxyisobutyric acid or (S) - or (R) -3-hydroxyisobutyric acid based polyhydroxyalkanoates and in processes for the preparation of malonate semialdehyde, of 3-hydroxypropionic acid or of

Hydroxypropionic acid based polyhydroxyalkanoates used. These process products are commercially valuable starting materials for chemical syntheses. This justifies, as in the present invention, the industrial applicability.

A disadvantage of the enzyme described lies in the relatively low activity of the enzyme, in particular with regard to the substrates S- and R-methylmalonyl-coenzyme A.

The object of the invention was to provide enzymes which have a greater specific activity in the reactions described above and thus in a shorter time more (S) - or (R) -Methylmalonatsemialdehyd or Malonatsemialdehyd from S- or R-methylmalonyl coenzyme A or Malonyl coenzyme A, in particular more (S) - or (R) -

Methylmalonatsemialdehyd from S- or R-methylmalonyl coenzyme A assets to make.

Description of the invention:

Surprisingly, it has been found that the polypeptides described below are able to solve the stated problem. The present invention therefore relates to those described in claim 1

Polypeptides with corresponding amino acid mutations compared to known

Wild-type sequences.

Another object of the invention are nucleic acids comprising nucleic acids encoding the polypeptides of the invention as well as cells containing the

Nucleic acids according to the invention were transformed. Another object of the

The invention relates to the use of the substances according to the invention for the preparation of (S) - or (R) -methylmalonate semialdehyde, of (S) - or (R) -3-hydroxyisobutyric acid, of (S) - or (R) -3-hydroxyisobutyric acid-based Polyhydroxyalkanoates, malonate semialdehyde, 3-hydroxypropionic acid and 3-hydroxypropionic acid based polyhydroxyalkanoates.

Advantages of the polypeptides according to the invention are, in addition to the described activities, also the very high stability. The increased stability leads to a long availability of the

Biocatalyst (long service life).

Another advantage is that bioprocesses are made possible, which can be carried out at elevated temperatures and thus require less cooling.

All percentages (%) are by mass unless otherwise specified. A contribution to the solution of the abovementioned objects is afforded by an isolated polypeptide having one of the following amino acid sequences:

A) an amino acid sequence having the SEQ ID NO. 2, SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ.ID.NO. 8, SEQ.ID.NO. 10, SEQ. ID. NO. 12, SEQ ID NO. 14, SEQ. ID. NO. 16 and SEQ ID NO. 18, in particular SEQ.-ID-No. 2,

B) an amino acid sequence obtained when at most 40 amino acids,

preferably at most 20 amino acids, more preferably at most 10 amino acids, and most preferably at most 5 amino acids in an amino acid sequence of group A) deleted, inserted, substituted or at the C and / or N-terminal end of an amino acid sequence of group A. ), wherein a protein with this amino acid sequence is capable of S- or R-

Methylmalonyl coenzyme A and malonyl coenzyme A to the corresponding

Semialdehydes ((S) - or (R) -methylmalonate semialdehyde or malonate semialdehyde), in particular S- or R-methylmalonyl-coenzyme A to (S) - or (R) - methylmalonatsemialdehyd, with preferably at least 80%, more preferably at least 90%, in particular at least 95%, especially more than 100% of the specific activity of the protein the respective reference sequence from group A),

C) an amino acid sequence having an amino acid sequence according to one of the groups A) and B), more preferably according to group A), at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 99% identical, being a protein with this

Amino acid sequence is capable of producing S- or R-methylmalonyl coenzyme A and malonyl

Coenzyme A to the corresponding semialdehydes ((S) - or (R) - methylmalonate semialdehyde or malonate semialdehyde), in particular S- or R-methylmalonyl coenzyme A to give (S) - or (R) -methylmalonate semialdehyde, preferably at least 80%, particularly preferably at least 90%, in particular at least 95%, very particularly more than 100% of the specific activity of a protein of the respective reference sequence from group A) and B), in particular group A), with the proviso that the amino acid sequence of groups A ) to C) at least one amino acid substitution selected from the group consisting of

V16T, K38R, K67R, L149I, L149K, L149M, L149Q, L149R, Y203F, Y203H, Y203L, Y203M, T207P, T207S, I208V, 121 1T, P282L, S310G, R315G, R315K, K317E, I330A, I330C, I330W and V334A,

preferably V16T, L149K, L149M, L149R, Y203L, T207P, T207S, I330A, I330C and I330W.

