Means and method for the microbial production of atactic poly(3-hydroxybutyrate)

Abstract

The present invention relates to a method for the production of atactic poly(3-hydroxybutyrate) (PHB) comprising (a) producing a mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA by (a1) reducing acetoacetyl-CoA to a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3-hydroxybutryl-CoA by contacting acetoacetyl-CoA with an enzyme catalyzing the reaction (R)-3-hydroxyacyl-CoA + NAD(P)+ = 3-oxoacyl-CoA + H+ + NAD(P)H and an enzyme catalyzing the reaction (3S)-3-hydroxybutyryl-CoA + NAD(P)+ = acetoacetyl-CoA + H+ + NAD(P)H in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), and / or (a2) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3- hydroxybutyryl-CoA with an enzyme catalyzing the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA, and / or (a3) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3- hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the reaction (3R)-3-hydroxybutyryl-CoA ↔ crotonyl-CoA + H2O to produce Crotonyl-CoA and contacting Crotonyl-CoA with an enzyme catalyzing the reversible hydration of crotonyl-CoA, and (b) polymerizing the mixture of (a) to atactic poly(3-hydroxybutyrate) by contacting the mixture of (a) with an enzyme catalyzing the reaction -3-hydroxybutyryl-CoA + poly(3-hydroxybutyrate)(n) = poly(3- hydroxybutyrate)(n+1) + CoA.

Description

[0001] Means and method for the microbial production of atactic poly(3-hydroxybutyrate)

[0002] The present invention relates to a method for the production of atactic poly(3-hydroxybutyrate) (PHB) comprising (a) producing a mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA by (al) reducing acetoacetyl-CoA to a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3-hydroxybutryl-CoA by contacting acetoacetyl-CoA with an enzyme catalyzing the reaction (R)-3-hydroxyacyl-CoA + NAD(P)+= 3-oxoacyl-CoA + H++ NAD(P)H and an enzyme catalyzing the reaction (3S)-3-hydroxybutyryl-CoA + NAD(P)+= acetoacetyl-CoA + H++ NAD(P)H in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), and / or (a2) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA, and / or (a3) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the reaction (3R)-3-hydroxybutyryl-CoA <-> crotonyl-CoA + H₂O to produce Crotonyl-CoA and contacting Crotonyl-CoA with an enzyme catalyzing the reversible hydration of crotonyl-CoA, and (b) polymerizing the mixture of (a) to atactic poly(3-hydroxybutyrate) by contacting the mixture of (a) with an enzyme catalyzing the reaction 3-hydroxybutyryl-CoA + poly(3-hydroxybutyrate)(n) = poly(3-hydroxybutyrate)(n+1) + CoA.

[0003] In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] PHB is a biodegradable polymer produced by microbial fermentation / 11While PHB is advertised as a sustainable alternative to petroleum-based plastics, its widespread adoption is hindered by high material costs and its material properties / 1-21The polymer's high crystallinity renders it brittle, causing breakage when stressed / 2-41Moreover, the proximity of melting and degradation temperatures leads to a narrow processing window due to polymer degradation / 1-31Introduction of the (S)-enantiomer into the polymer chain has been demonstrated to mitigate these drawbacks, reducing crystallinity and expanding the processing window with increasing contents.[5]

[0005] The biosynthesis of PHB initiates from the central metabolite acetyl-CoA and requires three enzymes.161The process begins by the condensation of two acetyl-CoA molecules to acetoacetyl-CoA by p-ketothiolase (PhaA) under the release of CoA. Acetoacetyl-CoA reductase (PhaB) then reduces acetoacetyl-CoA to (R)-3-hydroxybutyryl CoA using NADPH as a cofactor. Finally, PHA synthase (PhaC) elongates the (R)-isotactic polymer chain, releasing one CoA molecule for each repeating unit. The prevailing assumption in the prior art is that PHA synthases strictly favor (R)-3-hydroxybutyryl-CoA.[7-81

[0006] The appended examples surprisingly show that PHA synthases, in particular the ones as described herein deviate from strict stereoselectivity, enabling the production of atactic PHB.

[0007] Currently, microbially obtained PHB is exclusively isotactic and each stereocenter is in the (R)-configuration. Isotacticity in particular is the cause of the high crystallinity and poor processability. Using chemically synthesized, atactic PHB, it was shown that the undesirable material properties decrease with increasing proportion of (S)-enantiomer (Haslbbck, M. et al. (2018), Macromolecules, 51:5001-5010; and Haslbbck, M. et al. (2019), Macromolecules, 52:5407-5418).

[0008] The microbial production of atactic PHB as provided herein for the first time advantageously enables the sustainable synthesis of PHB based on renewable and biogenic raw materials (in the sense of a bioeconomy) with improved material properties.

[0009] Accordingly, the present invention relates in a first aspect to a method for the production of atactic poly(3-hydroxybutyrate) (PHB) comprising (a) producing a mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA by (al) reducing acetoacetyl-CoA to a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3-hydroxybutryl-CoA by contacting acetoacetyl-CoA with an enzyme catalyzing the reaction (R)-3-hydroxyacyl-CoA + NAD(P)+= 3-oxoacyl-CoA + H++ NAD(P)H and an enzyme catalyzing the reaction (3S)-3-hydroxybutyryl-CoA + NAD(P)+= acetoacetyl-CoA + H++ NAD(P)H in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), and / or (a2) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA, and / or (a3) the isomerization of (R)-3-hydroxybutyryl-CoAto (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the reaction (3R)-3-hydroxybutyryl-CoA <-> crotonyl-CoA + H₂O to produce Crotonyl-CoA and contacting Crotonyl-CoA with an enzyme catalyzing the reversible hydration of crotonyl-CoA, and (b) polymerizing the mixture of (a) to atactic poly(3-hydroxybutyrate) by contacting the mixture of (a) with an enzyme catalyzing the reaction 3-hydroxybutyryl-CoA + poly(3-hydroxybutyrate)(n) = poly(3-hydroxybutyrate)(n+1) + CoA.

[0010] In accordance with a preferred embodiment (al) the enzyme catalyzing the reaction (R)-3-hydroxyacyl-CoA + NAD(P)+= 3-oxoacyl-CoA + H++ NAD(P)H is acetoacetyl-CoA reductase (PhaB) and the enzyme catalyzing the reaction (3S)-3-hydroxybutyryl-CoA + NAD(P)+= acetoacetyl-CoA + H++ NAD(P)H is 3-hydroxybutyryl-CoA dehydrogenase (Hbd), and / or (a2) the enzyme catalyzing the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA is the enzyme 3-hydroxybutyryl-CoA epimerase (FadB), and / or (a3) the enzyme clatalyzing the reaction (3R)-3-hydroxybutyryl-CoA <-> crotonyl-CoA + H₂O is (3R)-3-hydroxybutyryl-CoA hydrolyase (PhaJ) and the enzyme that catalyzes the reversible hydration of crotonyl-CoA is (3S)-3-hydroxyacyl-CoA hydrolyase (Crt), and (b) the enzyme catalyzing the reaction 3-hydroxybutyryl-CoA+ poly(3-hydroxybutyrate)(n) = poly(3-hydroxybutyrate)(n+1) + CoA is polyhydroxyalkanoate synthase (PhaC).

[0011] Hence, and in accordance with the above preferred embodiment the present invention relates in a first aspect to a method for the production of atactic poly(3-hydroxybutyrate) (PHB) comprising (a) producing a mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA by (al) reducing acetoacetyl-CoA to a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3-hydroxybutryl-CoA by contacting acetoacetyl-CoA with the enzymes acetoacetyl-CoA reductase (PhaB) and 3-hydroxybutyryl-CoA dehydrogenase (Hbd) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), and / or (a2) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with the enzyme 3-hydroxybutyryl-CoA epimerase (FadB), and / or ((a3) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with the enzyme (3R)-3-hydroxybutyryl-CoA hydrolyase (PhaJ) to produce Crotonyl-CoA and contacting Crotonyl-CoA with the enzyme (3S)-3-hydroxyacyl-CoA hydrolyase (Crt), and (b) polymerizing the mixture of (a) to atactic poly(3-hydroxybutyrate) by contacting the mixture of (a) with polyhydroxyalkanoate synthase (PhaC).

[0012] Poly(3-hydroxybutyrate) is a polymer of the monomer hydroxybutyrate. Hydroxybutyrate is the base conjugate of hydroxybutyric acid, which is an organic compound having the chemical formula H₃CH(OH)CH₂CO₂H. PHB is a polyhydroxyalkanoate (PHA), a polymer belonging to the polyesters class that are of interest as bio-derived and biodegradable plastics. The poly(3-hydroxybutyrate) (PHB) form of PHB is the most common type of polyhydroxyalkanoate, but also other polymers of this class are produced by a variety of organisms: these include poly(4-hydroxybutyrate) (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO) and their copolymers.

[0013] PHB is produced by microorganisms (such as Cupriavidus necator, Methylobacterium rhodesianum or Bacillus megaterium), apparently in response to conditions of physiological stress; mainly conditions in which nutrients are limited. Microbial biosynthesis of PHB starts with the condensation of two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA. This latter compound is then used as a monomer to polymerize PHB. PHAs granules are then recovered by disrupting the cells. PHB is naturally produced by microorganisms exclusively in the (R)-configuration, which is, as explained above, brittle.

[0014] Acetoacetyl-CoA reductase (PhaB, EC:1.1.1.36) catalyzes the chiral reduction of acetoacetyl-CoAto (R)-3-hydroxybutyryl-CoA: (R)-3-hydroxyacyl-CoA + NAD(P)+= 3-oxoacyl-CoA + H++ NAD(P)H. It is involved in the natural biosynthesis of poly(3-hydroxybutyrate) (PHB). 3-hydroxybutyryl-CoA dehydrogenase (Hbd, EC:1.1.1.157) catalyzes the reaction (3S)-3-hydroxybutyryl-CoA + NAD(P)+= acetoacetyl-CoA + H++ NAD(P)H. It follows that in step (al) of the method of the first aspect a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3-hydroxybutryl-CoA from the educt acetoacetyl-CoA is produced. Hence, both PhaB and Hbd need as a coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH). They are NADPH / NADH dependent enzymes.

[0015] 3-Hydroxybutyryl-CoA epimerase (FadB, EC:5.1.2.3) is multifunctional and has the known functions of 3-hydroxybutyryl-CoA epimerase, delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase (N-terminal) 3-hydroxyacyl-CoA dehydrogenase (C-terminal). It follows that in step (a2) of the method of the first aspect (R)-3-hydroxybutyryl-CoA is isomerized to (S)-3-hydroxybutyryl-CoA by the enzyme 3-hydroxybutyryl-CoA epimerase (FadB).

[0016] (3R)-3-Hydroxybutyryl-CoA hydrolyase (PhaJ, (R)-specific enoyl-CoA hydratase, EC:4.2.1.55 and EC:4.2.1.119) catalyzes the reaction (3R)-3-hydroxybutyryl-CoA <-> crotonyl-CoA + H₂O. This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave or form carbon-oxygen bonds. (3S)-3-Hydroxyacyl-CoA hydrolyase (Crt, short-chain-enoyl-CoA hydratase, EC:4.2.1.150, medium-chain-enoyl-CoA hydratase, EC:4.2.1.74) catalyzes the reversible hydration of crotonyl-CoA (Crt.). It follows that in step (a3) of the method of the first aspect (R)-3-hydroxybutyryl-CoA isomerized into (S)-3-hydroxybutyryl-CoA by two steps: Contacting (R)-3-hydroxybutyryl-CoA with the enzyme (3R)-3-hydroxybutyryl-CoA hydrolyase to produce Crotonyl-CoA, and then contacting the Crotonyl-CoA with the enzyme (3S)-3-hydroxyacyl-CoA hydrolyase to produce (S)-3-hydroxybutyryl-CoA.

[0017] Steps (al), (a2) and (a3) can therefore be used in isolation or together in order to produce a mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA. Step (al) is preferred as compared to steps (a2) and (a3) because this route prevents the backwards reaction from (S)-3-hydroxybutyryl CoA to (R)-3-hydroxybutyryl CoA when all (R)-3-hydroxybutyryl CoA was consumed by a slightly (R)-selective PhaC.

[0018] Polyhydroxyalkanoate synthase (PhaC, EC:2.3.1.304) is the key enzyme involved in PHA biosynthesis and functions by polymerizing monomeric hydroxyalkanoate substrates. According to the common prior art knowledge and also according to the Uniport data base entry P73390- PHAC_SYNY3 PhaC catalyzes the reaction (R)-3-hydroxybutyryl-CoA + [(R)-hydroxybutyrate](n) = [(R)-hydroxybutyrate](n+1) + CoA.

[0019] As discussed herein above, it is surprisingly demonstrated herein that PhaC and in particular the specific PhaCs as described herein not only accept (R)-3-hydroxybutyryl-CoA as substrate but also a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3-hydroxybutryl-CoA thereby allowing the production of atactic poly(3-hydroxybutyrate) (PHB), i.e. PHB comprising as monomers both, (R)- and (S)-monomers. According to the best knowledge of the inventors the method of the present invention is the first method that enables the production of atactic PHB in cells. Compared to isotactic PHB, atactic PHB has the advantage that it has a lower crystallinity and is therefore suitable for a wider range of applications. The reduced crystallinity is also accompanied by improved biodegradability compared to isotactic PHB. Atactic PHB can also be processed more easily, as the melting and decomposition temperatures are further apart than with isotactic PHB. This means that during the thermal processing of atactic PHB, the polymers do not decompose as they do with isotactic PHB. Compared to the production of copolymer-based PHAs with improved material properties, the production of atactic PHB is simpler. The synthesis of atactic PHB for example, only requires glucose feed alone, which flows into PHB synthesis via glycolysis; the production of atactic PHB also takes place via this metabolic pathway. However, the production of PHAs other than PHB requires a carbon source such as glucose for the synthesis of the 3-hydroxybutyrate building block and, in addition, fatty acids for the synthesis of 3-hydroxyvalerate building blocks or 3-hydroxyhexanoate building blocks. However, feeding glucose and fatty acids leads to problems resulting in low productivity and uneven distribution of the building blocks. Compared to the chemical synthesis of atactic PHB, the enzymatic synthesis as provided opens up industrial production for the first time, which is not possible with current chemical processes. In the examples herein below the (S)-3-hydroxybutryl-CoA fraction (%) in the obtained mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA was between 0.3% and 21.0% dependent on the source of the enzymes that were used; see Tables 3 and 4. Hence, it is possible herein to adjust the (S)-3-hydroxybutryl-CoA fraction (%) in the obtained mixture within the range of 0.3% and 21.0% as needed. The highest fraction of 21.0% ± 4.9% was obtained in the E. coli BW25113 ΔfadB ΔfadJ strain with the operon AvPhaEC-CnPhaA-CkHbd-CnPhaB; see Table 4. Using methods of enzyme engineering PhaC variants might be produced to allow extending the range above the 21 %.

[0020] In accordance with a preferred embodiment of the first aspect of the invention

[0021] (i) PhaB has an amino acid sequence of SEQ ID NO: 1 or sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and / or

[0022] (ii) Hbd has an amino acid sequence of SEQ ID NO: 3 or sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

[0023] In accordance with the present invention, the term "percent (%) sequence identity" describes the number of matches ("hits") of identical nucleotides / amino acids of two or more aligned nucleic acid or amino acid sequences as compared to the number of nucleotides or amino acid residues making up the overall length of the template nucleic acid or amino acid sequences. In other terms, using an alignment, for two or more sequences or subsequences the percentage of amino acid residues or nucleotides that are the same (e.g. 70%, 75%, 80%, 85%, 90% or 95% identity) may be determined, when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. This definition also applies to the complement of any sequence to be aligned.

[0024] Nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402). BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST). The skilled person is aware of additional suitable programs to align nucleic acid sequences.

[0025] As defined herein, sequence identities of at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, and most preferred at least 95% are envisaged by the invention. However, also envisaged by the invention are with increasing preference sequence identities of at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100%. This applies to all SEQ ID NOs herein, not only to SEQ ID NOs 1 and 3.

[0026] SEQ ID NO: 1 is the amino acid sequence of PhaB of Cupriavidus necator and SEQ ID NO: 2 is the encoding nucleotide sequence.

[0027] > Cupriavidus necator, acetoacetyl coenzyme A reductase (phaB): SEQ ID NO: 1 MTQRIAYVTGGMGGIGTAICQRLAKDGFRVVAGCGPNSPRREKWLEQQKALGFDFIASEGNVADWDSTKTAFDK VKSEVGEVDVLINNAGITRDWFRKMTRADWDAVIDTNLTSLFNVTKQVIDGMADRGWGRIVNISSVNGQKGQF GQTNYSTAKAGLHGFTMALAQEVATKGVTVNTVSPGYIATDMVKAIRQDVLDKIVATIPVKRLGLPEEIASICAWLS SEESGFSTGADFSLNGGLHMG

[0028] > Cupriavidus necator, acetoacetyl coenzyme A reductase (phaB): SEQ ID NO: 2 ATGACTCAGCGCATTGCGTATGTGACCGGCGGCATGGGTGGTATCGGAACCGCCATTTGCCAGCGGCTGGCC AAGGATGGCTTTCGTGTGGTGGCCGGTTGCGGCCCCAACTCGCCGCGCCGCGAAAAGTGGCTGGAGCAGCAG AAGGCCCTGGGCTTCGATTTCATTGCCTCGGAAGGCAATGTGGCTGACTGGGACTCGACCAAGACCGCATTCG ACAAGGTCAAGTCCGAGGTCGGCGAGGTTGATGTGCTGATCAACAACGCCGGTATCACCCGCGACGTGGTGT TCCGCAAGATGACCCGCGCCGACTGGGATGCGGTGATCGACACCAACCTGACCTCGCTGTTCAACGTCACCAA GCAGGTGATCGACGGCATGGCCGACCGTGGCTGGGGCCGCATCGTCAACATCTCGTCGGTGAACGGGCAGAA GGGCCAGTTCGGCCAGACCAACTACTCCACCGCCAAGGCCGGCCTGCATGGCTTCACCATGGCACTGGCGCAG GAAGTGGCGACCAAGGGCGTGACCGTCAACACGGTCTCTCCGGGCTATATCGCCACCGACATGGTCAAGGCG ATCCGCCAGGACGTGCTCGACAAGATCGTCGCGACGATCCCGGTCAAGCGCCTGGGCCTGCCGGAAGAGATC GCCTCGATCTGCGCCTGGTTGTCGTCGGAGGAGTCCGGTTTCTCGACCGGCGCCGACTTCTCGCTCAACGGCG GCCTGCATATGGGCTGA

[0029] SEQ ID NO: 3 is the amino acid sequence of Hbd of Clostridium kluyveri and SEQ ID NO: 4 is the encoding nucleotide sequence.

