EA044562B1 - OBTAINING POLYHYDROXYBUTYRATE IN THE WOOD-LJUNGDAHL MICROORGANISM - Google Patents

Description

Cross reference to related application

This application claims priority to US patent application 62/568127, filed October 4

2017, which is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

This invention relates to genetically modified microorganisms and methods for producing polyhydroxybutyrate (PHB) through microbiological fermentation, in particular through microbiological fermentation of a gaseous substrate.

State of the art

Polymeric materials derived from petroleum have become vital to modern life, mainly due to their lightness, strength, durability and resistance to degradation. However, dependence on petroleum-derived polymer materials has led to many serious problems, including crude oil depletion, pollution and landfill accumulation. To reduce the environmental impact of polymeric materials, efforts are being made to replace conventional petroleum-derived polymeric materials with biopolymers, such as polylactide, polysaccharides, aliphatic polyesters and polyhydroxyalkanoates, which have physicochemical properties similar to those of conventional plastics (Anjum, Int J Biol Macromol, 89: 161-174, 2016). However, microorganisms and methods for producing such biopolymers are practically still not developed.

The essence of the invention

This invention provides a genetically modified microorganism capable of producing RNV. In particular, the present invention provides a non-naturally occurring Wood-Ljungdahl microorganism comprising (a) an enzyme that converts acetyl-CoA to acetoacetyl-CoA, (b) an enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and (c ) an enzyme that converts 3-hydroxybutyryl-CoA to RHB.

In one embodiment of the invention, the enzyme that converts acetyl-CoA to acetoacetyl-CoA is acetyl-CoA-C acetyltransferase (EC 2.3.1.9). For example, acetyl-CoAS acetyltransferase can be obtained from

Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Clostridium acetobutylicum, Cupriavidus necator, Escherichia coli, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudom onas oleovorans, Pseudomonas putida or Streptomyces coelicolor.

In one embodiment of the invention, the enzyme that converts acetoacetyl-CoA to 3hydroxybutyryl-CoA is acetoacetyl-CoA reductase (EC 1.1.1.36) or 3hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157). For example, acetoacetyl-CoA reductase can be obtained from

Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida or Stre ptomyces coelicolor.

In another example, 3-hydroxybutyryl-CoA dehydrogenase can be obtained from Clostridium beijerinckii, Clostridium acetobutylicum or Clostridium kluyveri.

In one embodiment of the invention, the enzyme that converts 3-hydroxybutyrylCoA to polyhydroxybutyrate is polyhydroxyalkanoate synthase (EC 2.3.1.-). For example, polyhydroxyalkanoate synthase can be obtained from

Aeromonas caviae, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida, Pseudom onas sp. 61-3, Rhodospirillum rubrum or Streptomyces coelicolor.

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In one embodiment of the invention, the microorganism is a member of a genus selected from the group consisting of: Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Clostridium,

Eubacterium, Moorella, Oxobacter, Sporomusa and Thermoanaerobacter. For example, the microorganism may be derived from a parent microorganism selected from the group consisting of:

Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium Ijungdahlii, Clostridium magnum, Clostridium ragsdalei, Clo stridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica , Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides and Thermoanaerobacter kiuvi.

In a preferred embodiment of the invention, the microorganism is derived from a parent bacterium selected from the group consisting of: Clostridium autoethanogenum, Clostridium coskatii, Clostridium ljungdahlii and Clostridium ragsdalei.

In one embodiment of the invention, the microorganism consumes gaseous substrates containing one or more of: CO, CO ₂ and H ₂ . In another embodiment of the invention, the microorganism is anaerobic. In yet another embodiment of the invention, the microorganism is not capable of degrading RNV.

This invention also provides a method for producing RNV, which includes cultivating a microorganism according to the invention in the presence of a gaseous substrate. For example, the gaseous substrate may contain one or more of: CO, CO ₂ and H ₂ . In one embodiment of the invention, cultivation is carried out under anaerobic conditions. In another embodiment of the invention, cultivation is carried out in the absence of carbohydrate substrates. In yet another embodiment of the invention, cultivation is carried out in the absence of light.

