Propylene production in cyanobacteria

Abstract

The present invention relates to a process for producing propylene and to a cyanobacterial cell for the production of propylene.

Description

[0001] Propylene production in cyanobacteria

[0002] Field of the invention

[0003] The present invention relates to the field of biochemistry, specifically to a process for producing propylene and to a cyanobacterial cell for the production of propylene.

[0004] Background of the invention

[0005] Numerous biotechnological processes make use of genetically engineered organisms in order to produce bulk or fine chemicals, proteins or antibiotics. In many cases, increased production has been obtained by improved gene expression and by optimization of growth conditions. In all processes we are aware of, the initial carbon-precursor has been and still is sugar (notably glucose, but many other mono- and polysaccharides are in use) or related organic substrates: solventogenesis (including butanol and ethanol) and organic acid production (e.g. lactic-, citric- or succinic acid) always starts from glucose, which makes it inefficient as the production process uses a high energy initial compound as substrate.

[0006] Propylene (or propene) is a colourless, flammable, gaseous hydrocarbon. Propylene is the second largest-volume chemical produced globally. It is an important raw material for the production of polymers, polymer intermediates and organic chemicals such as polypropylene, acrylonitrile, propylene oxide, and oxo alcohols, as well as for a large variety of industrial products.

[0007] Propylene is produced primarily as a by-product of petroleum refining and of ethylene production by steam cracking of hydrocarbon feedstocks. Typical hydrocarbon feedstocks are from non-renewable fossil fuels, such as petroleum, natural gas and to a much lesser extent coal. The fast consumption of fossil fuels is making conventional propylene production methods unsuitable to meet the growing demand for propylene, therefore, the development of more efficient and economical production methods is a major concern.

[0008] Alternative methods to produce propylene have been proposed, including the propane dehydrogenation (PDH), the methanol-olefin process and the Fischer-Tropsch olefin process. Propylene can be also produced via direct fermentation of sugars or other feedstocks to produce the alcohols 2-propanol (isopropanol) or 1 -propanol, which is separated, purified, and then dehydrated to propylene in a second step involving metal-based catalysis.

[0009] However, there exists a need for alternative methods for effectively producing commercial quantities of propylene, preferably without the need of expensive or complicated starting materials, which process does not have the drawbacks of existing processes. The present invention satisfies this need and provides related advantages as well.

[0010] Cyanobacteria have been reported for the production of various compounds. W02009078712 relates to the production of butanol, ethanol, ethylene, succinate, propanol, acetone and D-lactate in cyanobacteria. WO2011136639 relates to the production of L-lactate in cyanobacteria. WO2014092562 relates to the production of acetoin, 2,3-butanediol and 2-butanol in cyanobacteria. WO2016008883 relates to the production of various monoterpenes in cyanobacteria. W02016008885 relates to the production of various sesquiterpenes in cyanobacteria. Eisenhut et al. (2008) relate to the CO2 concentrating mechanism of cyanobacteria.

[0011] As depicted here above, one has succeeded in producing various compounds in cyanobacteria; however, the yields vary between products and for some products the yield appears still too low to be commercially relevant.

[0012] At present, there is no efficient bioprocess for the production of propylene available, neither on laboratory scale, nor on industrial scale, especially while using CO2 as carbon source. Thus, in view of the state of the art, there is still a need for an alternative, more sustainable and improved propylene production process, without the need for expensive and complicated starting materials, and with a commercially relevant yield.

[0013] Brief description of the drawings

[0014] Figure 1. Metabolic pathway to propylene (scheme).

[0015] Figure 2. Western Blotting for OleT-BM3R & OleTje. Western Blotting for detection of OleT- BM3R and OleTjE fused with a N-terminal 10x His tag, using Anti-His Tag Antibodies, as described in Example 3. Positive controls of the genes expressed in E coliwere included for reference.

[0016] Figure 3. Propylene formation using enzyme assays. Propylene formation measured by GC-FID of enzyme assays using lysates of a Synechocystis strain expressing OleT-BM3R (solid trace) or in absence of lysate (dashed trace) using supplemented butyric acid and NADPH, as described in Example 4.

[0017] Figure 4. Butyrate feeding to cyanobacteria for propylene production using OleT-BM3R. Propylene formation in vivo, as described in Example 5, of a Synechocystis strain expressing OleT- BM3R with supplemented butyrate (second), and a propylene standard sample (first) measured using GC-FID. Negative control of a Synechocystis expressing OleT-BM3R without supplemented butyrate (third). Negative control of a Synechocystis not expressing OleT-BM3R with supplemented butyrate (fourth).

[0018] Figure 5. Butyrate feeding to cyanobacteria for propylene production using OleT. Propylene formation in vivo, as described in Example 5, of a Synechocystis strain expressing OleT with supplemented butyrate (second) and a propylene standard sample (first) measured using GC-FID, as described in Example 5, resulting in production of 1.8 pg per liter of culture volume . Negative control of a Synechocystis expressing OleT without supplemented butyrate (third).

[0019] Figure 6. Butyric acid formation by cyanobacteria. Growth (OD730) and butyrate production (mM) of the following Synechocystis strains: wildtype control (black), and butyrate-producing strain (orange).

[0020] Description of the invention

[0021] Surprisingly, it has now been demonstrated that propylene can conveniently be produced from CO2 and light in a cyanobacterial cell. The inventors of the present invention have arrived at a scalable process for the production of propylene.

[0022] The biosynthesis of propylene according to the present invention is based on direct conversion of CO2 and light, using a photosynthetic organism by directing the metabolism of said photosynthetic organism towards butyrate formation and using the synthetic oxidative decarboxylase enzyme OleTjE for the formation of propylene, using O2 as the oxidant and NADPH as an electron donor (Figure 1).

[0023] A production pathway to butyric acid has already been established in cyanobacteria and Escherichia coli (see, for example, Lai et al. Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria. Biotechnology and bioengineering, vol. 116, issue 4, April 2019, pages 893- 903; Wang etal. Metabolic engineering of Escherichia coli for the production of butyric acid at high titer and productivity. Biotechnol Biofuels, Mar 22, 2019; 12:62).