Polypeptides having the polypeptide sequences of group A and the said

Amino acid exchanges are capable of forming S- or R-methylmalonyl coenzyme A to the corresponding semialdehyde ((S) - or (R) -methylmalonate semialdehyde) with preferably at least 1 10%, more preferably at least 150%, especially at least 200%, completely especially to convert at least 300% of the specific activity of the respective polypeptide with the polypeptide sequence of group A without said amino acid substitutions.

Preferred polypeptides according to the invention have at least one combination of

Amino acid substitution selected from the group consisting of A07S Y203F, V16T K38R, K38R L149M, K38R L149R, K38R T207P, K38R I208V, L149I T207P, L149M Y203M, L149M T207P, L149M R315K, L149M K317E and L149Q T207P, especially L149M T207P

on.

In particular, preferred polypeptides have at least one combination of

Amino acid substitution selected from the group consisting of

P108L D201V N268S, K67R L149M T207S, V16T E139G L149M, L149M S310G V334A, V16T S310G V334A, L149M Y203M K284T, L149M Q153H D201G, K67R L149M T207P, L149M T207P S310G,

K38R L149M T207P P282L, K38R L149M T207P Y298N, K38R L149M 121 1T V312F, L149Q T207P K284T K317E, L149R I 176T Y203H I208V, V16T L149K T236A N268S, V16T I80V T207P R315G, K38R

K38R K98N L149M T207P K317E, K38R L149M T207P I239M G314C, K38R Y267M 121 1T S310G V334A, N 106S L149M I 162L T207P I239V,

K38R K67R F1 12Y L149M T207P P307S, V92L D109Y L149M T207P A260V R270G, L149M F163S I208V E217G K317E I330W,

K38R K136I L149M I21 1T V222A R315G V334A, N26K K38R E139G L149M G160V K261E S310G V334A and V16T K38R R137G L149M M168I D169N T207P D329Y K254N S310G V334A

in particular the combinations K38R K67R F112Y L149M T207P P307S, K38R L149M T207P P282L, K38R L149M T207P N221S, K38R K98N L149M T207P K317E, K38R K67R F1 12Y L149M T207P P307S and N106S L149M I 162L T207P I239V are preferred. The "nucleotide identity" or "amino acid identity" is determined in the context of the present invention by known methods. In general, special computer programs are used with algorithms taking into account special requirements.

Preferred methods for determining identity initially produce the largest

Match between the sequences to be compared. Computer programs for

Determination of identity include, but are not limited to, the GCG program package, including GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387, Genetics Computer Group University of Wisconsin, Medicine (Wi), and BLASTP, BLASTN and FASTA (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410 The BLAST program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894; Altschul S. et al., Supra).

The well-known Smith-Waterman algorithm can also be used to determine nucleotide identity.

Preferred parameters for determining "nucleotide identity" are when using the BLASTN program (Altschul, S. et al., Journal of Molecular Biology 215 (1990), p. 403-

410:

Expect Threshold

Word size:

Match Score:

Mismatch Score:

Gap costs:

The above parameters are the default parameters in nucleotide sequence comparison.

The GAP program is also suitable for use with the above parameters. Preferred parameters for the determination of "amino acid identity" are when using the BLASTP program (Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410:

Expect Threshold: 10

Word size: 3

Matrix: BLOSUM62

Gap costs: Existence: 1 1; Extension: 1

Compositional adjustments: Conditional compositional score matrix adjustment

The above parameters are the default parameters in the amino acid sequence comparison. The GAP program is also suitable for use with the above parameters. The nomenclature for the amino acid substitutions defined above is well known to those skilled in the art. For example, V16T describes that the amino acid valine at position 16 has been replaced by a threonine. The numbering of the amino acid position always refers to the corresponding position in the considered amino acid sequence with SEQ. ID. NO. 2 for reference. For this purpose, the sequence to be considered with SEQ. ID no. 2 matched using the BlastP program using the above parameters; the numerical position indicated in the amino acid substitution is then found in the amino acid sequence under consideration in accordance with SEQ.ID.No. 2 assigned again.