These and other features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying claims. It should be noted that the scope of the claims is determined by the statements therein and not by specific consideration of the features and advantages set forth herein.

Brief description of graphic materials

In fig. 1 illustrates a diagram depicting the enzymatic pathway for the production of polyhydroxybutyrate (PHB).

In fig. Figure 2 illustrates a map of plasmid pPHB_01.

In fig. 3 is a graph depicting the production of RNV in C. autoethanogenum carrying the RNV biosynthesis plasmid (pPHB_01) or an empty plasmid (pMTL83157) as a negative control. The bacteria were grown anaerobically in pressurized bottles in which the carbon source was only CO and CO ₂ . The yield of RNF (expressed as wt.% dry cell mass (DMC)) was determined by HPLC. Values represent the means of biological triplets plus or minus standard deviations around these means. RNV was not detected in samples containing plasmid pMTL83157 (empty).

In fig. 4A-4D illustrate graphs depicting the growth of C. autoethanogenum under various growth conditions. The bacteria contained an empty plasmid (pMTL83157) or a plasmid containing the RNV synthesis pathway (pPHB_01). Each graph illustrates a different set of conditions. In fig. 4A (Condition 1) illustrates a repetition of the conditions used to obtain the data observed in FIG. 3, using a gas mixture containing 50/18/3/29 CO/CO ₂ /H ₂ /N ₂ as the sole carbon source. In fig. 4B (condition 2) illustrates conditions identical to condition 1, but with a new gas substrate (50/30/10/10 CO/CO ₂ /H ₂ /N ₂ ). In fig. 4C (condition 3) illustrates conditions identical to condition 2, but with extended incubation time. In fig. 4D (condition 4) illustrates conditions identical to condition 3, but with periodic renewal of the gas substrate. Values represent the means of biological triplets plus or minus standard deviations around these means.

In fig. 5 is a graph depicting the production of RNV in C. autoethanogenum under conditions 1-4, as described in conjunction with FIG. 4A-4D. Values represent the means of biological triplets plus or minus standard deviations around these means. RNV was not detected in samples containing plasmid pMTL83157 (empty).

In fig. 6 illustrates a graph depicting the production of RHB in C. autoethano- 2 044562 genum from gaseous carbon sources during continuous fermentation. Bacteria were conjugated to the pPHB_01 plasmid containing the RNV synthesis pathway. At the end of fermentation, RNV was measured. The gas substrates contained 20% hydrogen or 2% hydrogen in their mixtures of 50/20/20/10 CO/CO ₂ /H ₂ /Ar or 50/20/2/28 CO/CO2/H2/N2, respectively. The pH was maintained at 5. Values are the means of exactly replicated fermentations plus or minus standard deviations around these means.

In fig. 7 is a graph depicting the enhancement of RHB production in C. autoethanogenum from carbon gas sources during continuous fermentation. Bacteria were conjugated to the pPHB_01 plasmid containing the RNV synthesis pathway. At the end of fermentation, RNV was measured. The gas substrates contained 20% hydrogen or 2% hydrogen in their mixtures of 50/20/20/10 CO/CO ₂ /H ₂ /Ar or 50/20/2/28 CO/CO2/H2/N2, respectively. 20% hydrogen, low biomass had a reduced biomass concentration compared to other cycles. 20% hydrogen, pH change started at pH 6 and then dropped to 5.5. 20% hydrogen, pH 6 was maintained throughout the fermentation cycle until the cultures were reduced. Values represent the average of exactly replicated fermentations plus or minus standard deviations around these averages.

In fig. 8 illustrates a graph depicting RNV values for simulations using genome-wide metabolic model (GEM) reconstructions. Experimentally detected RNV was compared with RNV levels predicted by GEM using RNV yield maximization. Maximization of RNV yield was also tested with uptake of 2 mmol/gBM/h of either NADH, NADPH, Fd _red , or ATP. Conditions tested were RHB20 (control); PHBLowB (low biomass) and PHBpH5.5 (pH 5.5). ATP was found to limit PHB the most (the highest PHB value was achieved), followed by Fd _red , NADPH and then NADH. Data represent the mean ± standard deviation of two biological replicates of chemostats.