[0030] > Clostridium kluyveri, 3-hydroxybutyryl coenzyme A dehydrogenase (hbd): SEQ ID NO: 3 MKSVAVLGSGTMSRGIVQAFAEAGIDVIIRGRTEGSIGKGLAAVKKAYDKKVSKGKISQEDADKIVGRVSTTTELEKL ADCDLIIEAASEDMNIKKDYFGKLEEICKPETIFATNTSSLSITEVATATKRPDKFIGMHFFNPANVMKLVEIIRGMNTS QETFDIIKEASIKIGKTPVEVAEAPGFVVNKILVPMINEAVGILAEGIASAEDIDTAMKLGANHPMGPLALGDLIGLDV VLAVMDVLYSETGDSKYRAHTLLRKYVRAGWLGRKSGKGFFAY

[0031] > Clostridium kluyveri, 3-hydroxybutyryl coenzyme A dehydrogenase (hbd): SEQ ID NO: 4 ATGAAAAGTGTAGCGGTTTTAGGTAGTGGAACTATGTCTCGTGGAATTGTGCAGGCTTTTGCAGAAGCAGGTA TAGATGTAATTATCCGTGGAAGAACTGAAGGTAGTATTGGAAAAGGTCTAGCAGCAGTAAAGAAAGCTTATG ATAAAAAAGTATCAAAGGGGAAAATTTCCCAGGAAGATGCTGATAAAATAGTTGGAAGAGTAAGTACAACAA CTGAACTTGAAAAATTGGCTGATTGTGATCTTATAATAGAAGCAGCATCAGAGGATATGAATATAAAGAAAGA CTATTTTGGAAAATTAGAAGAAATATGCAAGCCTGAAACAATTTTTGCTACTAATACTTCTTCATTATCTATAAC TGAAGTAGCAACAGCTACAAAGAGGCCAGATAAATTCATAGGAATGCATTTCTTTAATCCAGCAAATGTTATG AAATTAGTTGAAATCATAAGAGGTATGAATACTTCACAAGAAACTTTTGATATTATAAAAGAAGCTTCCATTAA AATAGGAAAAACTCCTGTAGAAGTTGCAGAAGCTCCAGGATTTGTTGTAAACAAGATATTAGTACCAATGATC AATGAAGCAGTAGGAATTTTGGCAGAAGGAATAGCTTCAGCAGAAGATATCGATACAGCTATGAAATTAGGC GCTAATCACCCAATGGGTCCTTTAGCATTAGGAGATCTTATTGGACTTGATGTAGTTCTTGCAGTTATGGATGT ACTTTATAGTGAAACTGGAGATTCAAAATATAGAGCTCATACATTACTTAGAAAATATGTAAGAGCAGGATGG CTTGGAAGAAAATCAGGAAAAGGATTCTTCGCTTATTAA

[0032] The use of the above PhaB and Hbd sequences is illustrated by the appended examples.

[0033] In accordance with a further preferred embodiment of the first aspect of the invention FadB has an amino acid sequence of SEQ ID NO: 5 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

[0034] SEQ ID NO: 5 is the amino acid sequence of FadB of Escherichia coli and SEQ ID NO: 6 is the encoding nucleotide sequence.

[0035] > Escherichia coli, 3-hydroxyacyl-CoA epimerase, fatty acid oxidation complex subunit alpha (fadB): SEQ ID NO: 5 MLYKGDTLYLDWLEDGIAELVFDAPGSVNKLDTATVASLGEAIGVLEQQSDLKGLLLRSNKAAFIVGADITEFLSLFLV PEEQLSQWLHFANSVFNRLEDLPVPTIAAVNGYALGGGCECVLATDYRLATPDLRIGLPETKLGIMPGFGGSVRMP RMLGADSALEIIAAGKDVGADQALKIGLVDGVVKAEKLVEGAKAVLRQAINGDLDWKAKRQPKLEPLKLSKIEATM SFTIAKGMVAQTAGKHYPAPITAVKTIEAAARFGREEALNLENKSFVPLAHTNEARALVGIFLNDQYVKGKAKKLTK DVETPKQAAVLGAGIMGGGIAYQSAWKGVPVVMKDINDKSLTLGMTEAAKLLNKQLERGKIDGLKLAGVISTIHPT LDYAGFDRVDIVVEAVVENPKVKKAVLAETEQKVRQDTVLASNTSTIPISELANALERPENFCGMHFFNPVHRMPL VEIIRGEKSSDETIAKVVAWASKMGKTPIVVNDCPGFFVNRVLFPYFAGFSQLLRDGADFRKIDKVMEKQFGWPM GPAYLLDVVGIDTAHHAQAVMAAGFPQRMQKDYRDAIDALFDANRFGQKNGLGFWRYKEDSKGKPKKEEDAAV EDLLAEVSQPKRDFSEEEIIARMMIPMVNEVVRCLEEGIIATPAEADMALVYGLGFPPFHGGAFRWLDTLGSAKYLD MAQQYQHLGPLYEVPEGLRNKARHNEPYYPPVEPARPVGDLKTA > Escherichia coli, 3-hydroxyacyl-CoA epimerase, fatty acid oxidation complex subunit alpha (fadB): SEQ ID NO: 6 ATGCTTTACAAAGGCGACACCCTGTACCTTGACTGGCTGGAAGATGGCATTGCCGAACTGGTATTTGATGCCCC AGGTTCAGTTAATAAACTCGACACTGCGACCGTCGCCAGCCTCGGCGAGGCCATCGGCGTGCTGGAACAGCA ATCAGATCTAAAAGGGCTGCTGCTGCGTTCGAACAAAGCAGCCTTTATCGTCGGTGCTGATATCACCGAATTTT TGTCCCTGTTCCTCGTTCCTGAAGAACAGTTAAGTCAGTGGCTGCACTTTGCCAATAGCGTGTTTAATCGCCTG GAAGATCTGCCGGTGCCGACCATTGCTGCCGTCAATGGCTATGCGCTGGGCGGTGGCTGCGAATGCGTGCTG GCGACCGATTATCGTCTGGCGACGCCGGATCTGCGCATCGGTCTGCCGGAAACCAAACTGGGCATCATGCCTG GCTTTGGCGGTTCTGTACGTATGCCACGTATGCTGGGCGCTGACAGTGCGCTGGAAATCATTGCCGCCGGTAA AGATGTCGGCGCGGATCAGGCGCTGAAAATCGGTCTGGTGGATGGCGTAGTCAAAGCAGAAAAACTGGTTGA AGGCGCAAAGGCGGTTTTACGCCAGGCCATTAACGGCGACCTCGACTGGAAAGCAAAACGTCAGCCGAAGCT GGAACCACTAAAACTGAGCAAGATTGAAGCCACCATGAGCTTCACCATCGCTAAAGGGATGGTCGCACAAACA GCGGGGAAACATTATCCGGCCCCCATCACCGCAGTAAAAACCATTGAAGCTGCGGCCCGTTTTGGTCGTGAAG AAGCCTTAAACCTGGAAAACAAAAGTTTTGTCCCGCTGGCGCATACCAACGAAGCCCGCGCACTGGTCGGCAT TTTCCTTAACGATCAATATGTAAAAGGCAAAGCGAAGAAACTCACCAAAGACGTTGAAACCCCGAAACAGGCC GCGGTGCTGGGTGCAGGCATTATGGGCGGCGGCATCGCTTACCAGTCTGCGTGGAAAGGCGTGCCGGTTGTC ATGAAAGATATCAACGACAAGTCGTTAACCCTCGGCATGACCGAAGCCGCGAAACTGCTGAACAAGCAGCTTG AGCGCGGCAAGATCGATGGTCTGAAACTGGCTGGCGTGATCTCCACAATCCACCCAACGCTCGACTACGCCGG ATTTGACCGCGTGGATATTGTGGTAGAAGCGGTTGTTGAAAACCCGAAAGTGAAAAAAGCCGTACTGGCAGA AACCGAACAAAAAGTACGCCAGGATACCGTGCTGGCGTCTAACACTTCAACCATTCCTATCAGCGAACTGGCC AACGCGCTGGAACGCCCGGAAAACTTCTGCGGGATGGCGTTCTTTAACCCGGTCCACCGAATGCCGTTGGTAG AAATTATTCGCGGCGAGAAAAGCTCCGACGAAACCATCGCGAAAGTTGTCGCCTGGGCGAGCAAGATGGGCA AGACGCCGATTGTGGTTAACGACTGCCCCGGCTTCTTTGTTAACCGCGTGCTGTTCCCGTATTTCGCCGGTTTCA GCCAGCTGCTGCGCGACGGCGCGGATTTCCGCAAGATCGACAAAGTGATGGAAAAACAGTTTGGCTGGCCGA TGGGCCCGGCATATCTGCTGGACGTTGTGGGCATTGATACCGCGCATCACGCTCAGGCTGTCATGGCAGCAGG CTTCCCGCAGCGGATGCAGAAAGATTACCGCGATGCCATCGACGCGCTGTTTGATGCCAACCGCTTTGGTCAG AAGAACGGCCTCGGTTTCTGGCGTTATAAAGAAGACAGCAAAGGTAAGCCGAAGAAAGAAGAAGACGCCGC CGTTGAAGACCTGCTGGCAGAAGTGAGCCAGCCGAAGCGCGATTTCAGCGAAGAAGAGATTATCGCCCGCAT GATGATCCCGATGGTCAACGAAGTGGTGCGCTGTCTGGAGGAAGGCATTATCGCCACTCCGGCGGAAGCGGA TATGGCGCTGGTCTACGGCCTGGGCTTCCCTCCGTTCCACGGCGGCGCGTTCCGCTGGCTGGACACCCTCGGT AGCGCAAAATACCTCGATATGGCACAGCAATATCAGCACCTCGGCCCGCTGTATGAAGTGCCGGAAGGTCTGC GTAATAAAGCGCGTCATAACGAACCGTACTATCCTCCGGTTGAGCCAGCCCGTCCGGTTGGCGACCTGAAAAC GGCTTAA

[0036] In accordance with another preferred embodiment of the first aspect of the invention PhaJ has an amino acid sequence of SEQ ID NO: 7 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and / or Crt has an amino acid sequence of SEQ ID NO: 9 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

[0037] SEQ ID NO: 7 is the amino acid sequence of PhaJ of Aeromonas caviae and SEQ ID NO: 8 is the encoding nucleotide sequence.

[0038] > Aeromonas caviae, (R)-specific enoyl-CoA hydratase (phaJ), SEQ ID NO: 7 MSAQSLEVGQKARLSKRFGAAEVAAFAALSEDFNPLHLDPAFAATTAFERPIVHGMLLASLFSGLLGQQLPGKGSIY LGQSLSFKLPVFVGDEVTAEVEVTALREDKPIATLTTRIFTQGGALAVTGEAVVKLP

[0039] > Aeromonas caviae, (R)-specific enoyl-CoA hydratase (phaJ), SEQ ID NO: 8 ATGAGCGCACAATCCCTGGAAGTAGGCCAGAAGGCCCGTCTCAGCAAGCGGTTCGGGGCGGCGGAGGTAGC CGCCTTCGCCGCGCTCTCGGAGGACTTCAACCCCCTGCACCTGGACCCGGCCTTCGCCGCCACCACGGCGTTCG AGCGGCCCATAGTCCACGGCATGCTGCTCGCCAGCCTCTTCTCCGGGCTGCTGGGCCAGCAGTTGCCGGGCAA GGGGAGCATCTATCTGGGTCAAAGCCTCAGCTTCAAGCTGCCGGTCTTTGTCGGGGACGAGGTGACGGCCGA GGTGGAGGTGACCGCCCTTCGCGAGGACAAGCCCATCGCCACCCTGACCACCCGCATCTTCACCCAAGGCGGC GCCCTCGCCGTGACGGGGGAAGCCGTGGTCAAGCTGCCTTAA

[0040] SEQ ID NO: 9 is the amino acid sequence of Crt of Clostridium acetobutylicum and SEQ ID NO: 10 is the encoding nucleotide sequence.

[0041] > Clostridium acetobutylicum, (S)-specific enoyl-CoA hydratase (crt), SEQ ID NO: 9 MELNNVILEKEGKVAVVTINRPKALNALNSDTLKEMDYVIGEIENDSEVLAVILTGAGEKSFVAGADISEMKEMNTIE GRKFGILGNKVFRRLELLEKPVIAAVNGFALGGGCEIAMSCDIRIASSNARFGQPEVGLGITPGFGGTQRLSRLVGM GMAKQLIFTAQNIKADEALRIGLVNKVVEPSELMNTAKEIANKIVSNAPVAVKLSKQAINRGMQCDIDTALAFESEA FGECFSTEDQKDAMTAFIEKRKIEGFKNR

[0042] > Clostridium acetobutylicum, (S)-specific enoyl-CoA hydratase (crt), SEQ ID NO: 10 ATGGAACTAAACAATGTCATCCTTGAAAAGGAAGGTAAAGTTGCTGTAGTTACCATTAACAGACCTAAAGCAT TAAATGCGTTAAATAGTGATACACTAAAAGAAATGGATTATGTTATAGGTGAAATTGAAAATGATAGCGAAGT ACTTGCAGTAATTTTAACTGGAGCAGGAGAAAAATCATTTGTAGCAGGAGCAGATATTTCTGAGATGAAGGAA ATGAATACCATTGAAGGTAGAAAATTCGGGATACTTGGAAATAAAGTGTTTAGAAGATTAGAACTTCTTGAAA AGCCTGTAATAGCAGCTGTTAATGGTTTTGCTTTAGGAGGCGGATGCGAAATAGCTATGTCTTGTGATATAAG AATAGCTTCAAGCAACGCAAGATTTGGTCAACCAGAAGTAGGTCTCGGAATAACACCTGGTTTTGGTGGTACA CAAAGACTTTCAAGATTAGTTGGAATGGGCATGGCAAAGCAGCTTATATTTACTGCACAAAATATAAAGGCAG ATGAAGCATTAAGAATCGGACTTGTAAATAAGGTAGTAGAACCTAGTGAATTAATGAATACAGCAAAAGAAAT TGCAAACAAAATTGTGAGCAATGCTCCAGTAGCTGTTAAGTTAAGCAAACAGGCTATTAATAGAGGAATGCAG TGTGATATTGATACTGCTTTAGCATTTGAATCAGAAGCATTTGGAGAATGCTTTTCAACAGAGGATCAAAAGGA TGCAATGACAGCTTTCATAGAGAAAAGAAAAATTGAAGGCTTCAAAAATAGATAG

[0043] In accordance with a yet further preferred embodiment of the first aspect of the invention PhaC has an amino acid sequence of any one of SEQ ID NOs 11, 13, 15, 17 or 19 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical to any one of SEQ ID NOs 11, 13, 15, 17 or 19.

[0044] SEQ ID NO: 11 is the amino acid sequence of PhaC of Allochromatium vinosum and SEQ ID NO: 12 is the encoding nucleotide sequence.

[0045] > Allochromatium vinosum, poly(3-hydroxyalkanoate) synthase subunit C (phaC); SEQ ID NO: 11 MFPIDIRPDKLTQEMLDYSRKLGQGMENLLNAEAIDTGVSPKQAVYSEDKLVLYRYDRPEGAPEAQPVPLLIVYALV NRPYMTDIQEDRSTIKGLLATGQDVYLIDWGYPDQADRALTLDDYINGYIDRCVDYLREAHGVDKVNLLGICQGGA FSLMYSALHPDKVRNLVTMVTPVDFKTPDNLLSAWVQNVDIDLAVDTMGNIPGELLNWTFLSLKPFSLTGQKYVN MVDLLDDPDKVKNFLRMEKWIFDSPDQAGETFRQFIKDFYQNNGFLNGGVVLGGQEVDLKDITCPVLNIFALQDH LVPPDASRALKGLTSSPDYTELAFPGGHIGIYVSGKAQKEVTPAIGKWLNER

[0046] > Allochromatium vinosum, poly(3-hydroxyalkanoate) synthase subunit C (phaC), SEQ ID NO: 12 ATGTTCCCCATCGACATCCGGCCGGACAAACTCACCCAGGAGATGCTCGACTACAGCCGCAAGCTGGGTCAGG GCATGGAGAATCTGCTCAACGCCGAGGCCATCGACACCGGCGTCAGCCCCAAGCAGGCCGTCTACAGCGAGG ACAAGCTGGTCCTCTATCGCTATGACCGACCCGAGGGTGCGCCCGAAGCCCAGCCCGTGCCGCTCCTGATCGT CTACGCCCTGGTCAACCGACCCTACATGACCGACATCCAGGAAGACCGCTCCACCATCAAGGGACTGCTGGCC ACCGGCCAGGACGTCTATCTGATCGACTGGGGCTATCCGGATCAGGCCGACCGCGCGCTCACGCTCGACGACT ACATCAACGGCTACATCGACCGCTGCGTGGACTATCTGCGCGAGGCCCACGGCGTCGACAAGGTCAATCTGCT CGGTATCTGTCAGGGCGGCGCCTTCAGCCTGATGTACAGCGCGCTGCACCCGGACAAGGTGCGCAACCTCGTG ACCATGGTCACGCCGGTCGACTTCAAGACCCCGGACAACTTGCTCTCGGCTTGGGTGCAGAACGTCGACATCG ATCTGGCGGTCGACACCATGGGCAACATCCCCGGTGAGCTGCTGAACTGGACCTTCCTGTCGCTCAAGCCTTTC AGCCTGACGGGTCAGAAGTACGTCAACATGGTTGATCTGCTCGACGACCCGGACAAGGTCAAGAACTTCCTGC GCATGGAGAAATGGATCTTCGACAGCCCGGATCAGGCCGGCGAGACCTTCCGTCAGTTCATCAAGGACTTCTA CCAGAACAACGGCTTCCTCAATGGCGGCGTGGTGCTGGGCGGACAGGAAGTGGACCTGAAGGACATCACCTG CCCGGTGCTCAACATCTTCGCGCTCCAGGATCACCTGGTGCCGCCGGATGCCTCTAGGGCGCTCAAGGGACTG ACCAGCAGCCCGGACTACACCGAGCTGGCCTTCCCCGGCGGTCACATCGGCATCTATGTCAGCGGCAAGGCGC AGAAAGAAGTCACGCCGGCCATCGGTAAGTGGCTCAACGAGCGATAA

[0047] SEQ ID NO: 13 is the amino acid sequence of PhaC of Cupriavidus necator and SEQ ID NO: 14 is the encoding nucleotide sequence.