If the amino acid sequence under consideration at the indicated numerical position has a different amino acid than in the first place given in the amino acid exchange, this other amino acid is the last in the amino acid exchange

to replace specified.

In connection with the polypeptides of the invention derived from group B) or C) it should be noted that changes of amino acid residues of a given

Polypeptide sequence that do not lead to any significant changes in the properties and function of the given polypeptide are known in the art. Thus, for example, so-called conserved amino acids can be exchanged for each other; Examples of such suitable amino acid substitutions are: Ala versus Ser; Arg against Lys; Asn versus Gin or His; Asp against Glu; Cys versus Ser; Gin vs Asn; Glu vs Asp; Gly vs Pro; His against Asn or Gin; against Leu or Val; Leu vs Met or Val; Lys versus Arg or Gin or Glu; Mead against Leu or hell; Phe versus Met or Leu or Tyr; Ser against Thr; Thr against Ser; Trp against Tyr; Tyr against Trp or Phe; Val against hell or leu. It is also known that changes especially at the N- or C-terminus of a polypeptide in the form of, for example, amino acid insertions or deletions often do not exert a significant influence on the function of the polypeptide, such as an exchange of R2G.

To determine the specific activity of the protein under consideration with respect to the ability to convert S- or R-methylmalonyl-coenzyme A or malonyl-coenzyme A to the corresponding semialdehydes, the method described in Example 3 with correspondingly known enzyme concentrations can be used.

The polypeptide according to the invention is an enzyme which comprises both the conversion of (S) - or (R) -methylmalonyl-coenzyme A to (S) - or (R) - Methylmalonatsemialdehyd and the conversion of malonyl coenzyme A to Malonatsemialdehyd to catalyze. Such a polypeptide can be synthesized, for example, starting from the DNA sequence of SEQ. ID. NO. 01, SEQ ID NO. 03, SEQ ID NO. 05, SEQ ID NO. 07, SEQ ID NO. 09, SEQ ID NO. 1 1, SEQ.ID.NO. 13, SEQ ID NO. 15 or SEQ ID NO. 17, or by transformation of a suitable cell with a

suitable vector comprising the abovementioned nucleic acid sequences, expression of the protein encoded by these nucleic acid sequences in the cell, lysis of the cell to obtain a cell extract and subsequent purification of the enzyme by means of purification techniques known to those skilled in the art, for example by HPLC or others

chromatographic procedures. In addition to a chromatographic

Purification of the polypeptide from cell extracts can also take advantage of the fact that the polypeptide of the invention is heat stable up to a temperature of at least 75 ° C. Therefore, one can heat the cell extract to a temperature of, for example, 75 ° C, thereby causing coagulation and thus precipitation of the non-heat stable

Polypeptides in the cell extract comes. The polypeptide according to the invention remains in undenatured form in the cell extract.

Another object of the present invention are nucleic acids, in particular isolated nucleic acids encoding polypetide according to the invention.

By "coding for" is meant here the genetic code having a codon usage, as found for example in E. coli, but also constellations are conceivable in which an unconventional codon usage is used, as in Tetrahymena, for example.

Plasmodium, Mycobacterium pneumoniae or Candida tropicalis can lead to polypeptides according to the invention. These nucleic acids are also considered to be "coding for

polypeptide according to the invention ".

Nucleic acids preferred according to the invention encode preferred according to the invention

Polypeptides.

The invention further comprises the nucleotide sequences described

complementary nucleic acid molecules. A further contribution to the solution of the abovementioned objects is afforded by a vector, preferably an expression vector, comprising a nucleic acid according to the invention, as defined above. Suitable vectors are all vectors known to the person skilled in the art

Considerations that are commonly used to introduce DNA into a host cell. Preferred vectors are selected from the group comprising plasmids, such as the E. coli plasmids pTrc99A, pBR345 and pBR322, viruses such as bacteriophages, adenoviruses, vaccinia viruses, baculoviruses, measles viruses and retroviruses, cosmids or YACs, with plasmids being vectors most are preferred.

According to a preferred embodiment of the vector according to the invention, the nucleic acid according to the invention is under the control of a regulatable promoter which is used to express the polypeptide encoded by these DNA sequences in the cell of a

A microorganism, preferably a bacterial, yeast or pilin cell, more preferably a bacterial cell, most preferably one pound. co // cell, is suitable. Examples of such promoters are, for example, the trp promoter or the tac promoter.