Description of the invention

It has long been discovered that catalytic processes, such as the Fischer-Tropsch process, can be used to convert gases containing carbon dioxide (CO2), carbon monoxide (CO) and/or hydrogen (H2), such as industrial waste gas or synthesis gas. gas, into a variety of fuels and chemicals. However, recently, gas fermentation has emerged as an alternative platform for biological carbon fixation of such gases. In particular, acetogenic microorganisms (ie Wood-Ljungdahl) have been demonstrated to convert gases containing CO, CO2 and/or H2 into products such as ethanol and 2,3-butanediol. The feasibility of obtaining more complex polymer molecules, for example, RHB, from these gases is well substantiated (Drzyzga, J Chem Technol Biotechnol, 90: 1735-1751, 2015). However, the Wood-Ljungdahl pathway operates at the thermodynamic boundary of life (Schuchmann, Nat Rev Microbiol, 12: 809-821, 2014), which makes it difficult for Wood-Ljungdahl microorganisms to accumulate enough carbon even for cell growth and maintenance, producing far fewer complex carbon products. These metabolic problems are exacerbated by the poor dissolution of gaseous substrates (e.g. CO, CO2 and/or H2) in fermentation media compared to carbohydrate or sugar substrates. It therefore seems unlikely that Wood-Ljungdahl microorganisms could be engineered to synthesize RHB or other polyhydroxyalkanoates, especially since these polymers are initially produced by species such as Rhodospirillum rubrum and Cupriavidus necator as a means of storing excess carbon. Indeed, to date, attempts to create acetogenic microorganisms to produce RNF from CO2 and/or H2 have been unsuccessful (European project SYNPOL, biopolymers from syngas fermentation, 2012-2017).

However, after painstaking research and engineering, the inventors achieved the world's first synthesis of RNV in Wood-Ljungdahl microorganisms. This is an important milestone towards the production of renewable and sustainable biopolymers.

In a first aspect, the present invention provides a Wood-Ljungdahl microorganism capable of producing RNV. In a second aspect, the present invention provides a method for producing RNV by cultivating the aforementioned Wood-Ljungdahl microorganism in the presence of a gaseous substrate.

Path

Since Wood-Ljungdahl microorganisms do not natively produce RNV, production of RNV in a Wood-Ljungdahl microorganism requires the introduction of at least one heterologous enzyme. The microorganism of the invention typically contains three heterologous enzymes, namely: (a) an enzyme that converts acetyl-CoA to acetoacetyl-CoA, (b) an enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and (c ) an enzyme that converts 3-hydroxybutyryl-CoA to polyhydroxybutyrate. This path is illustrated in Fig. 1.

(1) Conversion of acetyl-CoA to acetoacetyl-CoA

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The conversion of acetyl-CoA to acetoacetyl-CoA can be catalyzed by any suitable enzyme. Although it is possible that native activity for this reaction may be present in some acetogenic bacteria, it is generally necessary to introduce a heterologous (ie, non-native) enzyme to catalyze this reaction. In a preferred embodiment, the enzyme is an acetyl-CoA-C-acetyltransferase (also known as a thiolase or 3-ketothiolase) which has the activity defined by EC 2.3.1.9 (ie 2-acetyl-CoA-C-CoA+acetoacetyl-CoA). Acetyl-CoA-C acetyltransferase can be obtained from any suitable host microorganism, e.g.

Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Clostridium acetobutylicum, Cupriavidus necator, Escherichia coli, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudom onas oleovorans, Pseudomonas putida or Streptomyces coelicolor.