[0024] The fatty acid decarboxylase OleTjE (from Jeotgalicoccus sp. ATCC 8456) has been shown to be functional as a catalyst for the production of linear a-alkenes, but it originally uses H2O2 as electron / oxygen donor (see, for example, Bauer et al. Production of Propene from n-Butanol: A Three- Step Cascade Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleTjE. Chembiochem 21 , 3273-3281). Bauer ef al. describes the use of OleT for the production of propene, but in an enzymatic catalysis reaction feeding butanol.

[0025] Fusing the decarboxylase OleTjE and the reductase domain of P450BM3 creates a self-sufficient protein, OleT-BM3R, which is able to efficiently catalyse oxidative decarboxylation of carboxylic acids into linear a-olefins (see Lu etal. An Engineered Self-Sufficient Biocatalyst Enables Scalable Production of Linear a-Olefins from Carboxylic Acids. ACS CataL 8, 2018, 5794-5798).

[0026] Although successful OleT expression has been reported in Chlamydomonas, no functional enzyme has been obtained (see Yunus et al. Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel. Metabolic Engineering, Volume 49, September 2018, pages 201-211).

[0027] The present invention combines metabolic properties of photoautotrophic and chemoorganotrophic microorganisms and is based on the employment of recombinant oxyphototrophs with high rates of conversion of Calvin cycle intermediates to a fermentative end product. Its beauty resides in the fact that its core chemical reactions use carbon dioxide as the sole carbon-containing precursor and light (preferably sunlight), as the sole energy source, to drive carbon dioxide reduction. Moreover, the cyanobacterial cell factory is more suitable for production of propylene than other microorganism used in fermentation processes such as E. coli, since the abundantly available co-factor in the cyanobacterial cell is NADPH, rather than NADH in most chemotrophic organisms used for fermentation. Alternatively, electrons can be derived directly from photosynthesis through the endogenous ferredoxins. Production may be controlled by a nutrient- or light-sensitive promoter. Using a nutrient-sensitive promoter, production is controlled by a medium component and can start at the most appropriate time, such as at the highest possible cell density. A light-mediated promoter is controlled by light intensity. Whereas in current production processes for biochemicals, organisms are substrate (e.g., crops in ethanol production) or product (e.g., microalgae as biodiesel), here microorganisms are used as highly specialized catalysts for the conversion of carbon dioxide as a substrate to a valuable end product. These catalysts can be subjected to further optimization strategies through physical- and chemical systems-biology approaches. The biochemical background of cyanobacterial cells for the production of valuable compounds is more extensively described in WO 2009 / 078712. The various aspects of the present invention are more extensively described below. In a first aspect, the present invention relates to a cyanobacterial cell capable of expressing, preferably expressing, at least one heterologous functional enzyme, wherein the at least one functional enzyme is an oxidative decarboxylase. Said cyanobacterial cell is herein further referred to as a cyanobacterial cell according to the invention or to the present invention. In the embodiments herein, the cyanobacterial cell is preferably capable of producing propylene, more preferably producing propylene.

[0028] In the embodiments herein, the oxidative decarboxylase may be a cytochrome P450 enzyme. In the embodiments herein, the oxidative decarboxylase preferably is an OleT decarboxylase. In the embodiments herein, the OleT decarboxylase preferably is from Jeotgalicoccus sp. ATCC 8456.

[0029] In the embodiments herein, the OleT decarboxylase may be linked to a redox partner. Preferably, the OleT decarboxylase is linked to a heterologous redox partner, such as BM3R or RhFRED. In the embodiments herein, the OleT decarboxylase may be linked (coupled) to the endogenous ferredoxin, receiving electrons directly from photosynthesis.

[0030] In the embodiments herein, at least one further endogenous or heterologous functional enzyme may be present and may be selected from the group consisting:

[0031] (i) a dehydratase, a reductase, an esterase, and a decarboxylase; preferably a 3-hydroxbutyryl- CoA dehydratase (crotonase), a trans-enoyl-coA reductase and / or a thioesterase; or

[0032] (ii) a 3-hydroxbutyryl-CoA dehydratase (crotonase), a trans-enoyl-coA reductase, phosphotransbutyrylase and a butyrate kinase; or

[0033] (iii) a malonyl CoA-ACP transacylase (fabD), p-ketoacyl-ACP synthase III (fabH), p-ketoacyl- ACP reductase (fabG), p-hydroxyacyl-ACP dehydratase (fabZ), enoyl-ACP reductase (fabl) and a thioesterase (TE); and optionally, an acetyl-CoA acetyltransferase (phaA), ketoacyl-ACP synthase III (NphT7) and an acetoacetyl-CoA reductase (phaB).

[0034] In the embodiments herein, a preferred 3-hydroxbutyryl-CoA dehydratase (crotonase) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0035] In the embodiments herein, a preferred 3-hydroxbutyryl-CoA dehydratase (crotonase) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6.

[0036] In the embodiments herein, a preferred 3-hydroxbutyryl-CoA dehydratase (crotonase) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 7.

[0037] In the embodiments herein, a preferred 3-hydroxbutyryl-CoA dehydratase (crotonase) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8.

[0038] In the embodiments herein, a preferred a trans-enoyl-coA reductase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:9, and SEQ ID NO: 10.

[0039] In the embodiments herein, a preferred a trans-enoyl-coA reductase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:9.

[0040] In the embodiments herein, a preferred a trans-enoyl-coA reductase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10.

[0041] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, and SEQ ID NO: 20.

[0042] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13.

[0043] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14.

[0044] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 15.

[0045] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 16.

[0046] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID

[0047] NO: 17.

[0048] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 18.

[0049] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 19.

[0050] In the embodiments herein, a preferred thioesterase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 20.

[0051] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27, and SEQ ID NO: 28.

[0052] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 25.

[0053] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 26.