The vector according to the invention should, in addition to a promoter, preferably a

Ribosome binding site and a terminator include. It is particularly preferred that the DNA according to the invention is incorporated into an expression cassette of the vector comprising the promoter, the ribosome binding site and the terminator. In addition to the structural elements mentioned above, the vector may further comprise selection genes known to those skilled in the art.

A contribution to the solution of the above-mentioned objects continue to make use of the above-described vector for the transformation of a cell as well as by

Transformation cell obtained with this vector. The cells which can be transformed with the vector according to the invention can

Be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), plant cells, or microorganisms such as yeasts, fungi or bacteria, with microorganisms being most preferred and bacteria and yeasts being most preferred.

As bacteria, yeasts or fungi especially those bacteria, yeasts or fungi are suitable, which in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany, as bacterial, yeast or fungal strains are deposited. Bacteria suitable according to the invention belong to the genera under

http://www.dsmz.de/species/bacteria.htm

Yeasts which are suitable according to the invention belong to those genera which are listed under http://www.dsmz.de/species/yeasts.htm

are listed and suitable fungi according to the invention are those under

http://www.dsmz.de/species/fungi.htm

are listed.

Particularly preferred cells according to the invention are those of the genera

Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia and Clostridium, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis,

Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha and Pichia pastoris are particularly preferred.

In an alternative embodiment of cells according to the invention, these are acetogenic microorganisms, such as, for example, species of the genus Acetobacterium, such as A. woodii and Clostridium aceticum. In particular, the acetogenic cells are selected from the group comprising, in particular consisting of, Acetoanaerobium notera, Acetobacterium woodii, Archaeoglobus fulgidus, Butyribacterium methylotrophicum, Butyribacterium methyltrophicum, Carboxydibrachium pacificus, Carboxydocella sporoproducens, Carboxydocella

thermoautotrophica, Carboxydothermus hydrogenoformans, Citrobacter sp. Y19, Clostridium aceticum, Clostridium acetobutylicum, Clostridium autoethanogenum, Clostridium

carboxidivorans, Clostridium ljungdahlii, Desulfotomaculum carboxydivorans,

Desulfotomaculum kuznetsovii, Desulfotomaculum thermobenzoicum subsp.

thermosyntrophicum, Eubacterium limosum, Methanosarcina acetivorans C2A, Methanosarcina barkeri, Methanothermobacter thermoautotrophicus, Moorella AMP, Moorella thermoacetica, Moorella thermoautotrophica, Oxobacter pfennigii, Peptostreptococcus productus,

Rhodopseudomonas palustris P4, Rhodospirillum rubrum, Rubrivivax gelatinosus, Thermincola carboxydiphila, Thermincola ferriacetica, Thermococcus AM4, Thermolithobacter carboxydivorans and Thermoanaerobacter kivui.

A particularly suitable cell in this context is Clostridium carboxidivorans, in particular such strains as "P7" and "P1 1". Such cells are described, for example, in US 2007/0275447 and US 2008/0057554. Another particularly suitable cell in this context is Clostridium ljungdahlii, in particular strains selected from the group comprising, in particular consisting of, Clostridium ljungdahlii PETC, Clostridium ljungdahlii ERI2, Clostridium IjungdahlW C0I and Clostridium ljungdahlii 0-52; these are described in WO 98/00558 and WO 00/68407.

The polypeptides, nucleic acids, vectors and cells according to the invention can be advantageously used for the preparation of (S) - or (R) -methylmalonate semialdehyde, of (S) - or (R) -3-hydroxyisobutyric acid, of (S) - or (R) 3-hydroxyisobutyric acid based

Polyhydroxyalkanoates, malonate semialdehyde, 3-hydroxypropionic acid or 3-hydroxypropionic acid based polyhydroxyalkanoates.

Instructions for methods for use according to the invention are found in the expert in DE102006025821.

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: Cloning of enzymes according to the invention

The Sulfolobus tokodaii mcr gene was isolated from the plasmid pTrc99A-mcr (Alber B et al., J. Bacteriol, 188: 8551-8559) with the two primers mcr_fwd_Ncol (5 ').