[0048] > Cupriavidus necator, poly(3-hydroxyalkanoate) synthase (phaC), SEQ ID NO: 13 MATGKGAAASTQEGKSQPFKVTPGPFDPATWLEWSRQWQGTEGNGHAAASGIPGLDALAGVKIAPAQLGDIQQ RYMKDFSALWQAMAEGKAEATGPLHDRRFAGDAWRTNLPYRFAAAFYLLNARALTELADAVEADAKTRQRIRFAI SQWVDAMSPANFLATNPEAQRLLIESGGESLRAGVRNMMEDLTRGKISQTDESAFEVGRNVAVTEGAVVFENEYF QLLQYKPLTDKVHARPLLMVPPCINKYYILDLQPESSLVRHVVEQGHTVFLVSWRNPDASMAGSTWDDYIEHAAIR AIEVARDISGQDKINVLGFCVGGTIVSTALAVLAARGEHPAASVTLLTTLLDFADTGILDVFVDEGHVQLREATLGGG AGAPCALLRGLELANTFSFLRPNDLVWNYVVDNYLKGNTPVPFDLLFWNGDATNLPGPWYCWYLRHTYLQNELK VPGKLTVCGVPVDLASIDVPTYIYGSREDHIVPWTAAYASTALLANKLRFVLGASGHIAGVINPPAKNKRSHWTNDA LPESPQQWLAGAIEHHGSWWPDWTAWLAGQAGAKRAAPANYGNARYRAIEPAPGRYVKAKA

[0049] > Cupriavidus necator, poly(3-hydroxyalkanoate) synthase (phaC), SEQ ID NO: 14 ATGGCGACCGGCAAAGGCGCGGCAGCTTCCACGCAGGAAGGCAAGTCCCAACCATTCAAGGTCACGCCGGGG CCATTCGATCCAGCCACATGGCTGGAATGGTCCCGCCAGTGGCAGGGCACTGAAGGCAACGGCCACGCGGCC GCGTCCGGCATTCCGGGCCTGGATGCGCTGGCAGGCGTCAAGATCGCGCCGGCGCAGCTGGGTGATATCCAG CAGCGCTACATGAAGGACTTCTCAGCGCTGTGGCAGGCCATGGCCGAGGGCAAGGCCGAGGCCACCGGTCCG CTGCACGACCGGCGCTTCGCCGGCGACGCATGGCGCACCAACCTCCCATATCGCTTCGCTGCCGCGTTCTACCT GCTCAATGCGCGCGCCTTGACCGAGCTGGCCGATGCCGTCGAGGCCGATGCCAAGACCCGCCAGCGCATCCG CTTCGCGATCTCGCAATGGGTCGATGCGATGTCGCCCGCCAACTTCCTTGCCACCAATCCCGAGGCGCAGCGCC TGCTGATCGAGTCGGGCGGCGAATCGCTGCGTGCCGGCGTGCGCAACATGATGGAAGACCTGACACGCGGCA AGATCTCGCAGACCGACGAGAGCGCGTTTGAGGTCGGCCGCAATGTCGCGGTGACCGAAGGCGCCGTGGTCT TCGAGAACGAGTACTTCCAGCTGTTGCAGTACAAGCCGCTGACCGACAAGGTGCACGCGCGCCCGCTGCTGAT GGTGCCGCCGTGCATCAACAAGTACTACATCCTGGACCTGCAGCCGGAGAGCTCGCTGGTGCGCCATGTGGTG GAGCAGGGACATACGGTGTTTCTGGTGTCGTGGCGCAATCCGGACGCCAGCATGGCCGGCAGCACCTGGGAC GACTACATCGAGCACGCGGCCATCCGCGCCATCGAAGTCGCGCGCGACATCAGCGGCCAGGACAAGATCAAC GTGCTCGGCTTCTGCGTGGGCGGCACCATTGTCTCGACCGCGCTGGCGGTGCTGGCCGCGCGCGGCGAGCAC CCGGCCGCCAGCGTCACGCTGCTGACCACGCTGCTGGACTTTGCCGACACGGGCATCCTCGACGTCTTTGTCG ACGAGGGCCATGTGCAGTTGCGCGAGGCCACGCTGGGCGGCGGCGCCGGCGCGCCGTGCGCGCTGCTGCGC GGCCTTGAGCTGGCCAATACCTTCTCGTTCTTGCGCCCGAACGACCTGGTGTGGAACTACGTGGTCGACAACTA CCTGAAGGGCAACACGCCGGTGCCGTTCGACCTGCTGTTCTGGAACGGCGACGCCACCAACCTGCCGGGGCC GTGGTACTGCTGGTACCTGCGCCACACCTACCTGCAGAACGAGCTCAAGGTACCGGGCAAGCTGACCGTGTGC GGCGTGCCGGTGGACCTGGCCAGCATCGACGTGCCGACCTATATCTACGGCTCGCGCGAAGACCATATCGTGC CGTGGACCGCGGCCTATGCCTCGACCGCGCTGCTGGCGAACAAGCTGCGCTTCGTGCTGGGTGCGTCGGGCC ATATCGCCGGTGTGATCAACCCGCCGGCCAAGAACAAGCGCAGCCACTGGACTAACGATGCGCTGCCGGAGT CGCCGCAGCAATGGCTGGCCGGCGCCATCGAGCATCACGGCAGCTGGTGGCCGGACTGGACCGCATGGCTGG CCGGGCAGGCCGGCGCGAAACGCGCCGCGCCCGCCAACTATGGCAATGCGCGCTATCGCGCAATCGAACCCG CGCCTGGGCGATACGTCAAAGCCAAGGCATGA

[0050] SEQ ID NO: 15 is the amino acid sequence of PhaC of Chromobacterium sp. and SEQ ID NO: 16 is the encoding nucleotide sequence.

[0051] > Chromobacterium sp., poly(3-hydroxyalkanoate) synthase (phaC), SEQ ID NO: 15 MQQFVNSLSLGQDQSDAPHPLTGAWSQLMSQTNQLLQLQSSLYQQQLGLWTQFLGQTAGNDASAPSAKPSDR RFASPEWDEHPFYSFLKQSYLQTSKWMMELVDKTQIDESAKDKLSFATRQYLDAMAPSNFMLTNPDVVKRAIETQ GESLVEGMKNMMEDIQKGHISMSDESKFQIGKNLVVTPGEVVFRNELIELIQYTPTTEKVHEKPLLFVPPCINKYYL MDLQPDNSMVRHFVGQGYRVFLVSWRSAVPEMKNFTWETYIEKGVFAAAEAVQKITKQPTMNALGFCVGGVIL TTALCVAQAKGLKYFDSATFMTSLIDHAEPGEISFFIDEALVASREAKMAAGGIISGKEIGRTFASLRANDLVWNYVV NNYLLGKTPAPFDLLYWNNDAVDLPLPMHTFMLRQFYINNALITPGAITLCGVPIDISKIDIPVYMFAAREDHIVLWS SAYSGLKYLSGTPSRRFVLGASGHIAGSINPVTKDKRNYWTNEQLPVNPEEWLEGAQSHPGSWWKDWDAWLAP QSGKQVPAPKMLGSKEFPPLQPAPGSYVLAKAMPPVAAALN

[0052] > Chromobacterium sp., poly(3-hydroxyalkanoate) synthase (phaC), SEQ ID NO: 16 ATGCAGCAGTTTGTTAATAGCCTGAGCCTGGGTCAAGATCAGAGTGATGCACCGCATCCGCTGACCGGTGCAT GGTCACAGCTGATGAGCCAGACCAATCAGCTGCTGCAACTGCAGAGCAGCCTGTATCAGCAGCAGTTAGGTCT GTGGACCCAGTTTCTGGGTCAGACCGCAGGTAATGATGCAAGCGCACCGAGCGCAAAACCGAGCGATCGTCG CTTTGCAAGTCCGGAATGGGATGAACATCCGTTTTATAGCTTTCTGAAACAGAGCTATCTGCAGACCAGCAAAT GGATGATGGAACTGGTTGATAAAACCCAGATTGATGAAAGCGCCAAAGATAAACTGAGCTTTGCAACCCGTCA GTATCTGGATGCTATGGCACCGAGCAATTTTATGCTGACCAATCCGGATGTTGTTAAACGTGCAATTGAAACCC AGGGTGAAAGCCTGGTTGAAGGTATGAAAAATATGATGGAAGATATCCAGAAAGGCCATATTAGCATGTCCG ATGAAAGCAAATTTCAGATCGGTAAAAATCTGGTTGTTACACCGGGTGAAGTTGTGTTTCGTAATGAACTGATC GAGCTGATTCAGTATACCCCGACCACCGAAAAAGTTCATGAAAAACCGCTGCTGTTTGTTCCGCCTTGCATTAA TAAGTATTATCTGATGGATCTGCAGCCGGATAATAGCATGGTTCGTCATTTTGTTGGTCAGGGTTATCGTGTTT TTCTGGTTAGCTGGCGTAGCGCAGTTCCGGAAATGAAAAACTTTACCTGGGAAACCTATATCGAAAAAGGTGT TTTTGCAGCCGCAGAAGCAGTTCAGAAAATTACCAAACAGCCGACCATGAATGCACTGGGTTTTTGTGTTGGT GGTGTTATTCTGACCACCGCACTGTGTGTTGCACAGGCAAAAGGTCTGAAATACTTTGATAGCGCAACCTTTAT GACCAGCCTGATTGATCATGCAGAACCTGGTGAAATTAGCTTTTTCATTGATGAAGCACTGGTTGCAAGCCGTG AAGCAAAAATGGCAGCCGGTGGTATTATTAGCGGTAAAGAAATTGGTCGTACCTTTGCAAGCCTGCGTGCAAA TGATCTGGTTTGGAATTATGTGGTGAACAATTATCTGCTGGGTAAAACACCGGCACCGTTTGATCTGCTGTATT GGAATAATGATGCCGTTGATCTGCCGCTGCCGATGCATACCTTTATGCTGCGTCAGTTTTATATCAACAACGCA CTGATTACTCCGGGTGCAATTACCCTGTGTGGTGTTCCGATTGATATTAGCAAAATTGATATCCCGGTGTACAT GTTTGCAGCACGTGAAGATCATATTGTTCTGTGGTCAAGCGCATATAGTGGCCTGAAATATCTGAGCGGCACC CCGAGCCGTCGTTTTGTTCTGGGTGCCAGCGGTCATATTGCAGGTAGCATTAATCCGGTGACCAAAGACAAAC GTAATTATTGGACCAATGAACAGCTGCCGGTTAATCCGGAAGAATGGCTGGAAGGTGCACAGAGCCATCCTG GTAGTTGGTGGAAAGATTGGGATGCATGGCTGGCACCGCAGTCAGGTAAACAGGTTCCGGCACCGAAAATGC TGGGTAGCAAAGAATTTCCGCCTCTGCAGCCTGCACCGGGTAGCTATGTTCTGGCAAAAGCAATGCCTCCGGT TGCCGCAGCACTGAATTAA

[0053] SEQ ID NO: 17 is the amino acid sequence of PhaC of Priestia megaterium and SEQ ID NO: 18 is the encoding nucleotide sequence.

[0054] > Priestia megaterium, poly(3-hydroxyalkanoate) synthase subunit C (phaC), SEQ ID NO: 17 MAIPYVQEWEKLIKSMPSEYKSSARRFKRAYEIMTTEAEPEVGLTPKEVIWKKNKAKLYRYTPVKDNLHKTPILLVYA LINKPYILDLTPGNSLVEYLLNRGFDVYLLDWGTPGLEDSNMKLDDYIVDYIPKAAKKVLRTSKSPDLSVLGYCMGGT MTSIFAALNEDLPIKNLIFMTSPFDFSDTGLYGAFLDDRYFNLDKAVDTFGNIPPEMIDFGNKMLKPITNFYGPYVTL VDRSENQRFVESWKLMQKWVADGIPFAGEAYRQWIRDFYQQNKLINGELEVRGRKVDLKNIKANILNIAASRDHI AMPHQVAALMDAVSSEDKEYKLLQTGHVSVVFGPKAVKETYPSIGDWLEKRSK

[0055] > Priestia megaterium, poly(3-hydroxyalkanoate) synthase subunit C (phaC), SEQ ID NO: 18 ATGGCAATTCCTTACGTGCAAGAGTGGGAAAAATTAATCAAATCAATGCCAAGTGAATATAAAAGTTCTGCAA GACGTTTTAAGCGTGCATATGAAATTATGACAACAGAAGCGGAACCGGAAGTTGGATTAACACCAAAAGAGG TTATTTGGAAAAAGAACAAAGCGAAATTATATCGCTATACGCCAGTAAAAGATAACCTGCATAAAACACCAAT CTTACTCGTATATGCATTGATCAATAAACCGTATATTTTGGATTTAACACCTGGAAACAGCCTTGTTGAATACTT ATTAAACCGCGGTTTTGACGTGTATTTGCTTGATTGGGGAACTCCTGGGCTTGAAGACAGCAATATGAAGCTA GATGATTATATTGTAGATTATATTCCAAAAGCGGCGAAAAAGGTGCTGCGCACTTCTAAATCTCCTGATTTGTC TGTTCTTGGTTACTGCATGGGCGGAACTATGACATCTATTTTTGCTGCATTAAATGAAGACTTGCCGATTAAAA ACTTAATTTTTATGACAAGTCCATTTGATTTTTCCGATACAGGTTTATACGGAGCATTCCTAGACGATCGCTACT TTAATTTAGATAAAGCAGTAGATACATTCGGAAACATCCCTCCAGAGATGATTGACTTTGGAAACAAGATGTTA AAGCCAATCACGAATTTCTACGGCCCGTATGTAACGTTGGTGGACCGTTCCGAAAATCAGCGGTTTGTTGAAA GCTGGAAGCTAATGCAAAAGTGGGTTGCTGACGGAATCCCATTTGCTGGTGAAGCTTATCGTCAGTGGATTCG TGACTTCTATCAACAAAATAAATTAATCAATGGTGAACTTGAAGTTCGCGGACGCAAAGTAGATTTGAAAAATA TTAAAGCTAATATTTTAAACATCGCTGCTAGCCGTGATCATATTGCGATGCCGCATCAGGTGGCAGCTTTAATG GACGCTGTTTCAAGTGAAGATAAAGAGTATAAATTGTTGCAAACAGGTCACGTATCTGTTGTATTTGGTCCAAA AGCAGTGAAGGAAACATATCCTTCAATCGGCGATTGGCTAGAAAAACGCTCTAAATAA

[0056] SEQ ID NO: 19 is the amino acid sequence of PhaC of Pseudomonas sp. and SEQ ID NO: 20 is the encoding nucleotide sequence.