In particular, the acetyl-CoA-C acetyltransferase may be or may be obtained from Acinetobacter baumannii PhaA (SCZ16966), Aeromonas hydrophilia PhaA (WP_043162470), Alcaligenes latus PhaA (AAC83659), Arthrospira platensis PhaA (WP_006617472), Bacillus subtilis PhaA ( CUB52080), Burkholderia cepacia PhaA (WP_043187452), Clostridium acetobutylicum Th1A (WP_0109661571), Cupriavidus necator PhaA (WP_013956452.1), Cupriavidus necator BktB (WP_011615089.1), Cupriavidus necator phaA ( WP_010810132.1), Escherichia coli AtoB (NP_416728. 1), Haloferax mediterranei PhaA (WP_004059344), Pseudomonas aeruginosa PhaA (WP_038823536), Pseudomonas fluorescens PhaA (WP_073525707), Pseudomonas mandelii PhaA (WP_019582144), Pseudomonas oleovorans PhaA ( WP_074859314), Pseudomonas putida PhaA (WP_058540218) or Streptomyces coelicolor PhaA (WP_011030221 ).

(2) Conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA

The conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA can be catalyzed by any suitable enzyme. Although it is possible that native activity for this reaction may be present in some acetogenic bacteria, it is generally necessary to introduce a heterologous (ie, non-native) enzyme to catalyze this reaction. In a preferred embodiment, the enzyme is an acetoacetyl-CoA reductase which has the activity defined by EC 1.1.1.36 (i.e. (R)-3-hydroxyacyl-CoA + NADP ⁺ θ3-oxoacyl-CoA + NADPH + H ⁺ ). Acetoacetyl-CoA reductase can be obtained from any suitable host microorganism, e.g.

Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida or Stre ptomyces coelicolor.

In particular, the acetoacetyl-CoA reductase may be Acinetobacter baumannii PhaB (WP_095389464), Aeromonas hydrophilia PhaB (WP_041216919), Alcaligenes latus PhaB (AAC83660), Arthrospira platensis PhaB (WP_043469113), Bacillus subtilis PhaB (WP_070 548955), Burkholderia cepacia PhaB ( WP_059234032), Cupriavidus necator PhaB (WP_010810131.1), Haloferax mediterranei PhaB (WP_004572392), Pseudomonas aeruginosa PhaB (WP_031690879), Pseudomonas fluorescens PhaB (WP_030141425), Pseudomona s mandelii PhaB (WP_094467462), Pseudomonas oleovorans PhaB (WP_074858624), Pseudomonas putida PhaB (BAB96554) or Streptomyces coelicolor PhaB (WP_011027734). In another preferred embodiment of the invention, the enzyme is 3-hydroxybutyryl-CoA dehydrogenase, which has the activity defined by EC 1.1.1.157 (i.e. (S)-3-hydroxybutanoyl-CoA+NADP ⁺ =3-acetoacetyl-CoA+ NADPH+H ⁺ ). The 3hydroxybutyryl-CoA dehydrogenase may be or may be obtained from any suitable host microorganism, for example Clostridium beijerinckii, Clostridium acetobutylicum or Clostridium kluyveri. In particular, the 3-hydroxybutyryl-CoA dehydrogenase may be Clostridium beijerinckii Hbd (WP_011967675.1), Clostridium acetobutylicum Hbd (NP_349314.1) or Clostridium kluyveri Hbd1 (WP_011989027.1).

Preferably, the enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA is β-specific, i.e. produces (R)-3-hydroxybutyryl-CoA, since (R)-3hydroxybutyryl-CoA is a typical substrate for the enzymatic production of RNV. However, in some cases, the enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA

- 4 044562 is (S)-specific, i.e. produces (S)-3-hydroxybutyryl-CoA. Without wishing to be bound by any particular theory, the inventors believe that native or introduced epimerase activity in an acetogenic bacterium may allow interconversion of (S)- and (R)-3hydroxybutyryl-CoA, e.g., (S)-3-hydroxybutyryl-CoA can be converted to (R)-3hydroxybutyryl-CoA, which can then be converted to RHB.