[0054] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 27.

[0055] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 28.

[0056] In the embodiments herein, a preferred CoA-ACP transacylase (fabD) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 29. In the embodiments herein, a preferred p-ketoacyl-ACP synthase III (fabH) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 30.

[0057] In the embodiments herein, a preferred p-ketoacyl-ACP reductase (fabG) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 31 .

[0058] In the embodiments herein, a preferred P-hydroxyacyl-ACP dehydratase (fabZ) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 32.

[0059] In the embodiments herein, a preferred thioesterase (TE) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27, and SEQ ID NO: 28.

[0060] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 34, 35, 36, and SEQ ID NO: 37.

[0061] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 34.

[0062] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 35.

[0063] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 36.

[0064] In the embodiments herein, a preferred butyrate kinase comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 37.

[0065] In the embodiments herein, a preferred acetyl-CoA acetyltransferase (phaA) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 , and SEQ ID NO: 2.

[0066] In the embodiments herein, a preferred acetyl-CoA acetyltransferase (phaA) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1 .

[0067] In the embodiments herein, a preferred acetyl-CoA acetyltransferase (phaA) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2.

[0068] In the embodiments herein, a preferred ketoacyl-ACP synthase III (NphT7) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 3.

[0069] In the embodiments herein, a preferred acetoacetyl-CoA reductase (phaB) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 4, and SEQ ID NO: 5.

[0070] In the embodiments herein, a preferred acetoacetyl-CoA reductase (phaB) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4.

[0071] In the embodiments herein, a preferred acetoacetyl-CoA reductase (phaB) comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 5.

[0072] In the embodiments herein, the at least one further functional enzyme may be one selected from the enzymes listed in Table 1 below.

[0073] Table 1. Enzymes and their origin

[0074] In the embodiments herein, the oxidative decarboxylase preferably comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 57.

[0075] In the embodiments herein, the oxidative decarboxylase preferably comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 54.

[0076] In the embodiments herein, the oxidative decarboxylase preferably comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 57.

[0077] In the embodiments herein, the oxidative decarboxylase is preferably encoded by a polynucleotide that has an nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO: 56.

[0078] In the embodiments herein, the oxidative decarboxylase is preferably encoded by a polynucleotide that has an nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 52.

[0079] In the embodiments herein, the oxidative decarboxylase is preferably encoded by a polynucleotide that has an nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 53.

[0080] In the embodiments herein, the oxidative decarboxylase is preferably encoded by a polynucleotide that has an nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 55.

[0081] In the embodiments herein, the oxidative decarboxylase is preferably encoded by a polynucleotide that has an nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 56.

[0082] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, and SEQ ID NO: 49.

[0083] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 38.

[0084] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 39.

[0085] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 40.

[0086] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 41 .

[0087] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 42.

[0088] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 43.

[0089] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 44.

[0090] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 45.

[0091] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 46.

[0092] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 47.

[0093] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 48.

[0094] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be under the control of a constitutive promoter, wherein the constitutive promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence as set forward in SEQ ID NO: 49.

[0095] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be underthe control of a nutrient-regulated promoter, wherein preferably a nutrient-regulated promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with SEQ ID NO: 50, or with SEQ ID NO: 51 .

[0096] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be underthe control of a nutrient-regulated promoter, wherein preferably a nutrient-regulated promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with SEQ ID NO: 50.

[0097] In the embodiments herein, the polynucleotide encoding the oxidative decarboxylase may be underthe control of a nutrient-regulated promoter, wherein preferably a nutrient-regulated promoter has at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with SEQ ID NO: 51 .

[0098] In the embodiments herein, the expression of said polynucleotide or polynucleotides preferably confers to the cell the ability to convert a butyrate intermediate into propylene. In the embodiments herein, a preferred butyrate intermediate is butyric acid.

[0099] In the embodiments herein, the cyanobacterial cell is capable of producing and preferably produces at least 1 .0 ug propylene per liter of culture volume when cultured under conditions conducive to the production of propylene. Preferably, the culture conditions include culturing in the presence of sunlight and feeding carbon dioxide for at least 1 day.

[0100] The person skilled in the art knows how to measure propylene in a liquid and in a gas.

[0101] The propylene produced within the cyanobacterial cell according to the invention may be present in a gas mixture. Assays for the detection of propylene are, but not limited to Gas Chromatography (GC) or Gas Chromatography-Mass Spectrometry (GC-MS). A preferred assay for the detection of propylene is Gas Chromatography (GC). A detectable amount for propylene is preferably at least 1 .0 pg per liter culture volume under said culture conditions and using GC. Preferably, a detectable amount (per liter culture volume) is at least 2.0pg. 3.0pg, 4.0pg, 5.0pg, 10.0pg, 20pg, 30pg, 40pg, 50pg, 60pg, 70pg, 80pg, 90pg, 100pg, 150pg, 200pg, 300pg, 400pg, 500pg, or at least 1000pg per liter culture volume.

[0102] Preferably, a cyanobacterial cell according to the present invention comprises at least one nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one of the at least one functional enzyme as defined herein above. Accordingly, a preferred cyanobacterial cell according to the invention comprises at least one nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one of the at least one functional enzyme as defined herein above.

[0103] The at least one functional enzyme as defined herein above is encoded by a polynucleotide. In all embodiments according to the invention, each encoding polynucleotide may be present on a separate nucleic acid molecule. Alternatively, two or more encoding polynucleotides may be present on a single nucleic acid molecule.

[0104] A preferred cyanobacterial cell according to the invention is a cyanobacterial cell wherein the at least one functional enzyme is encoded by a nucleic acid molecule comprising or consisting of a polynucleotide wherein said nucleic acid molecule is preferably present in the cyanobacterial cell as an episomal entity, preferably said episomal entity is a plasmid, more preferably a self-replicating plasmid. The episomal entity and plasmid can be any episomal entity and plasmid known to the person skilled in the art or can be based on any episomal entity and plasmid known to the person skilled in the art and modified to comprise any nucleic acid and / or polynucleotide described herein.