[0057] > Pseudomonas sp., poly(3-hydroxyalkanoate) synthase subunit C (phaC), SEQ ID NO: 19 MSNKNSDDLNRQASENTLGLNPVIGLRGKDLLTSARMVLTQAIKQPIHSVKHVAHFGIELKNVMFGKSKLQPESDD RRFNDPAWSQNPLYKRYLQTYLAWRKELHDWIGNSKLSEQDINRAHFVITLMTEAMAPTNSAANPAAVKRFFETG GKSLLDGLTHLAKDLVNNGGMPSQVDMGAFEVGKSLGTTEGAVVFRNDVLELIQYRPTTEQVHERPLLVVPPQIN KFYVFDLSPDKSLARFCLSNNQQTFIVSWRNPTKAQREWGLSTYIDALKEAVDVVSAITGSKDINMLGACSGGITCT ALLGHYAALGEKKVNALTLLVTVLDTTLDSQVALFVDEKTLEAAKRHSYQAGVLEGRDMAKVFAWMRPNDLIWNY WVNNYLLGNEPPVFDILFWNNDTTRLPAAFHGDLIEMFKNNPLVRANALEVSGTPIDLKQVTADIYSLAGTNDHIT PWKSCYKSAQLFGGKVEFVLSSSGHIKSILNPPGNPKSRYMTSTDMPATANEWQENSTKHTDSWWLHWQAWQ AERSGKLKKSPTSLGNKAYPSGEAAPGTYVHER

[0058] > Pseudomonas sp., poly(3-hydroxyalkanoate) synthase subunit C (phaC), SEQ ID NO: 20 ATGAGCAACAAAAACAGCGACGATCTGAATCGTCAGGCAAGCGAAAACACCCTGGGTCTGAATCCGGTTATTG GTCTGCGTGGTAAAGATCTGCTGACCAGCGCACGTATGGTTCTGACCCAGGCAATTAAACAGCCGATTCATAG CGTTAAACATGTGGCCCATTTTGGCATTGAACTGAAAAATGTGATGTTTGGCAAAAGCAAACTGCAGCCGGAA AGTGATGATCGTCGTTTTAATGATCCGGCATGGTCACAGAATCCGCTGTATAAACGTTATCTGCAGACCTATCT GGCATGGCGTAAAGAACTGCATGATTGGATTGGTAATAGCAAACTGAGCGAACAGGATATCAATCGTGCGCA TTTTGTTATTACCCTGATGACCGAAGCAATGGCACCGACCAATAGCGCAGCAAATCCGGCAGCAGTTAAACGT TTTTTTGAAACCGGTGGTAAAAGCCTGCTGGATGGCCTGACCCATCTGGCAAAAGATCTGGTTAATAATGGTG GTATGCCGAGCCAGGTTGATATGGGTGCATTTGAAGTTGGTAAAAGTCTGGGCACCACCGAAGGTGCAGTTG TTTTTCGTAATGATGTTCTGGAACTGATCCAGTATCGTCCGACCACCGAACAGGTGCATGAACGTCCGCTGCTG GTTGTTCCGCCTCAGATTAACAAATTCTATGTGTTTGATCTGAGTCCGGACAAAAGCCTGGCACGTTTTTGTCTG AGCAATAATCAGCAGACATTTATTGTGAGCTGGCGTAATCCGACCAAAGCACAGCGTGAATGGGGTCTGAGC ACCTATATTGATGCACTGAAAGAAGCAGTTGACGTTGTTAGCGCAATTACCGGTAGCAAAGATATTAACATGC TGGGTGCATGTAGCGGTGGTATTACCTGTACCGCACTGCTGGGTCATTATGCAGCACTGGGTGAAAAAAAAGT TAATGCACTGACCCTGCTGGTTACCGTTCTGGATACCACCCTGGATAGCCAGGTGGCACTGTTTGTTGATGAAA AAACCCTGGAAGCAGCAAAACGTCATAGCTATCAGGCAGGCGTTCTGGAAGGTCGTGATATGGCAAAAGTTTT TGCATGGATGCGTCCGAATGATCTGATTTGGAATTATTGGGTGAACAACTATCTGCTGGGTAATGAACCGCCT GTTTTCGATATTCTGTTTTGGAATAATGATACCACACGTCTGCCTGCAGCATTTCATGGCGATCTGATTGAAATG TTTAAAAACAATCCGCTGGTTCGTGCAAATGCACTGGAAGTTAGCGGCACCCCGATTGATCTGAAACAGGTTA CCGCAGATATCTATAGCCTGGCAGGCACCAATGATCACATTACCCCGTGGAAAAGCTGTTATAAAAGCGCACA GCTGTTTGGTGGTAAAGTTGAATTTGTTCTGAGCAGCAGCGGTCATATCAAAAGCATTCTGAATCCTCCGGGTA ATCCGAAAAGTCGTTATATGACCAGCACCGATATGCCTGCAACCGCAAATGAATGGCAAGAAAATAGCACCAA ACATACCGATAGTTGGTGGCTGCATTGGCAGGCATGGCAGGCCGAACGTAGCGGTAAACTGAAAAAAAGCCC GACCAGCCTGGGTAATAAAGCATATCCGAGCGGTGAAGCAGCACCGGGTACGTATGTTCACGAACGTTAA

[0059] The use of the above PhaC sequences is illustrated by the appended examples. For these PhaC it has been shown that they can produce atactic poly(3-hydroxybutyrate) in step (b) of the method of the first aspect.

[0060] In accordance with a more preferred embodiment of the first aspect of the invention the PhaC of SEQ ID NO: 11 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto is used together with the PhaE of SEQ ID NO: 21 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and / or

[0061] the PhaC of SEQ ID NO: 17 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto is used together with the PhaR of SEQ ID NO: 23 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

[0062] SEQ ID NO: 21 is the amino acid sequence of PhaE of Allochromatium vinosum and SEQ ID NO: 22 is the encoding nucleotide sequence. While PhaC of Allochromatium vinosum alone is enzymatically active and sufficient to produce atactic poly(3-hydroxybutyrate) in step (b) of the method of the first aspect, PhaE of Allochromatium vinosum further improves the enzymatic activity.

[0063] > Allochromatium vinosum, poly(3-hydroxyalkanoate) synthase subunit E (phaE), SEQ ID NO: 21 MSNTNFFNDDWLELQRKYWDNWTDMSRKAMGLDSASSSATTPWEAAIDQWWKAMAPAAPDLSRSFMEKM MEQGKNFFRLADTFAKRADEGNAGNGLELWTKTLEDMQKRFSGSLDDGGNTMQRLMSFWELPLDNWQRMM SSMSPMPGDMLRNMPHEQFKDSLDRALSAPGLGYTREEQSQYQELMRSAMEYQAALQEYTNVYTKLGMKSVEH MGSYIQGVIDSGKTIDSARALYDNWVACCEGAYADEVATPEYARIHGRLVNAQMALKKRMSILVDENLGALNMPT RSELRTLQDRLQETRRENKALRHSLHSLERRVAALAGEEPATKPATALRSPAPAAKAPARRRTTKTNPAD

[0064] > Allochromatium vinosum, poly(3-hydroxyalkanoate) synthase subunit E (phaE), SEQ ID NO: 22 ATGAGCAACACTAATTTCTTCAATGACGATTGGCTCGAACTGCAACGCAAGTACTGGGACAACTGGACCGACA TGAGCCGCAAGGCCATGGGTCTGGACAGCGCCTCCAGTTCGGCCACCACGCCCTGGGAGGCGGCCATCGATC AGTGGTGGAAAGCCATGGCGCCCGCCGCCCCGGATCTGTCGCGCTCGTTCATGGAGAAGATGATGGAGCAGG GCAAGAACTTCTTCCGTCTCGCCGACACCTTCGCCAAACGCGCCGACGAGGGCAACGCCGGCAACGGCTTGGA GCTCTGGACCAAGACGCTCGAAGACATGCAGAAGCGCTTCAGCGGCTCGCTCGACGACGGCGGCAACACCAT GCAGCGCCTGATGTCGTTCTGGGAGCTGCCGCTCGACAACTGGCAGCGCATGATGTCCTCGATGTCGCCGATG CCGGGCGACATGCTGCGCAACATGCCCCACGAGCAGTTCAAGGACAGTCTTGATCGAGCCCTCTCGGCGCCCG GTCTCGGCTACACCCGCGAGGAGCAGAGCCAGTATCAGGAGCTGATGCGCAGCGCCATGGAGTACCAGGCCG CGCTCCAGGAATACACCAACGTCTACACCAAGCTCGGCATGAAGTCGGTCGAGCACATGGGTTCCTACATCCA GGGTGTCATCGACAGCGGCAAAACCATCGACTCGGCGCGCGCGCTCTATGACAACTGGGTCGCCTGCTGCGA AGGCGCCTATGCCGACGAGGTCGCCACCCCGGAGTACGCCCGCATCCACGGCCGGCTGGTCAACGCCCAGAT GGCGCTCAAGAAGCGCATGTCGATCCTGGTCGACGAGAACCTGGGTGCATTGAACATGCCGACCCGCAGCGA ACTGCGCACCCTCCAGGACCGTCTCCAGGAGACCCGTCGCGAGAACAAGGCCCTGCGTCACAGCCTGCACAGT CTGGAACGTCGCGTCGCGGCCCTGGCCGGCGAGGAGCCGGCCACCAAGCCCGCCACCGCGCTCCGGTCGCCC GCCCCGGCCGCCAAGGCTCCGGCACGCCGGCGCACCACCAAGACCAATCCGGCCGATTGA

[0065] SEQ ID NO: 23 is the amino acid sequence of PhaR of Priestia megaterium and SEQ ID NO: 24 is the encoding nucleotide sequence. While PhaC of Priestia megaterium alone is enzymatically active and sufficient to produce atactic poly(3-hydroxybutyrate) in step (b) of the method of the first aspect, PhaR of Priestia megaterium further improves the enzymatic activity.

[0066] > Priestia megaterium, poly(3-hydroxyalkanoate) synthase subunit R (phaR), SEQ ID NO: 23 MEQQKVFDPFQAWKDVYDKTESYWGKVIGDNMNREEFSQLMGNVLNMNLQYQQAVNEVTGRYLHQVNVPTK EDVANVASLVINVEEKVELLEEQFDDRFDELEAQQESASALKKDVTKLKSDVKSLDKKLDKVLSLLEGQQKTQDELKE TIQKQIKTQGEQLQAQLLEKQEKLAEKPKAEAKSEAKPSNAQKTEQPARK

[0067] > Priestia megaterium, poly(3-hydroxyalkanoate) synthase subunit R (phaR), SEQ ID NO: 24 ATGGAACAGCAAAAAGTATTTGATCCGTTTCAAGCATGGAAAGACGTATATGACAAAACCGAATCTTACTGGG GTAAAGTTATTGGGGACAATATGAATCGTGAAGAATTTTCCCAGCTCATGGGAAATGTGCTAAATATGAACCT TCAATATCAACAAGCAGTAAATGAAGTAACGGGGCGCTATCTGCACCAAGTAAATGTACCAACAAAAGAAGAT GTAGCAAACGTTGCGTCATTAGTCATCAATGTGGAAGAAAAAGTAGAATTATTAGAAGAGCAATTTGACGATC GTTTTGACGAATTAGAAGCACAGCAAGAAAGTGCATCTGCTTTGAAAAAAGATGTCACTAAGCTGAAATCTGA TGTCAAATCGTTAGACAAAAAACTCGACAAAGTTTTATCTCTTCTTGAAGGGCAGCAAAAAACACAAGACGAG TTAAAAGAAACTATTCAAAAACAAATTAAAACTCAAGGTGAGCAGCTTCAGGCTCAGCTGTTAGAAAAACAAG AAAAATTAGCTGAAAAACCAAAGGCAGAAGCTAAATCTGAAGCAAAACCATCAAATGCTCAAAAAACTGAGC AGCCGGCTCGCAAGTAA In accordance with a preferred embodiment of the first aspect of the invention the method further comprises prior to step (a) (a') catalyzing acetyl-CoA into acetoacetyl-CoA by contacting acetyl-CoA with an enzyme catalyzing the reaction 2 acetyl-CoA = acetoacetyl-CoA + CoA, wherein the enzyme is preferably acetyl-CoA C-acetyltransferase (β-ketothiolase, PhaA).

[0068] Acetyl-CoA C-acetyltransferase (β-ketothiolase, PhaA; EC. 2.3.1.9) catalyzes the condensation of two acetyl-CoA units to form acetoacetyl-CoA: 2 acetyl-CoA = acetoacetyl-CoA + CoA. The enzyme is involved in the biosynthesis of poly-(3-hydroxybutyrate) in bacteria.

[0069] In order to provide acetyl-CoA, in particular in a cell, glucose can and is preferably used as initial feed. The cell then can metabolize glucose into 2x acetyl-CoA by glycolysis. During glycolysis, glucose is broken down into two three-carbon molecules of pyruvate. Glycolysis can occur in both aerobic and anaerobic states.

[0070] While glucose is preferred, alternative carbon sources can used. Such alternative carbon sources include other carbohydrates like starch, sucrose, fructose or lactose; food and agricultural by-products such as molasses, whey or corn steep liquor; agricultural waste products such as lignocellulose hydrolysates or crude glycerol; sewage sludge; organic acids and their esters such as acetic acid, butyric acid or longer chain fatty acids including triglycerides from waste cooking oil or animal fat; as well as C1-molecules such as methane, methanol, formaldehyde, formic acid, Carbon monoxide or Carbon dioxide. Because polymers are bulk chemicals, carbon sources are important to decrease the overall cost.

[0071] In accordance with a more preferred embodiment of the first aspect of the invention PhaA has an amino acid sequence of SEQ ID NO: 25 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

[0072] SEQ ID NO: 25 is the amino acid sequence of PhaA of Cupriavidus necator and SEQ ID NO: 26 is the encoding nucleotide sequence.

[0073] > Cupriavidus necator, beta-ketothiolase (phaA), SEQ ID NO: 25 MTDVVIVSAARTAVGKFGGSLAKIPAPELGAVVIKAALERAGVKPEQVSEVIMGQVLTAGSGQNPARQAAIKAGLP AMVPAMTINKVCGSGLKAVMLAANAIMAGDAEIVVAGGQENMSAAPHVLPGSRDGFRMGDAKLVDTMIVDGL WDVYNQYHMGITAENVAKEYGITREAQDEFAVGSQNKAEAAQKAGKFDEEIVPVLIPQRKGDPVAFKTDEFVRQ GATLDSMSGLKPAFDKAGTVTAANASGLNDGAAAVVVMSAAKAKELGLTPLATIKSYANAGVDPKVMGMGPVP ASKRALSRAEWTPQDLDLMEINEAFAAQALAVHQQMGWDTSKVNVNGGAIAIGHPIGASGCRILVTLLHEMKRR DAKKGLASLCIGGGMGVALAVERK

[0074] > Cupriavidus necator, beta-ketothiolase (phaA), SEQ. ID NO: 26 ATGACTGACGTTGTCATCGTATCCGCCGCCCGCACCGCGGTCGGCAAGTTTGGCGGCTCGCTGGCCAAGATCC CGGCACCGGAACTGGGTGCCGTGGTCATCAAGGCCGCGCTGGAGCGCGCCGGCGTCAAGCCGGAGCAGGTG AGCGAAGTCATCATGGGCCAGGTGCTGACCGCCGGTTCGGGCCAGAACCCCGCACGCCAGGCCGCGATCAAG GCCGGCCTGCCGGCGATGGTGCCGGCCATGACCATCAACAAGGTGTGCGGCTCGGGCCTGAAGGCCGTGATG CTGGCCGCCAACGCGATCATGGCGGGCGACGCCGAGATCGTGGTGGCCGGCGGCCAGGAAAACATGAGCGC CGCCCCGCACGTGCTGCCGGGCTCGCGCGATGGTTTCCGCATGGGCGATGCCAAGCTGGTCGACACCATGATC GTCGACGGCCTGTGGGACGTGTACAACCAGTACCACATGGGCATCACCGCCGAGAACGTGGCCAAGGAATAC GGCATCACACGCGAGGCGCAGGATGAGTTCGCCGTCGGCTCGCAGAACAAGGCCGAAGCCGCGCAGAAGGC CGGCAAGTTTGACGAAGAGATCGTCCCGGTGCTGATCCCGCAGCGCAAGGGCGACCCGGTGGCCTTCAAGAC CGACGAGTTCGTGCGCCAGGGCGCCACGCTGGACAGCATGTCCGGCCTCAAGCCCGCCTTCGACAAGGCCGG CACGGTGACCGCGGCCAACGCCTCGGGCCTGAACGACGGCGCCGCCGCGGTGGTGGTGATGTCGGCGGCCA AGGCCAAGGAACTGGGCCTGACCCCGCTGGCCACGATCAAGAGCTATGCCAACGCCGGTGTCGATCCCAAGG TGATGGGCATGGGCCCGGTGCCGGCCTCCAAGCGCGCCCTGTCGCGCGCCGAGTGGACCCCGCAAGACCTGG ACCTGATGGAGATCAACGAGGCCTTTGCCGCGCAGGCGCTGGCGGTGCACCAGCAGATGGGCTGGGACACCT CCAAGGTCAATGTGAACGGCGGCGCCATCGCCATCGGCCACCCGATCGGCGCGTCGGGCTGCCGTATCCTGGT GACGCTGCTGCACGAGATGAAGCGCCGTGACGCGAAGAAGGGCCTGGCCTCGCTGTGCATCGGCGGCGGCA TGGGCGTGGCGCTGGCAGTCGAGCGCAAATAA

[0075] The use of the above PhaA sequences is illustrated by the appended examples.

[0076] In accordance with a preferred embodiment of the first aspect of the invention the method is carried out in a host cell, wherein the host cell comprises genes encoding the enzymes as described herein above and the host cell expresses the enzymes as described herein above.

[0077] The method is preferably an ex vivo or in vitro method. Similarly, the method preferably excludes methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.

[0078] As discussed above, the method of the invention enables for the first time the production of atactic poly(3-hydroxybutyrate) in a cell. Hence, naturally occurring cells are not able to produce atactic poly(3-hydroxybutyrate) but only genetically engineered host cells that comprise genes encoding the enzymes as described herein above and the host cell expresses the enzymes as described herein above being required to produce atactic poly(3-hydroxybutyrate). The host may already in part naturally express the genes encoding the above-described enzymes being required for the production of atactic poly(3-hydroxybutyrate), so that only the remaining genes are to be introduced.

[0079] The term "genetically engineered cell" as used herein designates a genetically engineered cell that has been genetically engineered to express at least one enzyme that is not expressed by a corresponding (wild-type) cell that has been used to prepare the genetically engineered cell. It follows that the genetically engineered microorganism has been prepared by technical means and does not occur in nature.

[0080] In this respect, genetic engineering generally means the artificial manipulation, modification, or recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organisms. Genetic engineering can be accomplished using multiple art-established techniques. Non-limiting examples are transformation, transfection and transduction. Transformation is the direct alteration of a genetic component of a cell by passing the genetic material through the cell membrane. The cell membrane can be made amenable for the uptake of the genetic components to be transformed into the cell, for example, by divalent cations (e.g. calcium cations) or electroporation. The process used to insert foreign DNA into a cell is usually called transfection. For instance, liposomes and polymers can be used as vectors to deliver DNA into cultured animal cells via transfection. Positively charged liposomes bind with DNA, while polymers can be designed that interact with DNA. They form lipoplexes and polyplexes, respectively, which are then taken up by the cells. Other transfection techniques, e.g. including using electroporation and biolistics, are also known in the art.