ATAC C ATG G G AG AAC ATTAAAAG C-3 ', SEQ.ID.NO. 19) and mcr_rev_Sall (5-CTTGTCGACTTATTACTTTTCAATATATCC-3 ', SEQ ID NO: 20) PCR amplified (Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press). The 1088 bp PCR fragment was over the two

Restriction endonuclease cleavage Ncol and Sali into a pET-28a (+) E. coli

Expression vector (Merck KGaA, Darmstadt, Germany) (Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor

Laboratory Press). The pET-28a (+) - mcr expression construct was transformed into competent cells of the E. coli expression strain Rosetta ™ 2 (DE3) (Merck KGaA, Darmstadt, Germany).

Starting from the mcr gene, a SeSaM mutant library was prepared by the SeSaM method described in EP1670914 and labeled with the primers SEQ.ID.NO. 19 and 20 cloned. SDM mutant libraries were prepared using the Phusion ™ site-directed mutagenesis kit (Finnzymes Oy, Espoo, Finland) according to the manufacturer's instructions in the pET-28a (+) - mcr plasmid.

Example 2: Expression of enzymes according to the invention

Using 250 μί of a saturated 3.5 mL ZYM-505 (Studier 2005, Protein Expr. Purif, 41: 207-234) overnight, preculture 55 mL ZYM-5025 main culture in a 300 mL

Erlenmeyer flasks inoculated. Cholramphenicol (25 g / mL) and kanamycin (50 g / mL) were added to all media for selection. Protein expression was for 22 h at 37 ° C and 250 rpm. The cells were harvested by centrifugation for 15 min at 3,220 g and 4 ° C. The cell pellets were resuspended in 10 mL cell disruption buffer (100 mM TrisHCl pH 7.4 at 55 ° C, 5 mM MgCl ₂ ) and digested with a high-pressure homogenizer. E. coli

Proteins were heat-precipitated by incubation for 15 min at 75 ° C and 15 min on ice. The denatured proteins and cell debris were spun down for 20 min at 16,000 g and 4 ° C. The clear supernatant was used in the MCR activity detection.

Example 3: Activity determination of enzymes according to the invention in cuvettes

The proof of activity is based on the decrease of NADPH and is determined by measuring the

Traced absorption at 340 nm in the photometer. Procedure: 1. 430 μM buffer (100 mM TrisHCl pH 7.4 at 55 ° C, 5.32 mM MgCl ₂ , 5.86 mM DTT) are heated to 55 ° C in the heated cuvette

preheated; Second

Add 10 μM 25 mM NADPH (in 100 mM TrisHCl pH 7.4 at 55 ° C); 3. Addition of 50 μl cell extract (in cell disruption buffer); 4. Start the reaction by adding 10 15 mM malonyl-CoA or methylmalonyl-CoA (in 100 mM TrisHCl pH 7.4 at 55 ° C).

Final concentrations: 100 mM TrisHCl pH 7.4 at 55 ° C; 5 mM MgCl ₂ ; 5mM DTT, 0.5mM

NADPH; 0.3mM X-CoA. During the measurement, the decrease in absorbance at 340 nm is monitored for 8 min in the cuvette heated to 55 ° C.

It was compared with the enzyme prepared according to Example 1 without any

Amino acid mutation has a significant increase in specific activity on the substrate methylmalonyl-CoA found in such enzymes with amino acid substitutions of: Y203L, A07S Y203F, L149M, L149R, L149K, V16T, V16T, T207P, T207S, I330A, I330C and I330W.

In the following table, further enzymes according to the invention characterized by their amino acid exchanges compared to the cloned enzyme according to Example 1 are without

Amino acid exchange listed. The% activity represents the specific activity relative to

Monoded enzyme according to Example 1 without amino acid exchange based on methylmalonyl CoA, wherein the specific activity of the cloned enzyme according to Example 1 without

Amino acid exchange was set to 100%.