[0105] Another preferred cyanobacterial cell according to the invention is a cyanobacterial cell wherein the at least one functional enzyme is encoded by a nucleic acid molecule comprising or consisting of a polynucleotide wherein said nucleic acid molecule is preferably integrated in the cyanobacterial genome, preferably via homologous recombination.

[0106] A cyanobacterial cell according to the present invention may comprise a single copy but may also comprise multiple copies of a nucleic acid molecule comprising or consisting of an polynucleotide encoding a functional enzyme as defined herein above.

[0107] A preferred cyanobacterial cell according to the present invention is a cyanobacterial cell, wherein a polynucleotide encoding the at least one functional enzyme is under control of a regulatory system which responds to a change in the concentration of a nutrient when culturing said cyanobacterial cell.

[0108] A promoter that may be used for the expression of a polynucleotide encoding the at least one functional enzyme may be foreign to the polynucleotide, i.e. a promoter that is heterologous to the polynucleotide encoding the at least one functional enzyme to which it is operably linked. Although a promoter preferably is heterologous to the polynucleotide to which it is operably linked, it is also possible that a promoter is native to the cyanobacterial cell according to the present invention. Preferably, a heterologous (to the nucleotide sequence) promoter is capable of producing a higher steady state level of a transcript comprising a coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is a promoter that is native to the coding sequence. A suitable promoter in this context includes both a constitutive and an inducible natural promoter as well as an engineered promoter. A promoter used in a cyanobacterial cell according to the present invention may be modified, if desired, to affect its control characteristics.

[0109] A promoter used in a cyanobacterial cell according to the present invention may be selected from, but not limited to promoters listed in Table 2 below or a functional fragment thereof. Table 2. Promoters

[0110] A preferred promoter for constitutive expression is a Ptrc. The Ptrc promoter is a synthetic promoter, which is constructed as a chimera of the E. coli trp operon and / acUV5 promoters (Brosius et al, J Biol Chem 1985). The promoter is thus regulated by the Lac repressor, Lacl. In Synechocystis, the Lacl regulated repression and induction does not function efficiently, but the Ptrc promoter does show high constitutive expression levels in the absence of Lacl (Huang H-H, Camsund D, Lindblad P, Heidorn T: Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 2010, 38:2577-2593).

[0111] The cyanobacterial cell according to the present invention can conveniently be used for the production of propylene.

[0112] Accordingly, in a second aspect, the present invention relates to a process for producing propylene comprising culturing a cyanobacterial cell according to the present invention, preferably a cyanobacterial cell as defined in the first aspect of the present invention, under conditions conducive to the production of propylene-containing gas mixture and, optionally, separating propylene from the gas mixture.

[0113] In the embodiments herein, the culture conditions may comprise feeding carbon dioxide to the culture of cyanobacterial cells and / or subjecting the culture to light. In the embodiments herein, the culture conditions may further comprise feeding butyric acid to the culture of cyanobacterial cells.

[0114] In a preferred embodiment, carbon dioxide is fed to the culture of cyanobacterial cells. In a preferred embodiment, the culture of cyanobacterial cells is subjected to light. In a preferred embodiment, carbon dioxide is fed to the culture of cyanobacterial cells and the culture of cyanobacterial cells is subjected to light.

[0115] A preferred natural light is sunlight. Daylight (or sunlight) may have an intensity ranged between approximately 500 and approximately 1500 pEinstein / m 2 / s. The light used may be sunlight (direct or indirect) and / or may be artificial light. Such artificial light may have an intensity ranged between approximately 70 and approximately 800 pEinstein / m 2 / s. Preferably, the cells are continuously under the light conditions as specified herein above. However, the cells may also be exposed to high light intensities (such as e.g. daylight / sunlight) as defined herein above for a certain amount of time, after which the cells are exposed to a lower light intensity as defined elsewhere herein for a certain amount of time, and optionally this cycle is repeated. In an embodiment, the cycle is the day / night cycle.

[0116] In a preferred process, propylene is separated from the gas mixture. This may be realized continuously with the production process or subsequently to it. Separation may be based on any separation method known to the person skilled in the art.

[0117] In a third aspect, the present invention relates to propylene, obtainable by a process according to the present invention as described above.

[0118] Definitions

[0119] The term “functional enzyme” is herein preferably defined in the context of an oxidative decarboxylase as an enzyme from the family of lyases and able to catalyze the loss of carbon dioxide from a carboxylic acid by electron oxidation which results in propylene formation.

[0120] In the context of all embodiments of the present invention, the terms “a cyanobacterium”, “a cyanobacterium cell” and “a cyanobacterial cell” are used interchangeably and refer to a blue-green algae, a photosynthetic unicellular microorganism. Examples of cyanobacteria include the genera Aphanocapsa, Anabaena, Nostoc, Oscillatoria, Synechococcus, Synechocystis, Gloeocapsa, Agmenellum, Scytonema, Mastigocladus, Arthrosprira, Haplosiphon.

[0121] A cyanobacterial cell according to the present invention may be or may be derived from a Synechococcus, preferably Synechococcus elongatus PCC 7942, Synechococcus PCC7002, Synechococcus PCC11901, or from a Synechocystis, preferably a Synechocystis PCC 6803.

[0122] A preferred order of cyanobacteria is Chroococcales. A preferred cyanobacterium genus is Synechocystis. Another preferred cyanobacterium genus is Synechococcus. A more preferred species are Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803 species. Even more preferably, the Synechocystis is a Pasteur Culture Collection (PCC) 6803 Synechocystis, which is a publicly available strain via ATCC for example. PCC 6803 has been stored at ATCC under ATCC27184. The phototrophic Synechocystis PCC 6803 is a fast growing cyanobacterium with no specific nutritional demands. Its physiological traits are well-documented: it is able to survive and grow in a wide range of conditions. For example, Synechocystis sp. PCC 6803 can grow in the absence of photosynthesis if a suitable fixed-carbon source such as glucose is provided. Perhaps most significantly, Synechocystis sp. PCC 6803 was the first photosynthetic organism for which the entire genome sequence was determined (available via the internet world wide web at kazusa.or.jp / cyano / cyano). In addition, an efficient gene deletion strategy (Shestakov SV et al., 2002; and Nakamura Y et al., 1999) is available for Synechocystis sp. PCC 6803, and this organism is furthermore easily transformable via homologous recombination (Grigirieva GA et aL, 1982).