[0081] The term "expression" (or gene expression) designates the process by which information from a gene is used in the synthesis of a functional gene product, which product is in the present case a protein (more specifically an enzyme). Hence, expression comprises the steps of transcription of a gene into mRNA and the translation of the mRNA into protein.

[0082] The nature of the host cell will be further detailed herein below.

[0083] In accordance with a more preferred embodiment of the first aspect of the invention the genes are organized into a cluster of genes under the control of a single promoter or wherein each of the genes is under the control of a separate promoter. The first option of genes that are organized into a cluster of genes under the control of a single promoter is also designated "operon". An operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. Originally, operons were thought to exist solely in prokaryotes (which includes organelles like plastids that are derived from bacteria), but their discovery in eukaryotes was shown in the early 1990s, and are considered to be rare. In general, expression of prokaryotic operons leads to the generation of polycistronic mRNAs, while eukaryotic operons lead to monocistronic mRNAs. Although an operon only comprises one promoter for all genes, the strength of the expression can be modulated, noting that genes being close to the promoter are generally expressed stronger than genes being far away from the promoter. It is also possible to add more than one copy of a particular gene encoding a particular enzyme into an operon.

[0084] For the second option, wherein each of the genes is under the control of a separate promoter, the expression strength of the genes can be determined by using a stronger or weaker promoter, as needed.

[0085] If needed, it is a matter of routine to set the desired expression strength with the position in an operon or by different promoters.

[0086] In accordance with another more preferred embodiment of the first aspect of the invention the genes are in the genome of the host cell, or on one or more plasmids or vectors.

[0087] Hence, the genes as defined in connection with the first aspect of the invention can be expressed extrachromosomally from plasmids as well as intrachromosomally by introducing them into the genome of the host cell. While expression from plasmids and genomic expression both work, genomic expression of the enzymes is preferred since it is generally more robust as compared to expression from plasmids.

[0088] It is also possible and preferable to add more than one copy of the genes encoding the enzyme into the genome of the enzyme, such as with increasing preference at least 2, at least 5, at least 10 and at least 20 copies.

[0089] The term "vector or plasmid" in accordance with the invention means preferably a cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering which carries the nucleic acid molecule of the invention. The nucleic acid molecule(s) encoding the enzymes can, for example, be inserted into one or more of several commercially available vectors. Non-limiting examples include prokaryotic plasmid vectors, such as of the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXlN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

[0090] The nucleic acid molecules inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and / or to other amino acid encoding sequences can also be carried out using established methods. Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of transcription (e. g., translation initiation codon, promoters, such as naturally-associated or heterologous promoters and / or insulators; see above), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers.

[0091] The present invention relates in a second aspect to a nucleic acid molecule comprising or consisting of the genes encoding the enzymes according to the first aspect, wherein the genes are organized into a cluster of genes under the control of a single promoter or wherein each of the genes is under the control of a separate promoter.

[0092] In accordance with a preferred embodiment of the second aspect the nucleic acid molecule is a plasmid or vector or genome.

[0093] The definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second aspect of the invention as far as being amenable therewith. For instance, the nucleic acid molecule may be one of the particular vectors or plasmids as described herein above in connection with the first aspect.

[0094] The term "nucleic acid molecule" in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA. The nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001, 8: 1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. A nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like.

[0095] The genome is preferably a microbial genome and most preferably a bacterial genome.

[0096] With respect to the feature "comprising or consisting of the genes encoding the enzymes according to the first aspect" it is to be understood that the options "comprising" and "consisting of" apply each independently to the genes encoding the enzymes according to the first aspect of items (al) / (b), (a2) / (b) and (a3) / (b). For instance, the nucleic acid molecule, preferably the plasmid or vector or genome, may consist of the genes encoding the enzymes according to the first aspect of items (a3) / (b) or alternatively comprise the genes encoding the enzymes according to the first aspect of items (al) / (b) and (a2) / (b).

[0097] It is also preferred that the nucleic acid molecule, preferably plasmid or vector or genome, encodes the enzymes according to the first aspect but no further enzymes. Also this preferred embodiment may apply each independently to the genes encoding the enzymes according to the first aspect of items (al) / (b), (a2) / (b) and (a3) / (b). For instance, the nucleic acid molecule, preferably the plasmid or vector or genome, may encode the enzymes according to the first aspect of items (a3) / (b) but no further enzymes or alternatively the enzymes according to the first aspect of items (al) / (b) and (a2) / (b) optionally with further enzymes.

[0098] The present invention relates in a third aspect to a host cell which comprises genes encoding the enzymes as described in connection with the first aspect and being capable of expressing the enzymes as described in connection with the first aspect.

[0099] The definitions and preferred embodiments of the first and second aspect of the invention apply mutatis mutandis to the third aspect of the invention as far as being amenable therewith. For instance, the nucleic acid molecule may be one of the particular vectors or plasmids as described herein above in connection with the fist aspect.

[0100] The host cell herein (also in connection with the first aspect) may have deletions in genes involved in the p-oxidation cycle, in particular AfadB and AfadJ. It is known that such deletions improve the yield of PHA being produced by the host cell; see, for example, Agnew et al. (2012), Metab Eng; 14(6): 10.1016 / j.ymben.2012.08.003. For the production of atactic PHB it is advantageous that these deletions prevent the degradation of (S)-3-hydroxybutyryl-CoA. Under conditions of a somewhat stereoselective PhaC and the presence of PhaB and Hbd or PhaJ and Crt, these deletions can support higher (S)-3-hydroxybutyrate contents by preventing (S)-3-hydroxybutyryl-CoA isomerization. Hence, it was also found herein that the two knockouts increase the (S)-enantiomer content and this technical effect has not been observed before.

[0101] In accordance with a preferred embodiment of the third aspect the genes are in the genome of the host cell, or on one or more plasmids or vectors. Examples and further details on plasmids or vectors have been described herein above in connection with the first aspect.

[0102] In accordance with a preferred embodiment of the first and third aspect the host cell is a bacterial cel I, an archaeal cell, a fungal cell or a plant cell.

[0103] The nature of the host cell is not particularly limited. A suitable eukaryotic host cell may be a vertebrate cell, an insect cell, a fungal / yeast cell, a nematode cell or a plant cell. The host cell is more preferably a microbial host cell, even more preferably a fungal or bacterial host cell and most preferably a bacterial host cell. More specific examples of suitable host cells will be described herein below.

[0104] In accordance with another preferred embodiment of the first and third aspect the host cell is selected from the group consisting of Acinetobacter junii, Aeromonas hydrophila, Aeromonas hydrophila, Aeromonas sp., Alcaligens eutrophus, Alcaligens latus, Allochromatium vinosum, Aguitalea sp. USM4, Aspergillus eutrophus, Aulosira fertilissima, Azohydromonas lata, Azotobacter sp., Azotobacter vinelandii, Bacillus aerophilus, Bacillus amyloliguefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus laterosporus, Bacillus licheniformis, Bacillus macerans, Bacillus megaterium, Bacillus mycoides, Bacillus polymyxa, Bacillus sp., Bacillus sp. INT005, Bacillus sp. ISTVK1, Bacillus sp. JMa5, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Burkholderia cepecia, Burkholderia sacchari, Burkholderia sp., Caryphanon latum, Caulobacter Segnis, Chelatococcus thermostellatus, Chlorogloea fritschii, Chromobacterium sp., Chromobacterium sp. USM2, Chromobacterium violaceum, Comamonas sp., Corynebacterium matruchotii, Cupriavidus necator, Dechloromonas sp., Delftia acidovorans, Desulfococcus multivorans, Erwinia sp., Geobacillus sp. AY 946034, Haloferax mediterranei, Halomonas boliviensis, Halomonas campisalis, Halomonas sp., Halomonas sp. SF2003, Hydrogenophaga pseudoflava, Loktanella sp. SM43, Methylobacterium extorguens, Methylobacterium organophilum, Methylobacterium sp., Methylocystis parvus, Methylosinus trichosporium, Micrococcus luteus, Microlunatus phosphovorus, Nocordia Carolina, Nostoc muscorum, Novosphingobium nitrogenifigens, Pandoraea sp. ISTKB, Paracoccus denitrificans, Plataicicumulans acidivoransb, Priestia megaterium, Pseudomonas citronellolis, Pseudomonas hydrogenovora, Pseudomonas mendocina, Pseudomonas oleovorans, Pseudomonas pseudoflava, Pseudomonas putida, Pseudomonas resinovorans, Pseudomonas sp., Pseudomonas sp. 61-3, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus egui, Rhodococcus ruber, Rhodococcus sp. NCIMB, Rhodococcus sp. NCIMB 40126, Saccharophagus degradans, Schlegelella thermodepolymerans, Sphingobium yanoikuyae, Spirulina sp. LEB 18, Staphylococcus citreus, Streptomyces albus, Streptomyces aureofaciens, Streptomyces coelicolor, Streptomyces fradiae, Streptomyces grieseus, Streptomyces griseorubiginosus, Streptomyces lividans, Streptomyces nouresi, Streptomyces oliviacieous, Streptomyces parvus, Streptomyces sp. ATCC1238, Streptomyces sp. ATCC 21175, Streptomyces sp. NCIB9692, Streptomyces venezualae, Styphylococcus coelicolor, Synechoccus sp. MA19, Thermus thermophiles, Thiocapsa pfenningiii, Thiococcus pfennigii, Wautersia eutropha, and Zobellella denitrificans

[0105] The above non-limiting but preferred examples of host cells are host cells that are known to naturally produce PHB. Hence, these host cells generally comprise already genes encoding PhaA, PhaB and PhaC.

[0106] In accordance with another preferred embodiment of the first and third aspect the host cell is selected from the group consisting of Agrobacterium tumefaciens, Aliivibrio fischeri, Arabidopsis thaliana, Ashbya gossypii, Aspergillus nidulans, Aspergillus niger, Azotobacter vinelandii, Bacillus cereus, Bacillus subtilis, Bacteroides thetaiotaomicron, Candida albicans, Caulobacter crescentus, Chlamydomonas reinhardtii, Coprinus cinereus, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Cryptococcus neoformans, Escherichia coll, Halobacterium salinarum, Haloferax volcanii, Hansenula polymorpha, Komagataella phaffii, Lactobacillus rhamnosus, Methanococcus vannielii, Methanosarcina barker!, Mycoplasma genitalium, Neurospora crassa, Nicotiana tabacum, Pichia pastoris, Pseudomonas fluorescens, Pseudomonas putida, Pyrococcus abyss!, Pyrococcus furiosus, Rhizophagus irregularis, Rhizopus oryzae, Saccharomyces cerevisiae, Schizophyllum commune, Schizosaccharomyces pombe, Staphylococcus carnosus, Streptomyces coelicolor, Sulfolobus acidocaldarius, Sulfolobus beitou, Sulfolobus islandicus, Sulfolobus mongibelli, Sulfolobus neozealandicus, Sulfolobus solfataricus, Sulfolobus sp. A2, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus vallisabyssus, Synechocystis sp. PCC 6803, Thermococcus kodakarensis, Ustilago maydis, Vibrio natriegens, Yarrowia lipolytica, and Zymomonas mobilis, and is preferably an Escherichia coll cell.

[0107] The above non-limiting but preferred examples of host cells are host cells that do not naturally produce PHB. Hence, into these host cells all genes encoding the enzymes being required to produce atactic PHB need to be introduced.

[0108] The present invention relates in a fourth aspect to a method of producing the host cell of the third aspect comprising introducing into the host cell the genes encoding the enzymes as discussed in connection with the first aspect in an expressible form.

[0109] The definitions and preferred embodiments of the first to third aspect of the invention apply mutatis mutandis to the fourth aspect of the invention as far as being amenable therewith. For example, also in the fourth aspect the gene may be introduced into the genome of the host cells or extrachromosomally.

[0110] The term "in an expressible form" means that the genes introduced into the cells such that they can be expressed and translated into the enzymes.

[0111] The present invention relates in a fifth aspect to atactic poly(3-hydroxybutyrate) (PHB) obtained or obtainable by the method of the first aspect of the invention.

[0112] The definitions and preferred embodiments of the first to fourth aspect of the invention apply mutatis mutandis to the fifth aspect of the invention as far as being amenable therewith.

[0113] As discussed herein above, the atactic poly(3-hydroxybutyrate) (PHB) is obtained or is made obtainable by the method of the first aspect of the invention which is the first atactic poly(3-hydroxybutyrate) that can be obtained from cells. It is therefore expected that this atactic poly(3-hydroxybutyrate) has certain novel structural characteristics or certain novel properties.

[0114] As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0115] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0116] The figures show.

[0117] Figure 1: Metabolic pathways for the production of isotactic (top) and atactic (middle + bottom) PHB.

[0118] Figure 2: Degradation of fatty acids in Escherichia coli.

[0119] Figure 3: Plasmid-borne PHB production of E. coli BW25113 AfadB AfadJ during steady-state growth in minimal medium. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; n = 3

[0120] Figure 4: Quantification of RNA levels for phaA, phaB, phaC, and hbd in relation to 16S rRNA during steady-state growth. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; n = 3

[0121] Figure 5: Production of PHB in E. coli BW25113 AfadB AfadJ at 1 L scale in shake flasks. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; DCM, dry cell mass; RCM, real cell mass; n = 1

[0122] Figure 6: Peak splitting of the methyl group of microbially produced PHB into diads in a 13C NMR. Peak labelling according to Huang et al. (2023).6 A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; r, racemo; m, meso

[0123] Figure 7: Peak splitting of the methylene group of microbially produced PHB into triads in a 13C NMR. Peak labelling according to Huang et al. (2023).6 A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; r, racemo; m, meso

[0124] Figure 8: Melting and crystallization behavior of microbially produced PHB by E. coli BW25113 AfadB AfadJ expressing operon A (AvPhaEC-CnPhaAB).

[0125] Figure 9: Melting and crystallization behavior of microbially produced PHB by E. coli BW25113 AfadB AfadJ expressing operon B (AvPhaEC-CnPhaA-CkHbd-CnPhaB).

[0126] Figure 10: Melting and recrystallization behavior of microbially produced PHB by E. coli BW25113 AfadB AfadJ expressing operon C (AvPhaEC-CkHbd-CnPhaAB).

[0127] Figure 11: Melting and recrystallization behavior of microbially produced PHB by E. coli BW25113 AfadB AfadJ expressing operon D (CkHbd-AvPhaEC-CnPhaAB).

[0128] Figure 12: Suggested gene order in operons producing atactic PHB. The isotactic PHB producing reference is given at the top of each block. Numbers indicate nucleotide distance between stop and start codons.

[0129] Figure 13: Isotactic PHB production by dissolved oxygen-controlled glucose fed-batch processes in minimal medium by strain A

[0130] Figure 14: Atactic PHB production by dissolved oxygen-controlled glucose fed-batch processes in minimal medium by strain B.

[0131] Figure 15: Secretion of (S)-3-hydroxybutyrate during the fermentation of strain B.

[0132] Figure 16: Thermogravimetric analysis of PHB samples.

[0133] Figure 17:1H NMR spectra of PHB.

[0134] Figure 18:13C NMR spectrum of commercial isotactic PHB. 1 Ester carbon: 169.27 ppm; 2 methine carbon: 67.74 ppm; 3 methylene carbon: 40.92 ppm; 4 methyl carbon: 19.90 ppm

[0135] Figure 19:13C NMR spectra of microbial isotactic PHB produced by strain A. 1 Ester carbon: 169.27 ppm; 2 methine carbon: 67.74 ppm; 3 methylene carbon: 40.92 ppm; 4 methyl carbon: 19.90 ppm

[0136] Figure 20:13C NMR spectra of microbial atactic PHB produced by strain B and peak splitting into diads and triads. Diad and triad assignment according to Huang et al. (2023), Nature Catalysis 6, 720-728; 10.1038 / s41929-023-01001-7 (2023). Ester carbon: 169.36 ppm (rr + mr), 169.26 ppm (mm + rm); 2 methine carbon: 67.75 ppm (rm), 67.72 ppm (mm), 67.78 ppm (rr), 67.65 ppm (mr); 3 methylene carbon: 40.97 ppm (rm), 40.90 ppm (mm), 40.82 ppm (rr), 40.76 ppm (mr); 4 methyl carbon: 19.93 ppm (r), 19.88 ppm (m); m, meso; r, raceme

[0137] Figure 21: Images of isotactic and atactic PHB on a 1 cm grid produced by strain A and B, respectively. Figure 22: Molar mass decomposition of PHB during thermal processing. Isotactic and atactic PHB were processed at 170°C and 130°C, unless shown otherwise, n = 3

[0138] Figure 23: Thermal behavior of atactic PHB from strain B after different time sums of processing.

[0139] The examples illustrate the invention.

[0140] Example 1 - Results

[0141] Metabolic pathway design and knockout selection for the production of atactic PHB

[0142] We began by investigating ways how the production of atactic PHB could be implemented metabolically. As the native PHB-producing metabolic pathway only provides (R)-3-hydroxybutyryl CoA, an enzyme providing (S)-3-hydroxybutyryl CoA from a common precursor was needed (Figure 1 top). We settled for the enzyme class of (S)-3-hydroxybutyryl CoA dehydrogenases (Hbd), which perform a similar reaction to PhaB but provide the (S)-enantiomer. Hbd from Clostridium kluyveri DSM 555 was chosen because it requires NADPH unlike the other known family members (Table 2) and is therefore compatible with the native NADPH-requiring pathway.1The mixture of (R)- and (S)-3-hydroxybutyryl CoA supplied by PhaB and Hbd are then polymerized to atactic PHB by a suitable PhaC (Figure 1 middle).