(3) Conversion of 3-hydroxybutyryl-CoA to RHB

The conversion of 3-hydroxybutyryl-CoA to RHB can be catalyzed by any suitable enzyme. Although it is possible that native activity for this reaction may be present in some acetogenic bacteria, it is generally necessary to introduce a heterologous (ie, non-native) enzyme to catalyze this reaction. In a preferred embodiment, the enzyme is a polyhydroxyalkanoate synthase which has activity defined by EC 2.3.1.-, for example EC 2.3.1.B2 (type I) (i.e. 3-hydroxybutyryl-CoA+ [(R )-3-hydroxybutanoate] _n = [(R)-3-hydroxybutanoate] _n +1+ CoA), EC 2.3.1.B3 (type II) (i.e. 3-hydroxyacyl-CoA+[(R)- 3hydroxyacyl] _n =[(R)-3-hydroxyacyl] _n +1+CoA) or EC 2.3.1.B4 (type III) (i.e. 3-hydroxyacyl-CoA+[(R)3-hydroxyacyl] _n =[(R)-3-hydroxyacyl] _n +1+CoA). This enzyme may also be referred to as polyhydroxyalkanoate polymerase, polyhydroxybutyrate synthase, polyhydroxybutyrate polymerase, and similar terms. The polyhydroxyalkanoate synthase can be obtained from any suitable host microorganism, e.g.

Acinetobacter baumannii, Aeromonas caviae,

Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida, Pseudomonas sp. 61-3, Rhodospirillum rubrum or Streptomyces coelicolor.

In particular, the polyhydroxyalkanoate synthase may be or may be derived from Acinetobacter baumannii PhaC (SCY71072), Aeromonas caviae PhaC (WP_045524574), Aeromonas hydrophilia PhaC1 (WP_017780191) or PhaC2 (AAV41872), Alcaligenes latus PhaC (WP_084267317 ), Arthrospira platensis PhaC (WP_006617456), Bacillus subtilis PhaC (CUB58881), Burkholderia cepacia PhaC (WP_027784567), Cupriavidus necator PhaC (WP_011615085 or WP_013956451.1), Haloferax mediterranei PhaC (WP_004056138), Pseudom onas aeruginosa PhaC1 (WP_038823539) or PhaC2 (WP_025271419), Pseudomonas fluorescens PhaC1 (WP_057399292) or PhaC2 (WP_030141001), Pseudomonas mandelii PhaC1 (WP_094467460) or PhaC2 (WP_010465951), Pseudomonas oleovorans PhaC1 (AAL17611) or PhaC2 (WP_037049875), P seudomonas putida PhaC1 (BAB96552) or PhaC2 (WP_029886362), Pseudomonas sp. 61-3 PhaC1 (BAA36198) or PhaC2 (BAA36202), Rhodospirillum rubrum PhaC1 (WP_011388028), PhaC2 (WP_011390166), or PhaC3 (WP011398569), or Streptomyces coelicolor PhaC.

In some embodiments, one or more disruptive mutations may be introduced into one or more endogenous enzymes to reduce or eliminate competition with the introduced heterologous enzymes. In particular, a disruptive mutation is a mutation that reduces or eliminates (ie, destroys) the expression or activity of a gene or enzyme. A destructive mutation can partially inactivate, completely inactivate, or remove a gene or enzyme. The destructive mutation may be a knockout (KO) mutation. A destructive mutation can be any mutation that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. A disruptive mutation may include, for example, a mutation in a gene encoding an enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a nucleic acid that produces a protein that reduces or inhibits the activity of the enzyme, or the introduction of a nucleic acid ( eg antisense RNA, siRNA, guide RNA) and/or a protein (eg Cas protein) that inhibits enzyme expression. A destructive mutation can be introduced using any method known in the art.