[0123] “Capable of producing propylene” preferably means herein that detectable amounts of propylene can be detected in a culture of a cyanobacterial cell according to the present invention cultured, under conditions conducive to the production of propylene, preferably in the presence of light and dissolved carbon dioxide and / or bicarbonate ions, during at least 1 day using a suitable assay for detecting propylene. A preferred concentration of said dissolved carbon dioxide and / or bicarbonate ions is, the natural occurring concentration at neutral to alkaline conditions (pH 7 to 9) being approximately 1 mM. This is equivalent to 0.035% of carbon dioxide in ambient air. A more preferred concentration of carbon dioxide and / or bicarbonate ions is higher than this natural occurring concentration. Preferably, the concentration of bicarbonate ions is at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM... up to 200mM. A preferred method to increase the carbon dioxide and / or bicarbonate ions in solution is by enrichment with carbon dioxide, preferably waste carbon dioxide from industrial plants, sparged into the culture broth. The concentration of carbon dioxide is preferably increased to at least 0.04%, 0.05%, 0.1%, 0.15%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%.

[0124] "Sequence identity" or “identity” in the context of amino acid- or nucleic acid-sequence is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Within the present invention, sequence identity with a particular sequence preferably means sequence identity over the entire length of said particular polypeptide or polynucleotide sequence. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

[0125] "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0126] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403- 410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et aL, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et aL, J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0127] Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WL The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

[0128] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

[0129] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; lie to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

[0130] A polynucleotide is represented by a nucleotide sequence. A polypeptide is represented by an amino acid sequence. A nucleic acid construct is defined as a polynucleotide which is isolated from a naturally occurring gene or which has been modified to contain segments of polynucleotides which are combined or juxtaposed in a manner which would not otherwise exist in nature. Optionally, a polynucleotide present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of said peptide or polypeptide in a cell or in a subject. Polynucleotides described herein may be native or may be codon optimized. Codon optimization adapts the codon usage for an encoded polypeptide towards the codon bias of the organism where the polypeptide is to be produced in. Codon optimization generally helps to increase the production level of the encoded polypeptide in the host cell, such as in the preferred host herein: Cyanobacterium Synechocystis. Many algorithms are available to the person skilled in the art for codon optimization. A preferred method is the “guided random method based on a Monte Carlo alogorithm available via the internet world wide web genomes.urv.es / OPTIMIZER / (P. Puigbo, E. Guzman, A. Romeu, and S. Garcia-Vallve. Nucleic Acids Res. 2007 July; 35: W126-W131).

[0131] As used herein the term "heterologous" is one that is not naturally found. As used herein, the term “heterologous” may mean “recombinant”. "Recombinant" refers to a genetic entity distinct from that generally found in nature. As applied to a nucleotide sequence or nucleic acid molecule, this means that said nucleotide sequence or nucleic acid molecule is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in the production of a construct that is distinct from a sequence or molecule found in nature.

[0132] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production / expression of the peptide or polypeptide of the invention in a cell and / or in a subject. “Operably linked” may also be used for defining a configuration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and / or in a subject.

[0133] Expression will be understood to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification and secretion.

[0134] As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acid molecules, located upstream with respect to the direction of transcription of the transcription initiation site of the nucleic acid molecule, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation.

[0135] For expression of an enzyme in a cyanobacterial cell according to the inventions, as well as for additional genetic modification of a cyanobacterial cell according to the invention, the cell can be transformed with a nucleic acid or nucleic acid construct described herein by any method known to the person skilled in the art. Such methods are e.g. known from standard handbooks, such as Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of cyanobacterial cells are known from e.g. U.S. Pat. No. 6,699,696 or U.S. Pat. No. 4,778,759.

[0136] When a nucleic acid construct is used for expression of an enzyme in a cyanobacterial cell according to the invention, a selectable marker may be present in the nucleic acid construct comprising a polynucleotide encoding the enzyme. The term “marker” refers herein to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a cyanobacterial cell containing the marker. A marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Such markers include, but are not limited to, aminoglycoside phosphotransferases for kanamycin and streptomycin / spectinomycin, and chloramphenicol acetyltransferase In the embodiments of the invention, a non-antibiotic resistance marker may be used, such as an auxotrophic marker (URA3, TRP1 , LEU2). In the embodiments of the invention, the cyanobacterial cell, e.g. transformed with a nucleic acid construct, may be marker gene free. Methods for constructing recombinant marker gene free microbial host cells are described in (Cheah et al., 2013) and are based on the use of bidirectional markers. In the embodiments of the invention, a screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into a nucleic acid construct according to the invention allowing to screen for transformed cells.

[0137] Optional further elements that may be present in a nucleic acid construct according to the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and / or reporter genes, intron sequences, centromers, telomers and / or matrix attachment (MAR) sequences. A nucleic acid construct according to the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids / nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press.

[0138] Methods for inactivation and gene disruption in a cyanobacterial cell are well known in the art (see e.g. Shestakov S V et al, (2002), Photosynthesis Research, 73: 279-284 and Nakamura Y et al, (1999), Nucleic Acids Res. 27:66-68).

[0139] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0140] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.

[0141] The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the enzymes obtainable by expression of the genes as represented by SEQ ID Nos: 52, 53, 55 and 56 containing the enzyme encoding polynucleotide sequences should prevail.