[0143] Additionally, we examined gene knockouts that could elevate the fraction of (S)-3-hydroxybutyrate in the produced polymer. Our attention was drawn to the oxidation of acyl CoAs to acetyl CoAs by the fatty acid oxidation complex (Figure 2). The two homologs FadB and FadJ of the complex are annotated to perform three reactions: (1) hydration of trans-Δ2-enoyl CoA to (S)-3-hydroxyacyl CoA, (2) oxidation of (S)-3-hydroxacyl CoA to 3-ketoacyl CoA, and (3) epimerization between (R)- and (S)-3-hydroxyacyl CoAs. We hypothesized that the knockouts of fadB and fadJ could prevent degradation of (S)-3-hydroxybutyryl CoA by elimination of the oxidative reaction and increase the (S)-enantiomer fraction of PHB.2

[0144] Identification of PhaC producing atactic PHB

[0145] The production of atactic PHB requires PhaCs that are able to polymerize a mixture of (R)- and (S)-3-hydroxybutyryl CoA. Out of four PHA synthase classes, three are naturally able to produce PHB. From those three classes, four PhaCs were selected and analyzed for the production of atactic PHB: Cupriavidus necator H16 (class I), Chromobacterium sp. USM2 (class I), Allochromatium vinosum (class III), and Priestia megaterium 22-2 (class IV). The ability to produce atactic PHB was also investigated for PhaC from Pseudomonas sp. 61-3 as its STQK mutant is able to accumulate PHB despite being associated with class II.3PHB synthesis was encoded by a phaCAB operon from C. necator on a high copy number vector controlled by its native o70 promoter and the phaC gene from C. necator replaced to the other PhaCs of interest (Figure 12). GFP or Hbd were constitutively expressed on a second low copy number plasmid with different compatibility group.

[0146] All combinations of plasmids produced PHB and the PHA synthases from A. vinosum and Chromobacterium sp. showed to be the best producers of the polymer (Table 3). However, we only detected (S)-enantiomer when Hbd was provided as a second enzyme. Significantly more (S)-3-hydroxybutyrate was observed in the strains with the fadB fadJ genotype (p < 0.05; two-tailed, two-sample, equal variance t-test; except for P. megaterium and Pseudomonas sp. which had higher variance due to low PHB contents) indicating that (S)-3-hydroxybutyryl CoA was not degraded by the fatty acid oxidation complex. Surprisingly, PhaCs from all classes were able to incorporate the (S)-enantiomer into the polymer, indicating that the enzyme class is not as selective as previously assumed. As a result of the initial screening, we focused the remaining work on the double knockout strain and Hbd as the strategy to provide (S)-3-hydroxybutyryl CoA.

[0147] To decrease the complexity of the plasmid-based PHB production system, we combined PHB production and supply of (S)-3-hydroxybutyryl CoA via Hbd onto a single plasmid, hbd was cloned into the operons with A. vinosum and Chromobacterium sp. phaC at three different positions to obtain different expression levels of the gene (Figure 12). The new arrangement on a single plasmid contributed to much higher (S)-enantiomer fractions compared to the two-plasmid system (Table 4). The highest (S)-3-hydroxybutyrate fractions could be obtained with hbd at the second last position in the operon. hbd at the first position only produced the second highest (S)-enantiomer fractions -contrary to our expectation that expression should be higher closer to the promoter. (Reasons for this observation were investigated later.) The synthase from A. vinosum was still the best producer of atactic PHB reaching (S)-enantiomer fractions of 21.0% ± 4.8% but at the cost of producing lower PHB amounts. Generally, there was a clear correlation between (S)-3-hydroxybutyrate fraction and PHB content. Increasing fractions of (S)-enantiomer led to decreases in PHB content. It is assumed that this occurs because PhaCs prefer (R)- over (S)-3-hydroxybutyryl CoA and an accumulation of (S)-3-hydroxybutyryl CoA limits the overall flux towards PHB.

[0148] Characterization of metabolic pathways synthesizing atactic PHB

[0149] After identification of PHA synthases which could produce atactic PHB and choosing A. vinosum PhaC as the most promising candidate, we characterized the metabolic pathways arising from the different operon arrangements. Cells were analyzed during steady-state growth in minimal medium so that the productivity of PHB synthesis was a measure of metabolic flux.4Fitness of the three strains (denoted as: B, AvPhaEC-CnPhaA-CkHbd-CnPhaA; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB) was similar to the reference strain producing isotactic PHB (A, AvPhaEC-CnPhaAB) or even higher if hbd was expressed after phaC (C; Table 5). Incorporation of (S)-3-hydroxybutyrate in the strains C and D reduced PHB flux by a factor two compared to strain A (Figure 3). Strain B had flux reduced by around 'A compared to the reference, but generated the highest (S)-enantiomer fraction of 9.2% ± 0.1% and only a slight flux reduction to strains C and D with lower (S)-3-hydroxybutyrate fractions was observed. Again, flux towards atactic PHB had a somewhat negative correlation with (S)-3-hydroxybutyrate content. Strain C was an exception, which had similar flux compared to strain D but with a much lower (S)-enantiomer fraction.

[0150] To gain insight into how the operon arrangement affected flux, we determined transcript levels of the encoded genes. Adding the hbd gene to the second last position in the operon (strain B) increased transcripts of phaB in relation to the wildtype as well (A; Figure 4). Indeed, we could find promoterlike structures in the sequence of hbd explaining the increased transcription of phaB in this strain (data not shown). The arrangement of the operon in strain C resulted in lower expression levels than the other strains and probably explains its improved fitness because the metabolic burden of producing PHB is reduced. The highest transcript levels of hbd were observed in strain D but strong expression of this gene from the first position came at the cost of reducing transcripts of all other encoded genes. Despite the strong hbd expression, this probably induced a flux limitation towards PHB and decreased the (S)-enantiomer fraction to amounts lower than observed in strain B.

[0151] Production of PHB in shake flasks at 1-L-scale

[0152] To obtain a more detailed understanding of the four strains, we grew them in 1 L culture in minimal medium and followed dry cell mass, real cell mass, PHB content, and (S)-3-hydroxybutryate fraction over time (Figure 5). After around 30 h, all strains reached similar real cell masses between 1.0 g L-1and 1.5 g L-1. Except for strain B, growth stopped after approximately 13 h and increases in dry cell mass thereafter were predominately from accumulation of PHB. Strains producing atactic PHB did not benefited from cultivation for longer than 24 h as PHB accumulation came to a halt before. The fraction of (S)-3-hydroxybutyrate in the produced polymer stayed roughly similar over the whole cultivation and strain B again achieved the highest (S)-enantiomer content. Slightly more than % of the initial glucose was consumed and the viability and productivity of the strains could probably have been extended by pH control (data not shown).

[0153] Material properties of microbially synthesized polymers

[0154] We extracted the produced polymer from the biomass obtained during PHB production at 1-L-scale to determine its material properties. From strains A to D we were able to extract PHB with (S)-3-hydroxybutyrate fractions of 0.0%, 5.2%, 0.7%, and 3.4% respectively. The molar mass distribution of the extracted PHB was similar for all strains (Table 7). Weight average molar mass was >106g mol-1as is typical for microbially produced PHB.5Compared to commercially available microbial, isotactic PHB, the polymer produced by our strains and extracted in chloroform outperformed it in regard to molar mass and dispersity. Most importantly, incorporation of (S)-enantiomer in the polymer did not result in shortening of chains.

[0155] Additionally, we recorded 13C NMR spectra to verify if (S)-enantiomer was randomly distributed in the polymer or comprised two blocks of (R)- and (S)-enantiomer (Figure 6 & Figure 7). PHB extracted from the reference strain A was verified to be isotactic and only showed meso diads and triads. Polymers produced from strains B and D had clear signals for racemo diads and triads and can therefore be assumed to be mostly atactic. The (S)-3-hydroxybutyrate fraction of PHB extracted from strain C was probably too low to give detectable signals.

[0156] Lastly, we analyzed the thermal properties of the obtained polymers. The isotactic PHB from strain A had a melting temperature of 174°C and crystallinity of 51% (Table 8, Figure 8 to Figure 11). PHB from strain C, which had only low (S)-3-hydroxybutyrate fractions, demonstrated similar thermal properties. With increasing (S)-enantiomer fractions we observed decreases in melting temperature and strong reductions in crystallinity. The polymer produced by strain B, which had the highest (S)-enantiomer fraction, had reduced melting temperature by around 6°C and a pronounced reduction of crystallinity to half of the reference polymer (24%). Additionally, a much stronger glass transition, which is a property of amorphous and not of crystalline polymers, was observed. From this it can be assumed that this polymer has significantly lessened brittleness and degradation during melt processing is probably lower.

[0157] Upscaling and production of atactic PHB in laboratory-scale bioreactors

[0158] To obtain sufficient amounts of polymer for the analysis of its material properties and to demonstrate scalability, the production of PHB was transferred to laboratory-scale bioreactors. A dissolved oxygen-controlled glucose fed-batch process in minimal medium was employed, which could be separated in two phases: A growth phase in which oxygen was abundant for biomass production and an oxygen limiting phase, which halted biomass growth and diverted carbon flux to the production of PHB. Isotactic and atactic PHB were produced by strains A and B, respectively.

[0159] Both strains had identical growth rates during the batch phase (Table 8). With advancing time in the fed-batch phase, growth reduced in the atactic PHB producing strain. The fermentation of strain A could be clearly divided into a growth and a production phase, which was separated by oxygen limitation at 21 h as evidenced by off-gas analysis (Figure 13 & 14). Such a clear distinction between biomass accumulation and PHB production could not be observed in strain B despite oxygen limitation occurred at a similar time. After 25 h the fermentation of strain B was stopped because no additional growth and PHB production occurred. Contrary to that, the fermentation of strain A was halted after the maximum volume of the culture vessel was reached without any metabolic indicator that suggested PHB production could have diminished soon. This difference appeared in a considerable reduction in PHB titer and productivity compared to the reference strain (Table 8). While strain A had a similar PHB content compared to previous experiments (44.3% ± 0.2%), a threefold reduction was observed in strain B (4.6% ± 0.2%).

[0160] To identify the cause of reduced growth and low productivity in strain B, secreted metabolites were quantified using ion exclusion chromatography. A strong time-dependent signal in the chromatograms of the fermentation of strain B was not present in the reference and could be identified as 3-hydroxybutyrate. At the end of the fermentation, strain B accumulated an impressive amount of 17 g L-13-hydroxybutyrate, which proved to be almost exclusively made from the (S)-enantiomer (Figure 15). Because strong acid secretion typically inhibits growth, this likely explained the growth reduction at higher cell densities (Pinhal et al., Journal of Bacteriology 201; 10.1128 / jb.00147-19 (2019) and Luli et al., Applied and Environmental Microbiology 56, 1004-1011; 10.1128 / aem.56.4.1004-1011.1990 (1990)).

[0161] Given the previous insight that the used PHA synthase still prefers (R)- over (S)-3-hydroxybutyryl CoA, we wondered which metabolic consequences the accumulation of (S)-3-hydroxybutyryl CoA had on production and growth. Previously it was reported that E. coli contains native thioesterases which are capable of hydrolyzing large amounts of 3-hydroxybutyryl CoA (Tseng et al., Applied and Environmental Microbiology 75, 3137-3145; 10.1128 / AEM.02667-08 (2009)). Therefore, it seemed that strain B counteracted the accumulation of (S)-3-hydroxybutyryl CoA by hydrolysis, which released CoA to maintain metabolic activity. (S)-3-Hydroxybutyrate is then released as a byproduct into the culture medium via an unknown mechanism. A decreasing (S)-enantiomer fraction over the cultivation time of strain B might indicate the presence of intracellular (S)-3-hydroxybutyrate, which might have impacted the measurement. With advanced process time, more cells might hydrolyze releasing the monomer as suggested by a strong browning of the culture medium that was not observed during the cultivation of strain A.

[0162] These issues seemed to arise from the preference of the PHA synthase for the R-enantiomer. By engineering the stereoselectivity of PHA synthase or choice of less selective enzymes and / or deleting the gene of the (S)-3-hydroxybutyryl CoA hydrolysing enzyme these issues can be addressed.

[0163] Structural, thermal and mechanical polymer properties of fermentatively produced PHB

[0164] PHB was recovered from cells by chloroform extraction at an efficiency of around 80%. The extracted PHB proved to be free of detectable amounts of water, organic contaminants, salts and antifoam as evidenced by thermogravimetric analysis and nuclear magnetic resonance spectroscopy (Figures 16-20). The fraction of (S)-3-hydoxybutyrate monomers in the purified atactic polymer was quantified to be 6.84% ± 0.04% (Table 9). Distribution of the (S)-enantiomer along the polymer chains was determined from carbon-13 nuclear magnetic resonance spectra (Figure 20). The distribution proved to be nearly perfectly atactic, emphasizing that incorporation of (S)-3-hydroxybutyrate by PHA synthase is random.

[0165] The molar mass distribution of PHB samples was analyzed by size-exclusion chromatography (Table 9). While the isotactic PHB showed similar distributions, the molar mass distribution of atactic PHB was slightly shifted to shorter chain length but with a considerably less pronounced high-chain-length fraction. This was reflected in a lower polydispersity (commercial, 4.1 ± 0.3; strain A, 4.4 ± 0.6; strain B, 2.8 ± 0.2), moderately lower number average molar mass (commercial, (346 ± 23) kg mol-1; strain A, (369 ± 43) kg mol-1; strain B, (252 ± 25) kg mol-1) and mass average molar mass reduced to more than one half (commercial, (1402 ± 82) kg mol-1; strain A, (1590 ± 130) kg mol-1; strain B, (704 ± 64) kg mol-1). These results indicate that chain termination kinetics of PHA synthase are considerably altered when producing atactic PHB. Again, it can be assumed that a PHA synthase with higher activity on the S-Enantiomer would alleviate this.

[0166] Differential scanning calorimetry was utilized to quantify the consequences of altered tacticity on the thermal behavior of the polymer. The first sign that atactic PHB was more amorphous than the reference polymers was a more pronounced glass transition (Table 9). Secondly, a reduction in melting temperature by around 20-25°C verified that crystal formation was impaired (commercial, 174.7°C ± 1.3°C; strain A, 179.3°C ± 0.7°C; strain B, 154.3°C ± 1.1°C). A weaker tendency towards nucleation and crystal propagation was evidenced by a distinct cold crystallization exotherm and a more heterogeneous composition of crystalline regions was indicated by a broader melt transition. The superior thermal behavior of atactic PHB was summarized by a reduced enthalpy of fusion and degree of crystallinity (commercial, 54.7% ± 0.7%; strain A, 55.0% ± 1.7%; strain B, 39.1% ± 3.2%; Table 9). The bulk of industrial PHB processing is performed via thermal techniques - mainly extrusion and injection molding. Despite the low amount of atactic PHB that could be obtained, we were looking for a method to accurately simulate thermal processing: Comparable amounts of PHB were compacted and hot pressed into discs. With similar pressure and time, equivalent thickness and diameter of the discs could be produced at 170°C and 130°C for isotactic and atactic PHB, respectively. At a material thickness of around 0.2-0.3 mm, the atactic PHB appeared optically transparent whereas isotactic PHB was opaque (Figure 21). This emphasizes that the reduced crystallinity is accompanied with additional favorable properties, which are highly advantageous, for example, for consumer packaging. How the lower crystallinity affected mechanical polymer properties was determined from strips cut from the prepared discs.

[0167] A reduced Young's modulus of the atactic polymer compared to its isotactic counterpart displayed its higher ductility (Table 9; commercial, (1.2 ± 0.3) GPa; strain A, (1.1 ± 0.2) GPa; strain B, (0.6 ± 0.1) GPa). Interruption of crystal formation by the (S)-enantiomer seemed to be the most plausible cause, but the altered molar mass distribution might also play a role. The higher ductility was also demonstrated by a markedly improved elongation at break (commercial, 5.3% ± 1.3%; strain A, 4.8% ± 1.0%; strain B, 10.5% ± 1.5%) without compromises in tensile strength. The enhanced mechanical properties of microbial, atactic PHB were summarized by a higher fracture work (commercial, (1.4 ± 0.4) MJ m“3; strain A, (1.1 ± 0.3) MJ m“3; strain B, (2.5 ± 0.4) MJ m“3).

[0168] Molar mass decomposition of PHB begins at temperatures between 175°C and 180°C. Since the melting temperature of atactic PHB was significantly lower, we investigated how repeated processing cycles affect its molar mass. Processing was performed at temperatures comparable to those used for the production of tensile test specimens. A pronounced reduction of molar mass was observed in both isotactic polymers, number average molar mass decreased by 53% over 25 min and by 56% over 30 min at 170°C for commercial isotactic PHB and isotactic PHB from strain A, respectively (Figure 22). In contrast, the decomposition of atactic PHB was significantly lower (17% reduction of number average molar mass over 42.5 min) with prolonged processing time, despite a stepwise increase of processing temperature to 150°C. (We believe that at the processing temperature for atactic PHB, melting occurs differently between two polymer phases. Amorphous (S)-enantiomer rich regions melt at lower temperatures than (R)-enantiomer rich crystalline regions, which over time leads to separation of the melt peak into two (Figure 23). To be able to produce foils, increasing temperatures are required to melt the crystalline regions of the polymer. Nevertheless, it should be noted that the required melting temperature did not reach values similar to isotactic PHB and was still outside of the critical window of molar mass decomposition. This is analogous to the description by Bossu et al., Polymer 229, 123987; 10.1016 / j.polymer.2021.123987 (2021) for poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate]. Although isotactic PHB initially has a favorable molar mass distribution, prolonged heat treatment diminished its advantage below the molar mass of atactic PHB. Our findings indicate that atactic PHB can be processed with less compromises in its material properties. Moreover, they suggest that atactic PHB may be recycled more frequently than isotactic PHB before its properties deteriorate.