For example, a microorganism of the invention may have a destructive mutation in the endogenous thioesterase enzyme. Three putative thioesterases have been identified in Clostridium autoethanogenum: (1) thioesterase 1 (AGY74947.1; annotated as palmitoyl-CoA hydrolase), (2) thioesterase 2 (AGY75747.1; annotated as 4-hydroxybenzoyl-CoA thioesterase), and ( 3) thioesterase 3 (AGY75999.1; annotated as a putative thioesterase). Three putative thioesterases have also been identified in Clostridium ljungdahlii: (1) thioesterase 1 (ADK15695.1; annotated as predicted acyl-CoA thioesterase 1), (2) thioesterase 2 (ADK16655.1; annotated as predicted thioesterase), and (3) thioesterase 3 (ADK16959.1; annotated as a predicted thioesterase). A destructive mutation may affect any of these thioesterases or any other thioesterases that may be endogenous to the microorganism of the invention.

Microorganism

A microorganism is a microscopic organism, such as a bacterium, archaea, virus or fungus. The microorganism of this invention is typically a bacterium. In the context of this invention, the term microorganism should be considered to include the term bacterium.

The microorganism of the invention does not occur in nature. The term non-naturally occurring when applied to a microorganism is intended to mean that said microorganism has at least one genetic modification not found in a naturally occurring strain of the listed species, including wild-type strains of the listed species. Non-naturally occurring microorganisms are typically developed in a laboratory or research facility. In contrast, the term wild type refers to the typical form of an organism, strain, gene, or characteristic as it occurs in nature.

The terms genetic modification, genetic alteration, or genetic engineering broadly refer to the manipulation of the genome or nucleic acids of a microorganism by human hands. Likewise, the terms genetically modified, genetically altered or genetically engineered refer to a microorganism containing such a genetic modification, genetic alteration or genetic engineering. These terms can be used to distinguish a laboratory-created microorganism from a naturally occurring microorganism. Genetic modification methods include, for example, heterologous gene expression, insertion or deletion of a gene or promoter, nucleic acid mutation, expression or inactivation of an altered gene, enzyme engineering, directed development, knowledge-based design, random mutagenesis techniques, gene shuffling, and codon optimization. .

Recombinant means that a nucleic acid, protein, or microorganism is the product of genetic modification, engineering, or recombination. In general, the term recombinant refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material obtained from multiple sources, such as two or more different strains or species of microorganisms. The microorganism of this invention is typically recombinant.

The term derived from means that a nucleic acid, protein or microorganism is modified or adapted from a different (eg, parent or wild type) nucleic acid, protein or microorganism to produce a new nucleic acid, protein or microorganism. Such modifications or adaptations generally include insertion, deletion, mutation or substitution of nucleic acids or genes. In general, the microorganism of the invention is obtained from a parent microorganism, which is the microorganism used to create the microorganism of the invention. The parent microorganism may be a naturally occurring microorganism (ie, a wild-type microorganism) or a microorganism that has been previously modified (ie, a mutant or recombinant microorganism). The microorganism of the invention may be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parent microorganism. Likewise, a microorganism of the invention may be modified to include one or more genes that were not included by the parent microorganism. The microorganism of the invention may also be modified not to express or to express lesser amounts of one or more enzymes that were expressed in the parent microorganism. In one embodiment of the invention, the microorganism of the invention is derived from a parent microorganism selected from the group consisting of: Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment of the invention, the microorganism of the invention is derived from the parent microorganism Clostridium autoethanogenum LZ1561, which was deposited on June 7, 2010 in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), located at Inhoffenstrae 7B, D-38124 Braunschwieg, Germany on June 7, 2010 in accordance with the terms of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the supplied accession number DSM23693. This strain is described in international patent application No. PCT/NZ2011/000144, which is published as WO 2012/015317.

The microorganism of this invention can be further classified based on functional characteristics. For example, the microorganism of this invention may be or may be derived from a Wood-Ljungdahl microorganism, a C1-fixing microorganism, an anaerobic, an acetogen, an ethanologen, and/or a carboxydotroph. In table 1 provides a representative list of microorganisms and their functional characteristics.