[0142] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0143] Table 3. Sequences

[0144] Examples

[0145] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0146] Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2ndedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

[0147] Example 1. Culture conditions

[0148] Escherichia coli strains XL-1 blue (Stratagene), Turbo (NEB) or CopyCutter EPI400 (Epicentre biotechnologies) were used for plasmid amplification and manipulation, grown at 37°C in Lysogeny Broth (LB) or on LB agar. Escherichia coli strains BL21 (DE3) were used for expression of recombinant proteins, grown at 37 °C in LB or on LB agar. Strains of Synechocystis sp. PCC 6803 were cultivated either on BG-11 plates or in 2xBG-11 medium (Sigma-Aldrich) optionally supplemented with 10 mM TES-KOH (pH = 8), and / or 50 mM sodium hydrogencarbonate. BG-11 agar plates were supplemented with 10 mM TES-KOH (pH = 8) and 0.3 % (w / v) sodium thiosulfate. When appropriate, the following antibiotics were used: ampicillin (100 pg / ml), kanamycin (20 or 50 pg / ml, for Synechocystis and E. coli, respectively), spectinomycin (25 or 50 pg / ml, for Synechocystis and E. coli, respectively), streptomycin (10 pg / ml), and chloramphenicol (20 pg / ml). Strains were grown in Erlenmeyer flasks in an enriched CO2atmosphere (0.7%) at 30 °C or 35 °C, shaking 120 rpm. At several time points samples were taken from the culture vessel to analyze cell density and product formation by HPLC analysis, using a UV and Rl detector, or GC analysis, using a FID detector.

[0149] Example 2. General protocols

[0150] Restriction endonucleases were purchased from Thermo Scientific. Cloning was performed in E. coli using CaCh-competent XL1-blue, Turbo or CopyCutter EPI400 cells, according to manufacturer protocol.

[0151] Natural transformation for genomic integration of exogenous genes or deletion of endogenous genes in Synechocystis was performed using plates with increasing concentrations of antibiotic for growing the transformants to drive segregation. In the case of making markerless mutants, we used the mazF gene, encoding an endoribonuclease, driven by a Ni2+inducible promoter system that allows for counter-selection (Cheah et aL, 2013). This PBRS-mazF cassette was synthesized at a gene synthesis company (GenScript) and then combined with different antibiotic markers. This cassette was used in combination with homologous regions targeting a specific part of the genome, first introducing and fully segregating it, before removing the marker based on Ni2+selection.

[0152] Conjugation of RSF1010-based plasmids from E. coli XL-1 to Synechocystis was performed by tri-parental mating using E. coli J53 (pRP4) as the helper strain. Correct insertion of the genes and full segregation, as well as insertion of conjugation plasmids, were verified by colony PCR with MyTaq DNA polymerase (Bioline).

[0153] The nucleotide sequence encoding OleT and OleT-BM3R were synthesized using the native nucleotide sequence or with codon-optimization (IDT) [SEQ ID NOs: 52, 53, 55, 56], and inserted with either a Ptrcl [SEQ ID NO: 45] or PcpcBA promoter [SEQ ID NO: 42] into the broad host range RSF1010 derivative plasmid pAVO+ (van der Woude et aL, 2016). An N-terminal His10-tag [SEQ ID NO: 58] and a linker sequence were inserted between the promoter and the gene to allow for protein detection using Western Blotting. The resulting constructs were introduced through conjugation into a markerless Synechocystis strain with a PhaEC deletion.

[0154] The nucleotide sequences encoding phaJ, TER and TE [SEQ ID NOs: 6, 9, 10, 15, 19, 34, 35, 36, 37] were synthesized using the native nucleotide sequence or with codon-optimization, and inserted with a Ptrcl promoter [SEQ ID NO: 45] into the broad host range RSF1010 derivative plasmid pAVO+ (van der Woude et aL, 2016). The resulting constructs were introduced through conjugation into a markerless Synechocystis strain with a phaEC deletion.

[0155] GC-FID analysis was carried out using a Shimadzu GC-2010 system equipped with a Rt-Q- BOND 30 m x 0.32 mm column and a HS-20 headspace autosampler. After equilibration at 60 °C for 2 minutes in the autosampler, the samples were pressurized to 100 kPa during 1 minute, and further equilibrated for 0.5 min. The headspace was then drawn to a transfer line heated at 150 °C for 0.5 minutes, and after 0.1 minutes, the samples were injected during 1 minute with a 1 :20 split mode. The carrier gas was H2with a column flow of 2.5 mL / min in a flow control mode using constant linear velocity. The FID detector temperature was set at 260 °C. The column was heated from 40 °C to 90 °C at a rate of 21 °C min-1, after which heating proceeded at a rate of 10 °C min-1until a temperature of 120 °C was reached and kept constant for 2 min.

[0156] Samples for HPLC were analysed with a Shimadzu LC-20AT Prominence system equipped with a UV-vis detector (Shimadzu SPD-20A) set at 210 nm and a refractive index detector (Shimadzu RID- 20A). Samples and standards (15 pL) were injected using an autosampler (Shimadzu SIL-20AC) onto a 300x 7.8 mm column (Ion exclusion Rezex ROA-Organic Acid H+(8%)) purchased from Phenomenex. The mobile phase comprised 5 mM sulfuric acid with a total flow rate of 0.9 ml min-1at 55 °C. Quantifications are performed with the external standard method through a 5-point calibration line (linear, not forced through 0, weighing 1 / C2, ranging from 0.5-10 mM (LLOQ-HLOQ), R2=0.9999) using TES as an internal standard.

[0157] Example 3. Detection of OleT and OleT-BM3R expression

[0158] Briefly, Synechocystis cells expressing OleT-BM3R were cultivated as described above, followed by pelleting of 50 OD units and discarding of the supernatant. The pellet was kept at -80 °C until further use. The cells were resuspended in 600 pL of lysation buffer (100 mM Tris-HCI, pH 7.5; 10% glycerol, complete Protease Inhibitor Cocktail EDTA-free) and lysed by bead beating (3x 20 sec 6000 rpm). Soluble lysate was obtained by centrifuging for 30 minutes at 15 000 rpm.