Amino Acid Exchange% activity

K38R K136I L149M I21 1T V222A R315G V334A 607

K38R K67R F1 12Y L149M T207P P307S 1395

K38R K98N L149M T207P K317E 1489

K38R L149M 389 K38R L149M 121 "IT V312F 444

K38R L149M T207P I239M g314c 718

K38R L149M T207P N221S 1320

K38R L149M T207P P282L 1557

K38R Y267M 121 1 T S310G V334A 171

K67R L149M T207S 226

L149M F163S I208V E217G K317E I330W 587

L149M Q153H D201 G 403

L149M S310G V334A 201

L149M T207P 651

N106S L149M I 162L T207P I239V 1227

V92L D109Y L149M T207P A260V R270G 534

It could thus be characterized enzymes that have up to a nearly 15-fold increased activity based on methylmalonyl-CoA.

Claims

Claims:

1 . Isolated polypeptide having one of the following amino acid sequences:

A) an amino acid sequence having the SEQ ID NO. 2, SEQ. ID. NO. 4, SEQ. ID. NO. 6, SEQ.ID.NO. 8, SEQ. ID. NO. 10, SEQ. ID. NO. 12, SEQ ID NO. 14, SEQ. ID. NO. 16 and SEQ ID NO. 18, in particular SEQ.-ID-No. 2,

B) an amino acid sequence obtained when at most 40 amino acids in an amino acid sequence of group A) are deleted, inserted, substituted or attached to the C- and / or N-terminal end of an amino acid sequence of group A), wherein a protein with this amino acid sequence, S- or R-methylmalonyl coenzyme A and malonyl coenzyme A to the corresponding semialdehydes ((S) - or (R) -Methylmalonatsemialdehyd

Malonate semialdehyde),

C) an amino acid sequence having an amino acid sequence according to any one of

Groups A) and B) is at least 80% identical, wherein a protein having this amino acid sequence is capable of forming S- or R-methylmalonyl-coenzyme A and malonyl-coenzyme A to the corresponding semialdehydes ((S) - or ( R) - Methylmalonatsemialdehyd or Malonatsemialdehyd) implement.

with the proviso that the amino acid sequence of groups A) to C) at least one

Amino acid exchange selected from the group consisting of

V16T, K38R, K67R, L149I, L149K, L149M, L149Q, L149R, Y203F, Y203H, Y203L,

Y203M, T207P, T207S, I208V, 121 1T, P282L, S310G, R315G, R315K, K317E, I330A,

I330C, I330W and V334A

having.

2. Polypeptide according to claim 1, characterized in that it has at least one

Combination of amino acid substitutions selected from the group consisting of A07S Y203F, V16T K38R, K38R L149M, K38R L149R, K38R T207P, K38R I208V, L149I T207P, L149M Y203M, L149M T207P, L149M R315K, L149M K317E and L149Q T207P.

3. Polypeptide according to claim 1 or 2, characterized in that it comprises at least one combination of amino acid substitutions selected from the group consisting of P108L D201V N268S, K67R L149M T207S, V16T E139G L149M, L149M S310G V334A, V16T S310G V334A, L149M Y203M K284T, L149M Q153H D201G, K67R L149M T207P, L149M T207P S310G,

K8R L149M T207P P282L, K38R L149M T207P Y298N, K38R L149M I21 1T V312F, L149Q T207P K284T K317E, L149R I 176T Y203H I208V, V16T L149K T236A N268S, V16T I80V T207P R315G, K38R

K168E, K38R K98N L149M T207P K317E, K38R L149M T207P I239M G314C, K38R Y267M I21 1T S310G V334A, N106S L149M I 162L T207P I239V,

K38R K136I L149M I21 1T V222A R315G V334A, N26K K38R E139G L149M G160V K261 E S310G V334A and V16T K38R R137G L149M M168I D169N T207P D329Y K254N S310G V334A

having.

4. Isolated nucleic acid encoding a polypeptide according to at least one of

Claims 1 to 3.

5. Vector comprising a nucleic acid according to claim 4.

6. cell transformed with a nucleic acid according to claim 4 or a vector after

Claim 5.

7. Use of a polypeptide according to at least one of claims 1 to 3, a nucleic acid claim 4, a vector according to claim 5 or a cell according to claim 6 for the preparation of for the production of (S) - or (R) -methylmalonatsemialdehyd, from (S ) or (R) -3-hydroxyisobutyric acid, of (S) - or (R) -3-hydroxyisobutyric acid based polyhydroxyalkanoates, of malonate semialdehyde, of Hydroxypropionic acid or polyhydroxyalkanoates based on 3-hydroxypropionic acid.