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Table 1

Wood-Ljungdahl C1 - fixing microorganism Anaerobe Acetogen Ethanologen Autotroph Carboxydotroph Acetobacterium woodii + + + + +/- ¹ + - Alkalibaculum bacchii + + + + + + + Blautia producta + + + + - + + Butyribacterium methylotrophicum + + + + + + + Clostridium aceticum + + + + - + + Clostridium autoethanogenum + + + + + + + Clostridium carboxidivorans + + + + + + + Clostridium coskatii + + + + + + + Clostridium drakei + + + + - + + Clostridium formicoaceticum + + + + - + + Clostridium Ijungdahlii + + + + + + + Clostridium magnum + + + + - + +/- ² Clostridium ragsdalei + + + + + + + Clostridium scatologenes + + + + - + + Eubacterium limosum + + + + - + + Moorella thermoautotrophica + + + + + + + Moorella thermoacetica (formerly Clostridium thermoaceticum) + + + + 3 + + Oxobacter pfennigii + + + + - + + Sporomusa ovata + + + + - + +/- ⁴ Sporomusa silvacetica + + + + - + +/- ⁵ Sporomusa sphaeroides + + + + - + +/- ⁶ Thermoanaerobacter kiuvi + + + + - + -

Acetobacterium woodi can produce ethanol from fructose, but not from gas.

² It has not been studied whether Clostridium magnum can be grown on CO.

³ It has been reported that one strain from Moorella thermoacetica, Moorella sp. HUC22-1, produces ethanol from gas.

⁴ It has not been studied whether Sporomusa ovata can be grown on CO.

⁵ It has not been studied whether Sporomusa silvaceca can be grown on CO.

⁶ It has not been investigated whether Sporomusa sphaeroides can be grown on CO.

The term Wood-Ljungdahl refers to the Wood-Ljungdahl carbon fixation pathway described, for example, in Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008. Wood-Ljungdahl microorganisms predictably refer to the microorganisms that make up the Wood-Ljungdahl pathway. In general, the microorganism of the invention constitutes the native Wood-Ljungdahl pathway. In the context of the present invention, the Wood-Ljungdahl pathway may be the native, unmodified Wood-Ljungdahl pathway, or it may be the Wood-Ljungdahl pathway with some degree of genetic modification (e.g., overexpression, heterologous expression, knockout, etc.) provided that it still functions to convert CO, _CO2 and/or _H2 to acetyl-CoA.

C1 refers to a molecule with one carbon atom, such as CO, CO ₂ or CH ₃ OH. Cloxygenate refers to a molecule with one carbon atom that also contains at least one oxygen atom, such as CO, CO ₂ or CH ₃ OH. A Cl-carbon source refers to a single carbon molecule that serves as a partial or sole carbon source for the microorganism of the present invention. For example, the C1 carbon source may include one or more compounds selected from CO, CO ₂ , CH ₃ OH or CH ₂ O ₂ . The C1 carbon source preferably includes one or more compounds selected from CO and CO ₂ . A C1-fixing microorganism is a microorganism capable of producing one or more products from a C1 carbon source. Typically, the microorganism of this invention is a C1-fixing bacterium. In a preferred embodiment of the invention, the microorganism of this invention is obtained from the C1-fixing microorganism shown in table. 1. For the purposes of this invention, methane (CH ₄ ) could be considered as a source of C1-carbon, but only if the bacterium of this invention was engineered to constitute a methane metabolic pathway, as described for example in WO 2016/138050, since acetogenic bacteria are not capable of natively using methane as a carbon source.

An anaerobe is a microorganism that does not require oxygen to grow. An anaerobe can react negatively or even die when oxygen is present above a certain threshold. However, some anaerobes are able to tolerate low levels of oxygen (eg, 0.0000015% oxygen). Typically, the microorganism of this invention is an anaerobic. In a preferred embodiment of the invention, the microorganism of this invention is obtained from the anaerobes given in table. 1.