[0159] A bicinchoninic acid (BCA) assay was performed on a flat bottom 96 wells plate using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Reagents A and B were mixed to a ratio of 49:1 and equilibrated for ~1 min. 90uL was used per sample. 10uL of samples was added with known standard solution and samples were measured in duplo. A set of standard proteins (BSA) was added of 1 ug, 2.5ug, 5ug, 7.5ug, 10ug. Using a SPECTROstar Nano Microplate reader at 37°C, absorbance 562 nm a set BCA protocol was run. The protein concentration was normalized for dilutions and the samples were used further for Western Blotting.

[0160] S / D buffer (5X) was prepared containing final concentrations of 250 mM Tris-HCI (pH 6.8), 500 mM dithiothreitol (DTT), 10% sodium dodecyl sulphate (SDS), 25 mM EDTA and 50% in distilled water. MOPS (Genscript) buffer was added to deionized water. Precast gels from Genscript, with Express Plus gradient of 8-16% were used. Before loading, protein samples (3.3 pg) were mixed with S / D buffer and boiled at 90 °C for 10 minutes.

[0161] Samples were loaded, as well as a PageRuler™ Prestained Protein Ladder (10 to 180 kDa), and the gel ran at 110 V for 45-60 min. After running, gels were removed from the cassette.

[0162] For Western Blotting the gel was put into a sandwich that contained the nitrocellulose membrane in direct contact with the gel, blotting papers on both sides and sponges on the outsides. The sandwich was run in a blotting chamber with blotting buffer in ice. Blotting transfer buffer was prepared from 3.0 g Tris base, 4.1 g Bicine 100 mL of methanol and 900 mL of water. The blotting chamber ran for 350 mA for 90 minutes or 50 mA overnight. The nitrocellulose membrane was taken out from the sandwich and shaken in 5% milkpowder (low fat) PBS-T blocking buffer (25mL) for 60 minutes at room temperature. 10X PBS-T was previously prepared in a 1 L volume which contained 4.28 g sodium phosphate monobasic monohydrate, 10.5 g sodium phosphate dibasic dihydrate, 90 g sodium chloride, 0.1 g merthiolate, 7 mL Tween and as remainder MilliQ water. The pH was set to 7.2 with NaOH (12g / 50mL). Afterwards the primary antibody was added in a new 5% milkpowder PBS-T solution. For proteins with the histidine tag, Sigma antibody (A7058, 1 :10.000 dilution) was used. Afterwards the gel was washed three times in PBS-T for 10 minutes. Following, the secondary antibody Goat anti Mouse Peroxidase (GAMPO, 1 :3000) was added to a new 5% milkpowder PBS-T for another 60 minutes. The gel was washed again two times in PBS-T for 10 minutes and a final time in PBS (no Tween added). The ECL substrates from SuperSignal West Femto Maximum Sensitivity were mixed in a 1 :1 ratio and put on the membrane and spread with a roller. The membrane was immediately exposed with the Odyssey® XF imaging system (Li-Cor Biosciences). 730 nm measurements revealed the ladder whilst the chemi measurements revealed immunofluorescent proteins (Figure 2).

[0163] Example 4. Enzyme assays with OleT-BM3R lysates with supplemented butyrate and NADPH Synechocystis cells expressing OleT-BM3R were cultivated as described above, followed by pelleting of 50 OD units and discarding of the supernatant. The pellet was kept at -80 °C until further use. The cells were resuspended in 600 pL of lysation buffer (100 mM Tris-HCI, pH 7.5; 10% glycerol, complete Protease Inhibitor Cocktail EDTA-free) and lysed by bead beating (3x 20 sec 6000 rpm). Soluble lysate was obtained by centrifuging for 30 minutes at 15 000 rpm.

[0164] The buffer employed for the enzyme consisted of Tris-HCI (100 mM, pH 7.5) and KCI (300 mM) in glycerol (20%). After addition of the required co-factors (butyrate, 10 mM; NADPH, 10 mM) the pH was re-adjusted to 7.5. Buffer (2.9 mL) and soluble lysate (100 pL) were mixed in a closed-vial reactor and incubated overnight at 22 °C, shaking 110 rpm. The headspace was sampled for propylene analysis via GC-FID (Figure 3).

[0165] Example 5. Butyrate feeding to cyanobacteria for propylene production using OleT-BM3R or OleTjE

[0166] Strains expressing codon-optimized OleT-BM3R and OleTjE were cultivated as described above until an OD of 1 - 2 was reached. The culture was transferred to a closed-vial reactor, supplemented with additional NaHCOa (100 mM) and butyric acid (10 - 70 mM). As negative controls, no butyric acid was supplemented to the strains, and the parental strain lacking the decarboxylase genes was tested. The cultures were further incubated during 96 h, followed by sampling of the headspace for propylene analysis via GC-FID (Figures 4 and 5). The native DNA sequence for OleTjE was also used with the same result (data not shown).

[0167] Example 6. Butyric acid formation from CO2

[0168] For butyric acid formation (in mM) from CO2, Synechocystis strains expressing heterologous phaJ, TER and TE were cultivated as described above. During the course of the 8-day experiment, samples were taken for cell density analysis and butyrate quantification via HPLC (Figure 6).

[0169] Example 7. Propylene formation from CO2

[0170] Strains expressing heterologous phaJ, TER, TE and OleT-BM3R are cultivated as described above. During the course of the 8-day experiment, cultures at different cell densities are supplemented with additional NaHCOs (100 mM) and transferred to a closed reactor. The cultures are further incubated during 96 h, followed by sampling of the headspace for propylene analysis via GC-FID and butyrate quantification via HPLC. An amount of 1 .8 pg propylene per liter of culture volume is measured.