[0169] Example 2 - Outlook

[0170] This work demonstrated for the first time that PHB containing (S)-3-hydroxybutyrate cannot only be synthesized chemically but also produced microbially. Against common believes, we could show that the enzyme polymerizing PHB is not selective for the (R)-enantiomer but also incorporates the (S)-enantiomer into the polymer chain. Our data suggests, that all PHA synthases are at least to some extend able to synthesize atactic PHB. The metabolic pathways producing (S)-enantiomer containing PHB were analyzed, the recombinant E. coli strains characterized, and genotypes identified which led to higher (S)-enantiomer fractions. PHB extracted from the strain which provided the highest (S)-3-hydroxybutyrate fraction, showed random distribution of stereocenters and proved not to be blocks of two enantiomers. Its molar mass distribution was found to be similar to microbially produced isotactic PHB and routinely outperforms (S)-enantiomer containing PHB synthesized chemically by ring-opening polymerization.6-8The polymer had clear signs that its material properties had improved. This work successfully lowered the melting temperature of PHB away from the critical temperature range of 175°C-180°C where molecular weight decomposition occurs and created a less brittle material. Our results show that the incorporation of (S)-3-hydroxybutyrate into the polymer chain interrupted crystal formation and propagation, which led to improved optical, thermal and mechanical properties. At an (S)-enantiomer fraction of 6.84% ± 0.04% and racemo diad probability of 11.8% ± 0.7%, the limitations of isotactic PHB were successfully resolved. Lower (S)-enantiomer fractions are likely sufficient to prevent molecular weight decomposition. Comparison between microbially and chemically synthesized atactic PHB suggest that its properties are moderately better, which is likely attributed to its higher molar mass distribution (Table 10). All in all, this work significantly enhances our understanding about microbial production of atactic PHB and helps to overcome the common disadvantages associated with the material properties of PHB. By applying commonly known methods of enzyme engineering PHA-Synthases can be modified such that the production of atactic PHB and other PHAs with contents of the (R)-enantiomer between 0 and 100 % and correspondingly of the (S)-enantiomer between 100 and 0 % will be possible.

[0171] Example 3 - Material and Methods

[0172] Microbial growth

[0173] All E. coli strains used in this work are shown in Table. Routinely, cells were grown at 37°C in LB Miller medium (10 g L-1sodium chloride, 10 g L-1tryptone, 5 g L-1yeast extract) for cloning purposes and in LB Miller with 20 g L-1glucose or MR medium for PHB production. MR medium (pH 6.9) contained 22 g L-1potassium dihydrogen phosphate, 20 g L-1glucose, 3 g L-1diammonium hydrogen phosphate, 0.8 g L-1citric acid, 0.7 g L-1magnesium sulfate heptahydrate, and 5 mL L-1trace metal solution.9The trace metal solution was prepared in 5 M hydrochloric acid and consisted of 10 g L-1iron sulfate heptahydrate, 2.7 g L-1calcium chloride dihydrate, 2.2 g L-1zink sulfate heptahydrate, 1.0 g L-1copper sulfate pentahydrate, 360 mg L-1manganese sulfate monohydrate, 100 mg L-1ammonium heptamolybdate tetrahydrate, and 20 mg L-1sodium tetraborate decahydrate. Media were supplemented with 25 pg mL-1kanamycin sulfate and / or 34 pg mL-1chloramphenicol to maintain plasmids.

[0174] Plasmid and strain construction

[0175] Cloning was performed according to standard procedures.10The ampicillin resistance of pUC19 was replaced via Gibson assembly to a kanamycin resistance amplified from pET-28a(+). A 5253 bp fragment containing the phaCAB operon, between Smal to EcoRI restriction sites, was amplified from the genome of C. necator H16 and placed into pUC19-Kan in inverse orientation also by Gibson assembly. In a similar way, the native phaC gene was replaced by phaC from Chromobacterium sp. USM2 (codon-optimized), phaEC from A. vinosum, phaCl STQK from Pseudomonas sp. 61-3 or phaRC from P. megaterium subcloned from gene strings, plasmids, plasmids or genomic DNA, respectively. Position of hbd in the operons was varied by Gibson assembly. pACYC184 vectors were cloned in a similar manner. Sequence integrity was verified by whole plasmid sequencing.

[0176] Knockout strains were constructed by the method of Datsenko & Wanner (2000)11or taken from the Keio collection.12Knockouts were combined by Pl phage transduction with and antibiotic resistance genes were removed as described previously.6-7

[0177] Screening for PHB operons producing atactic PHB

[0178] E. coli BW25113 or E. coli BW25113 ISfadB ISfadJ was transformed with pUC-based and pACYC184-based plasmids and grown overnight. 100 mL baffled shake flasks with 10 mL prewarmed LB medium with 20 g L-1glucose were inoculated with fresh colonies in triplicate. After 48 h growth cells were harvested by centrifugation, lyophilized, and biomass was used for DCM and PHB quantification. RCM was defined as DCM subtracted by PHB mass.

[0179] Growth rate determination and steady-state PHB production

[0180] E. coli BW25113 EfadB E adJ was freshly transformed with the corresponding pUC-based plasmids and grown overnight. Single colonies were inoculated in triplicate into 14 mL cell culture tubes with 5 mL prewarmed LB medium and grown for 10-11 h. 0.5 mL was transferred to 14 mLcell culture tubes with 5 mL prewarmed MR medium and grown again for 10-11 h. 500 mL baffled shake flasks with 50 mL prewarmed MR medium were inoculated to ODgoo = 0.02. Samples for ODgoo measurements were taken at regular intervals and growth rate was determined after In-transformation. When the exponentially growing cultures reached ODgoo = 2.0 samples for transcriptional profiling were taken and frozen at -80°C. The remaining biomass was used for DCM and PHB quantification as described below. Transcriptional characterization of PH B synthesis operons

[0181] RNA was extracted using the Monarch Total RNA Miniprep Kit (New England BioLabs) including an on-column DNase I digest of genomic and plasmid DNA. 60 ng RNA was subjected to cDNA synthesis via the LunaScript RT SuperMix (New England BioLabs). 5 pL of a 100-fold dilution of the cDNA synthesis reaction was used as template for qPCR with the Luna Universal qPCR Master Mix (New England BioLabs). qPCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). phaA, phaB, phaC, and hbd were quantified in relation to 16S rRNA (rrsA). Prior to relative quantification, sufficient amplification efficiency for each primer pair was validated using a dilution of PCR amplified template. Primer specificity was verified by performing melt curve analysis after each run.

[0182] PHB production in shake flask and extraction from biomass

[0183] E. coli BW25113 bfadB bfadJ was freshly transformed with the corresponding pUC-based plasmids and grown overnight. Single colonies were inoculated into 14 mL cell culture tubes with 5 mL prewarmed LB medium and grown for 10-11 h. Then, 500 mL baffled shake flasks with 50 mL prewarmed MR medium were inoculated from the previous culture and grown for the same duration. The final production culture in 5 L baffled shake flasks with 1 L prewarmed MR medium was inoculated to ODgoo = 0.2. Samples were taken at regular intervals, washed with ice-cold water, and lyophilized to determine dry cell mass, PHB content, and (S)-3-hydroxybutyrate fraction. Cultures were harvested after 30 h by centrifugation (4000xg, 10 min, 4°C) and lyophilized for PHB extraction.

[0184] PHB was extracted from lyophilized biomass by stirring one part biomass in twenty parts chloroform at 40°C and for 2 days. Cell debris was removed via a Buchner funnel and the filtrate was dripped into two volumes of ice-cold ethanol. The precipitated PHB was separated by another filtration step and dried under vacuum.

[0185] Dissolved oxygen-stat fed-batch fermentation

[0186] Strains maintained as glycerol stocks were streaked on LB agar plates and grown at 37°C for 24 h. A fresh colony was inoculated into a 14 mL cell culture tube containing 5 mL LB medium and grown at 37°C for 8 h. 50 mL MR medium in a 500 mL baffled Schott shake flask was inoculated to OD600 = 0.01 and grown at 30°C for 24 h. The bioreactor with an initial volume of 1.0 L MR medium was inoculated to OD600 = 0.1. Benchtop fermentation was carried out with a 2 L Univessel Glass cultivation vessel and Biostat B controller (Sartorius). Temperature was controlled at 30°C. Dissolved oxygen was maintained at 30% by varying stirrer speed (500 rpm to 1500 rpm) and constant air sparging (1.0 L min-1). PHB production was induced upon oxygen limitation when the controller was unable to sustain the setpoint. pH was controlled on-sided at 6.80 by addition of 25% (v / v) ammonia-water. A dissolved oxygen-stat feeding strategy was employed and a solution of 600 g L-1glucose and 12 g L-1magnesium sulfate heptahydrate was feed when dissolved oxygen rose > 45%. The feeding pulse added approximately 20 g glucose. Foam suppression was achieved by automatic addition of a 10% Antifoam B emulsion (Sigma Aldrich). Growth was monitored using a BioPAT Fundalux probe (Sartorius). Processes were stopped after dissolved oxygen start to rise. Data was evaluated with BioPAT MFCS 4.12.0 (Sartorius).

[0187] Approximately 10 mL samples were drawn from the reactor at regular intervals from which OD600 was determined and clarified supernatant obtained by centrifugation at 17,000xg for 20 min. Supernatant samples were immediately stored at -20°C until analysis. From the OD600 measurement volumes equivalent to 20 mg biomass were washed twice (7000xg, 5 min) with 10 mL phosphate buffered saline (pH 7.4) and transferred to a weighted vial for freeze drying and GC analysis.

[0188] Gas chromatographic analysis of PHB stereocomposition

[0189] Weighted biomass or extracted PHB was resuspended in 0.5 mL chloroform and 0.25 mL derivatization solution with the internal standard benzoic acid (85% methanol, 15% sulfuric acid, 0.7% (w / v) benzoic acid) was added. Derivatization to 3-hydroxybutyric acid methyl esters was performed at 95°C for 3 h. After derivatization, samples were washed trice with 0.5 mL ddHjO and the chloroform phase was used for measurement.

[0190] The enantiomeric composition of PHB was analyzed on a GC-2010 Plus gas chromatograph (Shimadzu, Japan). 1 pL sample was injected in split mode with injector temperature set to 215°C. Flow rate and linear velocity of helium through the 25 m MEGA-DEX DAC-Beta column (MEGA, Italy) were set to 1 mL min-1and 28 cm s'1, respectively. After injection, oven temperature was increased from 70°C to 95°C over 5 min. 95°C was maintained for 5 min and then increased to 215°C over 12 min and 215°C was held for 3 min. FID-detector temperature was maintained at 250°C.

[0191] Ion exclusion chromatography of carbohydrates and organic acids

[0192] Acetate, ethanol, formate, glucose, 3-hydroxybutyrate, lactate, pyruvate and succinate were quantified with ion exclusion chromatography. Samples were thawed at room temperature and centrifuged at 17,000xg for 20 min, supernatant was diluted in 2.5 mM sulfuric acid, filtered through a 0.22 pm PVDF filter and analyzed on an UltiMate 3000 system (Thermo Fisher Scientific) equipped with an UltiMate 3000 RS Diode Array Detector (Thermo Fisher Scientific) and RI-101 refractive index detector (Shodex). 10 pL samples were injected into a flow of 0.5 mL min-12.5 mM sulfuric acid on a Rezex ROA-Organic Acid H+ (8%) ION Exclusion HPLC Column (Phenomenex) at 70°C. Data analysis was performed in Chromeleon 6.80 (Thermo Fisher Scientific). Nuclear magnetic resonance spectroscopy

[0193] Spectra were acquired using an ECS 400 MHz machine from JEOL in deuterochloroform. The deuterochloroform signal at a chemical shift 6 of 77.16 ppm served as the reference. All measurements were conducted at 23°C, employing the JEOL-preset13C-acquisition method with added force tune.

[0194] Analysis of facticity by nuclear magnetic resonance spectroscopy

[0195] Spectra were acquired using an Avance Neo 500 MHz NMR spectrometer (Bruker). Samples were dissolved in deuterated chloroform and chemical shifts (6) were reported relative to the solvent signal as an internal reference (1H: 6 = 7.26 ppm, 13C: 6 = 77.16 ppm). 1H NMR spectra were recorded with 16 scans and 1.0 s delay and 13C NMR spectra with 256 scans, 2.0 s delay and broadband 1H decoupling. Probability of racemo diads (Pr) was calculated from 13C spectra by fitting methyl group signals at 19.87 ppm and 19.93 ppm after Gaussian apodization with a window size of 2.0 Hz and linear interpolation to 217 data points: Pr = Ar / (Ar + Am) x 100% where Ar and Am are the area of racemo and meso diads according to Huang et al. (2023).9 Data were processed using MestReNova 14.2.3 (Mestrelab Research).

[0196] Gel permeation chromatographic analysis of molar mass distribution

[0197] PHB was dissolved to 5 mg mL-1in chloroform by gentle heating and filtered through a 0.2 μm PTFE filter. Analysis was performed with a SECcurity (SEC, PSS Polymer, Germany) setup: Set of SDV columns at 23°C (5 μm (5 cm) precolumn, 100,000 A column (30 cm), 1000 A column (30 cm)) and a refractive index detector at 35°C (1260 Infinity, Agilent, USA). Flow rate was set to 0.7 mL min-1chloroform and injection volume to 50 μL. Polystyrene standards from 1250 g mol-1to 1,670,000 g mol-1were used for calibration.

[0198] Differential scanning calorimetry

[0199] Melting and recrystallization behavior was analyzed by differential scanning calorimetry on a DSC 1 STAR System (Mettler Toledo). Samples (5-10 mg) were weighted into aluminum crucibles. The program was run from -30°C to 200°C at a rate of 10 K min-1. Heating and cooling was performed twice with 2 min isotherms between. Analysis was performed under nitrogen at a flowrate of 50 mL min-1. Crystallinity was calculated from the equation Xc= ΔHm / AH°mx 100% where ΔHmis the measured melting enthalpy and ΔH°mis the melting enthalpy of PHB with 100% crystallinity in the ideal state. ΔH°mwas assumed to be 146 J g-1from the literature (Barham et al. 1994).14

[0200] Thermal analysis

[0201] Melting and recrystallization behavior was analyzed by differential scanning calorimetry on a DSC 1 STAR System (Mettler Toledo). 10 mg to 20 mg samples were weighted into aluminum crucibles. Heating and cooling were performed twice from -30°C to 200°C at a rate of 10 K min-1with 2 min isotherms and nitrogen gas flowrate of 50 mL min-1. Crystallinity was calculated from the equation Xc = ΔHm / ΔH0m × 100% where ΔHm is the sum of measured melting enthalpies during heating and cooling and ΔH0m is the melting enthalpy of PHB with 100% crystallinity in the ideal state. ΔH0m was given as 146 J g-1(Barham et al., Journal of Materials Science 19, 2781-2794; 10.1007 / BF01026954 (1984).). Thermogravimetric analysis was used to analyze thermal decomposition of PHB according to the manufacturer instructions. (TGA PT 1600, Linseis Messgerate)

[0202] Production of test specimens and tensile testing

[0203] Approximately 0.6 g of PHB was compacted at 50 bar and room temperature in a cylindrical mold with a diameter of 2 cm. Unless noted otherwise, compacted isotactic and atactic PHB was hot pressed for 2.5 min into discs at 10 bar and 170°C or 130°C, respectively. Discs were left to crystallize at 23°C and 50% relative humidity for at least one week. Strips between 8 and 10 cm width were cut from the material, its dimensions measured and analyzed on a universal testing machine equipped with contacting displacement transducers (smarTens 010, KARG Industrietechnik). Data was recorded and analyzed with LabMaster 2.5.11.3 (Hegewald & Peschke Mess- und Prüftechnik).

[0204] Tables

[0205] Table 1: Escherichia coli strains used in this work.