[0171] Reference list

[0172] 1. Lai et al. Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria. Biotechnology and bioengineering, , April 2019, pages 893-903;

[0173] 2. Wang et al. Metabolic engineering of Escherichia coli for the production of butyric acid at high titer and productivity. Biotechnol Biofuels, Mar 22, 2019; 12:62;

[0174] 3. Bauer et al. Production of Propene from n-Butanol: A Three-Step Cascade Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleTJE. Chembiochem 21 , 3273-3281 ;

[0175] 4. Lu et al. An Engineered Self-Sufficient Biocatalyst Enables Scalable Production of Linear a- Olefins from Carboxylic Acids. ACS Catal. 8, 2018, 5794-5798;

[0176] 5. Yunus et al. Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel. Metabolic Engineering, Volume 49, September 2018, pages 201-211 ;

[0177] 6. Shestakov SV et al, (2002), Photosynthesis Research, 73: 279-284;

[0178] 7. Nakamura Y et al, (1999), Nucleic Acids Res. 27:66-68;

[0179] 8. Grigirieva GA et al, (1982), FEMS Microbiol. Lett. 13: 367-370;

[0180] 9. Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;

[0181] 10. Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;

[0182] 11. Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;

[0183] 12. Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987;

[0184] 13. Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073, 1988;

[0185] 14. Carillo, H„ and Lipman, D„ SIAM J. Applied Math., 48:1073 (1988);

[0186] 15. Devereux, J., et al., Nucleic Acids Research 12 (1): 387, 1984;

[0187] 16. Altschul, S. F. et al., J. Mol. Biol. 215:403-410, 1990;

[0188] 17. BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S„ et al., J. Mol. Biol. 215:403-410, 1990;

[0189] 18. Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970;

[0190] 19. Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919, 1992;

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[0192] 21. Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press;

[0193] 22. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York, 1987;

[0194] 23. Cheah et al, (2013) Biotechnol Prog 2013, 29:23-30; 24. Shestakov S V et al, (2002), Photosynthesis Research, 73: 279-284;

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[0196] 26. Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2ndedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; 27. Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current

[0197] Protocols, USA;

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[0199] 29. Brosius et al, J Biol Chem 1985; 30. Huang H-H, Camsund D, Lindblad P, Heidorn T: Design and characterization of moleculartools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 2010, 38:2577-2593.

[0200] 31 . van der Woude et al., Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol, Microb Cell Fact, 2016,15:60

Claims

Claims1. A cyanobacterial cell capable of expressing, preferably expressing, at least one heterologous functional enzyme, wherein the at least one functional enzyme is an oxidative decarboxylase.

2. A cyanobacterial cell according to claim 1 , wherein the oxidative decarboxylase is a cytochrome P450 enzyme, preferably an OleT decarboxylase.

3. A cyanobacterial cell according to claim 2, wherein the OleT decarboxylase is linked to a redox partner, wherein preferably the OleT decarboxylase is:(i) operably linked to a heterologous redox partner, such as BM3R or RhFRED, or(ii) coupled to the endogenous ferredoxin, receiving electrons directly from photosynthesis.

4. A cyanobacterial cell according to any one of the preceding claims, wherein at least one further endogenous or heterologous functional enzyme is present and is selected from the group consisting of:(i) a dehydratase, a reductase, an esterase and a decarboxylase; preferably a 3-hydroxbutyryl- CoA dehydratase (crotonase), a trans-enoyl-coA reductase and / or a thioesterase; or(ii) a 3-hydroxbutyryl-CoA dehydratase (crotonase), a trans-enoyl-coA reductase, phosphotransbutyrylase and a butyrate kinase; or(iii) a malonyl CoA-ACP transacylase (fabD), p-ketoacyl-ACP synthase III (fabH), p-ketoacyl- ACP reductase (fabG), p-hydroxyacyl-ACP dehydratase (fabZ), enoyl-ACP reductase (fabl) and a thioesterase (TE); and optionally, an acetyl-CoA acetyltransferase (phaA), ketoacyl-ACP synthase III (NphT7) and / or an acetoacetyl-CoA reductase (phaB).

5. A cyanobacterial cell according to any of the preceding claims, wherein at least one further functional enzyme of the enzymes as listed in Table 1.

6. A cyanobacterial cell according to any of the preceding claims, wherein the oxidative decarboxylase comprises or consists of a polypeptide that has an amino acid sequence with at least 30% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 57.

7. A cyanobacterial cell according to any of the preceding claims, wherein the oxidative decarboxylase is encoded by a polynucleotide that has an nucleic acid sequence with at least 30% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO: 56.

8. A cyanobacterial cell according to claim 7, wherein the polynucleotide encoding the oxidative decaboxylase is under the control of a constitutive promoter, preferably a promoter that has a nucleicacid sequence that has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, and SEQ ID NO: 49.

9. A cyanobacterial cell according to claim 7, wherein the polynucleotide is under the control of a nutrient-regulated promoter, preferably a nutrient-regulated promoter that has a nucleic acid sequence that has at least 80% sequence identity with SEQ ID NO: 50, or with SEQ ID NO: 51 .

10. A cyanobacterial cell according to any of claims 7 to 9, wherein expression of said polynucleotide or polynucleotides confers to the cell the ability to convert a butyrate intermediate into propylene.

11. A cyanobacterial cell according to claim 10, wherein the butyrate intermediate is butyric acid.

12. A cyanobacterial cell according to any of the preceding claims, wherein the cyanobacterial cell is or is derived from a Synechococcus, preferably Synechococcus elongatus PCC 7942, Synechococcus PCC7002, Synechococcus PCC11901, or from a Synechocystis, preferably a Synechocystis PCC 6803.

13. A process for producing propylene comprising culturing a cyanobacterial cell according to any one of claims 1 to 12 under conditions conducive to the production of propylene-containing gas mixture and, optionally, separating propylene from said gas mixture.

14. A process according to claim 13, wherein the culture conditions comprise feeding carbon dioxide to the culture of cyanobacterial cells and / or subjecting the culture to light.

15. A process according to claim 14, wherein the culture conditions further comprise feeding butyric acid to the culture of cyanobacterial cells.

16. Propylene, obtainable by a process according to any of claims 13 - 15.

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