[0206] Name Genotype Source

[0207] XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' Agilent proAB lacIqΔM15 Tn10 (TetR)]

[0208] BW25113 F“ rrnB3 DEIacZ4787 hsdR514 DE(araBAD)BB7 Datsenko and Wanner DE(rhaBAD)BB8 rph-1 et al. (2000) BW25113 A adB F“ rrnB3 DEIacZ4787 hsdR514 DE(oroBAD)567 This work

[0209] A / adJ DE(rhaBAD)BB8 rph-1 fadB EfadJ

[0210] Table 1: Specific activity of Hbd enzymes from Clostridium acetobutylicum and Clostridium kluyveri of acetoacetyl CoA reduction via NADH or NADPH. pH = 7.8, T = 30°C, n = 3

[0211] Enzyme vNADH(U mg-1) vNADPH(U mg-1)

[0212] CaHbd 673 ± 37 8.87 ± 0.92

[0213] CkHbd 4.62 ± 0.17 509 ± 6 Table 2: Screening for PHA synthases producing atactic PHB in two strain background. DCM, dry cell mass; n = 3

[0214] Strain Operon, 1st Supplemented DCM (g PHB (S)-3-HB plasmid enzyme, 2nd L-1) content fraction plasmid (g g-1) (%) E. coli BW25113 CnPhaCAB GFP 1.9 ± 0.0 0.04 ± 0.0 ± 0.0

[0215] 0.02

[0216] E. coli BW25113 CnPhaCAB CkHbd 1.8 ± 0.1 0.01 ± 0.0 ± 0.0

[0217] 0.00

[0218] E. coli BW25113 CsPhaC-CnPhaAB GFP 5.9 ± 1.8 0.39 ± 0.0 ± 0.0

[0219] 0.14

[0220] E. coli BW25113 CsPhaC-CnPhaAB CkHbd 10.2 ± 0.41 ± 0.3 ± 0.1

[0221] 0.1 0.03

[0222] E. coli BW25113 PsPhaCl-CnPhaAB GFP 0.04 ±

[0223] 1.2 ± 0.1 0.01 0.0 ± 0.0 E. coli BW25113 PsPhaCl-CnPhaAB CkHbd 0.04 ± 10.7 ±

[0224] 1.2 ± 0.1 0.02 8.8 E. coli BW25113 AvPhaEC- GFP 8.1 ± 0.9 0.64 ± 0.0 ± 0.0

[0225] CnPhaAB 0.04

[0226] E. coli BW25113 AvPhaEC- CkHbd 5.7 ± 0.2 0.57 ± 1.4 ± 0.1

[0227] CnPhaAB 0.07

[0228] E. coli BW25113 PmPhaRC- GFP 1.3 ± 0.0 0.05 ± 0.0 ± 0.0

[0229] CnPhaAB 0.01

[0230] E. coli BW25113 PmPhaRC- CkHbd 1.3 ± 0.0 0.02 ± 3.3 ± 0.8

[0231] CnPhaAB 0.00

[0232] E. coli BW25113 A / adB CnPhaCAB GFP 1.9 ± 0.1 0.07 ± 0.0 ± 0.0 A adJ 0.02

[0233] E. coli BW25113 A / adB CnPhaCAB CkHbd 2.3 ± 0.6 0.03 ± 6.9 ± 1.9 A / adJ 0.01

[0234] E. coli BW25113 A / adB CsPhaC-CnPhaAB GFP 10.9 ± 0.53 ± 0.0 ± 0.0 A / adJ 0.1 0.10

[0235] E. coli BW25113 A / adB CsPhaC-CnPhaAB CkHbd 9.7 ± 0.5 0.46 ± 0.5 ± 0.0 A / adJ 0.04

[0236] E. coli BW25113 ΔfadB PsPhaCl-CnPhaAB GFP 0.08 ± A / adJ 1.2 ± 0.1 0.04 0.0 ± 0.0 E. coli BW25113 A / adB PsPhaCl-CnPhaAB CkHbd 0.05 ± 14.2 ± A / adJ 1.3 ± 0.1 0.02 3.9 E. coli BW25113 ΔfadB AvPhaEC- GFP 5.9 ± 0.1 0.50 ± 0.0 ± 0.0 ΔfadJ CnPhaAB 0.05 E. coli BW25113 ΔfadB AvPhaEC- CkHbd 5.5 ± 0.2 0.67 ± 1.8 ± 0.1 ΔfadJ CnPhaAB 0.06 E. coli BW25113 ΔfadB PmPhaRC- GFP 1.4 ± 0.0 0.06 ± 0.0 ± 0.0 ΔfadJ CnPhaAB 0.00 E. coli BW25113 ΔfadB PmPhaRC- CkHbd 1.4 ± 0.1 0.03 ± 5.9 ± 1.9 ΔfadJ CnPhaAB 0.00

[0237] Table 3: Effect of operon structure on PHB content and the fraction of (S)-3-hydroxybutyrate in the synthesized polymer. DCM, dry cell mass; n = 3

[0238] Strain Operon DCM (g PHB (S)-3-HB L-1) content fraction (g g-1) (%) E. coli BW25113 ΔfadB CsPhaC-CnPhaA-CkHbd-CnPhaB 2.0 ± 0.14 ± 1.8 ± 0.4 ΔfadJ 0.3 0.02

[0239] E. coli BW25113 ΔfadB CsPhaC-CkHbd-CnPhaAB 4.0 ± 0.44 ± 0.4 ± 0.1 ΔfadJ 0.0 0.02

[0240] E. coli BW25113 ΔfadB CkHbd-CsPhaC-CnPhaAB 3.2 ± 0.41 ± 0.6 ± 0.3 ΔfadJ 0.3 0.05

[0241] E. coli BW25113 ΔfadB AvPhaEC-CnPhaA-CkHbd-CnPhaB 2.1 ± 0.01 ± 21.0 ± ΔfadJ 0.2 0.00 4.8 E. coli BW25113 ΔfadB AvPhaEC-CkHbd-CnPhaAB 3.9 ± 0.29 ± 2.3 ± 1.2 ΔfadJ 0.8 0.09

[0242] E. coli BW25113 ΔfadB CkHbd-AvPhaEC-CnPhaAB 1.9 ± 0.03 ± 12.9 ± ΔfadJ 0.0 0.00 2.7

[0243] Table 4: Characteristics of plasmid carrying E. coli BW25113 ΔfadB ΔfadJ producing PHB during steady-state growth in minimal medium. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; td, doubling time; qp, time specific productivity; rp, biomass specific productivity; YPX, product yield; n = 3

[0244] Operon td(hh:mm) qP(mg L-1h-1) rP(mg g“1h“1) ^ (g g-1) A 1:49 ± 0:05 11.0 ± 0.2 36.7 ± 3.4 0.48 ± 0.03 B 1:46 ± 0:01 2.8 ± 0.5 12.6 ± 0.2 0.15 ± 0.01 C 1:15 ± 0:01 6.2 ± 0.4 18.5 ± 1.2 0.16 ± 0.01 D 1:54 ± 0:02 5.0 ± 0.2 19.0 ± 1.8 0.23 ± 0.02

[0245] Table 5: Dry cell mass and PHB extracted from a 1 L cultivation of E. coli BW25113 ΔfadB ΔfadJ. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd- AvPhaEC-CnPhaAB; DCM, dry cell mass; (S)-3-HB, (S)-3-hydroxybutyrate fraction; n = 1

[0246] Operon DCM (g) PHB (g) (S)-3-HB (%) A 2.32 1.22 0.0 B 1.61 0.42 5.2

[0247] C 1.24 0.39 0.7

[0248] D 1.03 0.26 3.4

[0249] Table 6: Molar mass distribution of microbially produced PHB. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA-CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; Mn, number average molar mass; Mw, weight average molar mass; Đ, dispersity; n = 1

[0250] Operon Mn(g mol-1) Mw(g mol-1) Đ

[0251] Commercial 3.38 x 1051.07 x 1063.18

[0252] A 5.08 x 1051.41 x 1062.76

[0253] B 4.64 x 1051.25 x 1062.70 C 4.02 x 1051.18 x 1062.92 D 5.36 x 1051.26 x 1062.35

[0254] Table 7: Thermal properties of microbially produced PHB. A, AvPhaEC-CnPhaAB; B, AvPhaEC-CnPhaA- CkHbd-CnPhaB; C, AvPhaEC-CkHbd-CnPhaAB; D, CkHbd-AvPhaEC-CnPhaAB; (S)-3-HB, (S)-3-hydroxybutyrate; Tm, melting temperature; ACP, glass transition step height; Xc, crystallinity; n = 1 Operon (S)-3-HB (%) Tm(°C) ACP(J g-1K-1) Xc(%) A 0.0 174.3 0.13 51 B 5.2 168.6 0.25 24 C 0.7 173.1 0.14 42 D 3.4 168.1 0.15 35

[0255] Table 8: Process metrics for the production of isotactic and atactic PHB. Data are given for biological duplicates.

[0256] Metric Strain A, isotactic Strain B, atactic

[0257] Process time (h) 28.90 28.93 25.18 25.20

[0258] Batch growth rate (h-1) 0.35 0.34 0.36 0.36

[0259] Doubling time (min) 117 122 116 116

[0260] PHB titer (g L-1) 38.8 40.6 1.76 1.61

[0261] RCM titer (g L-1) 49.2 50.9 35.7 35.2

[0262] DCM titer (g L-1) 88.0 91.5 37.5 36.8

[0263] PHB productivity (g L-1h-1) 1.34 1.40 0.07 0.06 RCM productivity (g L-1h-1) 1.70 1.76 1.42 1.40

[0264] DCM productivity (g L-1h-1) 3.04 3.16 1.49 1.46

[0265] PHB yield (g g1) 0.20 0.20 0.012 0.011

[0266] RCM yield (g g-1) 0.25 0.25 0.24 0.23

[0267] DCM yield (gg-1) 0.45 0.45 0.25 0.25

[0268] PHB content (%) 44.1 44.4 4.7 4.4

[0269] Table 9: Structural, thermal, and mechanical properties of fermentatively produced PHB.

[0270] Data are given as mean and standard deviation. (S)-HB, (S)-3-hydroxybutyrate fraction; Pr, racemo diad probability; Mn, number average molar mass; Mw, weight average molar mass; Đ, polydispersity; Tg, glass transition temperature; Tm, melting temperature; ACP, specific heat capacity of glass transition; ΔHf, enthalpy of fusion; ΔHc, enthalpy of crystallization; Xc, degree of crystallinity; E, Young's modulus; o, tensile strength; s, elongation at break; W, fracture work; n, sample size

[0271] Commercial, Strain A, isotactic Strain B, atactic n isotactic

[0272] (S)-HB (%) 0.00 ± 0.00 0.00 ± 0.00 6.84 ± 0.04 4 Pr(%) 0.0 ± 0.0 0.0 ± 0.0 11.8 ± 0.7 2 Mn(104g mol-1) 34 ± 2 36 ± 4 25 ± 2 ≥ 6 Mw(104g mol-1) 126 ± 15 141 ± 18 66 ± 6 ≥ 6 Đ 3.7 ± 0.5 4.0 ± 0.7 2.7 ± 0.2 ≥ 6 Tg(°C) 5.7 ± 1.3 8.2 ± 1.5 6.7 ± 0.5 ≥ 3 Tm(°C) 174.7 ± 1.3 179.3 ± 0.7 154.3 ± 1.1 ≥ 3 ΔCP(J g-1K-1) 0.04 ± 0.06 0.13 ± 0.01 0.70 ± 0.06 ≥ 3 ΔHf(J g-1) −89 ± 4 −85 ± 3 −50 ± 3 ≥ 3 ΔHc(J g-1) 80 ± 1 79 ± 3 57 ± 5 ≥ 3 Xc(%) 54.7 ± 0.7 54.0 ± 1.7 39.1 ± 3.2 ≥ 3 E (GPa) 1.2 ± 0.3 1.1 ± 0.2 0.6 ± 0.1 ≥ 9 σ (MPa) 40 ± 1 36 ± 2 36 ± 1 ≥ 9 ε (%) 5.3 ± 1.3 4.8 ± 1.0 10.5 ± 1.5 ≥ 9 W (MJ m-3) 1.4 ± 0.4 1.1 ± 0.3 2.5 ± 0.4 ≥ 9

[0273] Table 10: Structural, thermal and mechanical comparison between microbially and chemically produced atactic PHB. (S)-HB, (S)-3-hydroxybutyrate fraction; Pr, racemo diad probability; Mn, number average molar mass; Mw, weight average molar mass; D, polydispersity; Tg, glass transition temperature; Tm, melting temperature; Xc, degree of crystallinity; E, Young's modulus; o, tensile strength; s, elongation at break; W, fracture work; n.a., not available This work Bloembergen Abe (1994)* Haslbbck (2018

[0274] (1989)9& 2019** Origin Microbial Chemical Chemical Chemical (S)-HB (%) 6.84 ± 0.04 n.a. n.a. 7

[0275] Pr(%) 11.8 ± 7 15 16 13

[0276] Mn(104g mol-1) 34 ± 2 n.a. 11 9.3 ± 1.3 Mw(104g mol-1) 126 ± 15 n.a. 20 n.a.

[0277] D 3.7 ± 0.5 n.a. 1.8 1.12 ± 0.05 Tg(°C) 6.7 ± 0.5 5 6 5 ± 2

[0278] Tm(°C) 154.3 ± 1.1 165 132 141 ± 8 Xc(%) 39.1 ± 3.2 34 49 38.6 ± 0.4 £ (GPa) 0.6 ± 0.1 n.a. 1.2 1.5 ± 0.1 o (MPa) 36 ± 1 n.a 15 30 ± 1 E (%) 10.5 ± 1.5 n.a. 7 5 ± 2

[0279] 1 / 1 / (MJ m-3) 2.5 ± 0.4 n.a. n.a. 0.47 ± 0.07 * Abe, H., Matsubara, I., Doi, Y., Hori, Y. & Yamaguchi, A. Physical Properties and Enzymic Degradability of Poly(3-hydroxybutyrate) Stereoisomers with Different Stereoregularities.

[0280] Macromolecules 27, 6018-6025; 10.1021 / ma00099a013 (1994).

[0281] ** Haslbbck, M. et al. Structures of Mixed-Tacticity Polyhydroxybutyrates. Macromolecules 51, 5001-5010; 10.1021 / acs.macromol.8b01047 (2018); and Haslböck, M. et al. Mechanical and Thermal Properties of Mixed-Tacticity Polyhydroxybutyrates and Their Association with Iso- and Atactic Chain Segment Length Distributions. Macromolecules 52, 5407–5418; 10.1021 / acs.macromol.9b00931 (2019) References of the Introduction

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Claims

CLAIMS1. A method for the production of atactic poly(3-hydroxybutyrate) (PHB) comprising(a) producing a mixture of (R)-3-hydroxybutryl-CoA and (S)-3-hydroxybutryl-CoA by (al) reducing acetoacetyl-CoA to a mixture of (R)-3-hydroxybutyryl-CoA and (S)-3- hydroxybutryl-CoA by contacting acetoacetyl-CoA with an enzyme catalyzing the reaction (R)-3-hydroxyacyl-CoA + NAD(P)+= 3-oxoacyl-CoA + H++ NAD(P)H and an enzyme catalyzing the reaction (3S)-3-hydroxybutyryl-CoA + NAD(P)+= acetoacetyl-CoA + H++ NAD(P)H in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), and / or(a2) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the isomerization of (R)- 3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA, and / or(a3) the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3-hydroxybutyryl-CoA by contacting (R)-3-hydroxybutyryl-CoA with an enzyme catalyzing the reaction (3R)-3-hydroxybutyryl-CoA ↔ crotonyl-CoA + H2O to produce Crotonyl-CoA and contacting Crotonyl-CoA with an enzyme catalyzing the reversible hydration of crotonyl-CoA, and (b) polymerizing the mixture of (a) to atactic poly(3-hydroxybutyrate) by contacting the mixture of (a) with an enzyme catalyzing the reaction 3-hydroxybutyryl-CoA + poly(3- hydroxybutyrate)(n) = poly(3-hydroxybutyrate)(n+ij + CoA.

2. The method of claim 1, wherein(al) the enzyme catalyzing the reaction (R)-3-hydroxyacyl-CoA + NAD(P)+= 3-oxoacyl-CoA + H++ NAD(P)H is acetoacetyl-CoA reductase (PhaB) and the enzyme catalyzing the reaction (3S)-3- hydroxybutyryl-CoA + NAD(P)+= acetoacetyl-CoA + H++ NAD(P)H is 3-hydroxybutyryl-CoA dehydrogenase (Hbd), and / or(a2) the enzyme catalyzing the isomerization of (R)-3-hydroxybutyryl-CoA to (S)-3- hydroxybutyryl-CoA is enzyme 3-hydroxybutyryl-CoA epimerase (FadB), and / or(a3) the enzyme catalyzing the reaction (3R)-3-hydroxybutyryl-CoA ↔ crotonyl-CoA + H2O is (3R)-3-hydroxybutyryl-CoA hydrolyase (PhaJ) and the enzyme that catalyzes the reversible hydration of crotonyl-CoA is (3S)-3-hydroxyacyl-CoA hydrolyase (Crt), and(b) the enzyme catalyzing the reaction 3-hydroxybutyryl-CoA + poly(3-hydroxybutyrate)(n)= poly(3-hydroxybutyrate)(n+1)+ CoA is polyhydroxyalkanoate synthase (PhaC).

3. The method of claim 2, wherein(i) PhaB has an amino acid sequence of SEQ ID NO: 1 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and / or(ii) Hbd has an amino acid sequence of SEQ ID NO: 3 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

4. The method of claim 2 or 3, wherein FadB has an amino acid sequence of SEQ ID NO: 5 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

5. The method of any one of claims 2 to 4, wherein PhaJ has an amino acid sequence of SEQ ID NO:7 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and / or Crt has an amino acid sequence of SEQ ID NO: 9 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

6. The method of any one of claims 1 to 5, wherein PhaC has an amino acid sequence of any one of SEQ ID NOs 11, 13, 15, 17 or 19 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical to any one of SEQ ID NOs 11, 13, 15, 17 or 19.

7. The method of any one of claims 1 to 6 further comprising prior to step (a)(a') catalyzing acetyl-CoA into acetoacetyl-CoA by contacting acetyl-CoA with an enzyme catalyzing the reaction 2 acetyl-CoA = acetoacetyl-CoA + CoA, wherein the enzyme is preferably acetyl-CoA C-acetyltransferase (β-ketothiolase, PhaA).

8. The method of claim 7, wherein PhaA has an amino acid sequence of SEQ ID NO: 25 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

9. The method of any one of claims 1 to 8, wherein the method is carried out in a host cell, wherein the host cell comprises genes encoding the enzymes according to any one of claims 1 to 8 and the host cell expresses the enzymes according to any one of claims 1 to 8.

10. The method of claim 9, wherein the genes are organized into a cluster of genes under the control of a single promoter or wherein each of the genes is under the control of a separate promoter.

11. The method of claims 10 or 11, wherein the genes are in the genome of the host cell, or on one or more plasmids or vectors.

12. A nucleic acid molecule comprising the genes encoding the enzymes according to any one of claims 1 to 8, wherein the genes are organized into a cluster of genes under the control of a single promoter or wherein each of the genes is under the control of a separate promoter, wherein the nucleic acid molecule is preferably a plasmid, vector or genome.

13. A host cell comprising genes encoding the enzymes according to any one of claims 1 to 8 and being capable of expressing the enzymes according to any one of claims 1 to 8.

14. The host cell of claim 13, wherein the genes are in the genome of the host cell, or on one or more plasmids or vectors.

15. A method of producing the host cell of claim 13 or 14 comprising introducing into the host cell the genes encoding the enzymes according to any one of claims 1 to 8 in an expressible form.

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