Acetogens are obligate anaerobic bacteria that use the Wood-Ljungdahl pathway as their main mechanism to conserve energy and synthesize acetyl-CoA and acetyl-CoA-derived products such as acetate (Ragsdale, Biochim Biophys Acta, 1784: 1873-1898, 2008 ). Acetogens use the Wood-Ljungdahl pathway as (1) a mechanism for the reductive synthesis of acetyl-CoA from CO2, (2) a terminal electron-withdrawing, energy-saving process, (3) a mechanism for the fixation (assimilation) of CO2 during cellular carbon synthesis (Drake, Acetogenic Prokaryotes, In: The Prokaryotes, ^3rd edition, p. 354, New York, NY, 2006). All naturally occurring acetogens are C1-fixing, anaerobic, autotrophic and non-methanotrophic. Typically, the microorganism of this invention is an acetogen. In a preferred embodiment of the invention, the microorganism of this invention is obtained from the acetogen given in table. 1.

An ethanologen is a microorganism that produces or is capable of producing ethanol. Typically, the microorganism of this invention is an ethanologen. In a preferred embodiment of the invention, the microorganism of this invention is obtained from the ethanol given in table. 1.

An autotroph is a microorganism that can grow in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources, such as CO and/or CO ₂ . Typically, the microorganism of this invention is an autotroph. In a preferred embodiment of the invention, the microorganism of this invention is obtained from the autotroph shown in table. 1.

A carboxydotroph is a microorganism capable of using CO as the sole source of carbon and energy. Typically, the microorganism of this invention is a carboxydotroph. In a preferred embodiment of the invention, the microorganism of this invention is obtained from the carboxydotroph shown in table. 1.

In a broader sense, the microorganism of this invention can be obtained from microorganisms of any kind or species listed in table. 1. For example, the microorganism may be a member of a genus selected from the group consisting of: Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Clostridium, Eubacterium, Moorella, Oxobacter, Sporomusa and Thermoanaerobacter. In particular, the microorganism may be derived from a parent bacterium selected from the group consisting of:

Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta,

Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermauto trophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica , Sporomusa sphaeroides and Thermoanaerobacter kiuvi.

In a preferred embodiment, the microorganism of this invention is obtained from the Clostridia cluster, which includes the species Clostridium autoethanogenum, Clostridium coskatii, Clostridium ljungdahlii and Clostridium ragsdalei. Such species were first described and characterized by Abrini, Arch Microbiol, 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, Int J System Bacteriol, 43: 232-236, 1993 (Clostridium ljungdahlii) and Huhnke, WO 2008/028055 ( Clostridium ragsdalei).

These species have a lot in common. Specifically, all of these species are C1-fixing, anaerobic, acetogenic, ethanologous, and carboxidotrophic members of the genus Clostridium. Such species have similar genotypes and phenotypes and modes of energy conservation and enzymatic metabolism. In addition, these species are clustered in group I of clostridial rRNA homology with 16S rRNA DNA, more than 99% identical, have a G+C DNA content of about 2230 mol%, are gram-positive, have similar morphology and size (logarithmically growing cells in the range between 0.5-0.7x3-5 µm), are mesophilic (grow optimally at a temperature of 30-37°C), have similar pH ranges of about 4-7.5 (with an optimal pH value of about 5.5 -6), do not contain cytochromes and store energy through the Rnf complex. Moreover, how

- 8 044562 it has been shown that in these species, carboxylic acids are reduced to form their corresponding alcohols (Perez, Biotechnol Bioeng, 110:1066-1077, 2012). It is important to note that all of these species also exhibit strong autotrophic growth on CO-containing gases, produce ethanol and acetate (or acetic acid) as major fermentation products, and under certain conditions produce small amounts of 2,3-butanediol and lactic acid.

However, these types also have a number of differences. These species were isolated from different sources: Clostridium autoethanogenum from rabbit intestines, Clostridium ljungdahlii from chicken coop waste, and Clostridium ragsdalei from freshwater sediments. These species differ in their utilization of various sugars (eg, rhamnose, arabinose), acids (eg, gluconate, citrate), amino acids (eg, arginine, histidine), and other substrates (eg, betaine, butanol). In addition, these species differ in their auxotrophy for certain vitamins (eg, thiamine, biotin). These species have differences in the nucleic acid and amino acid sequences of genes and proteins using the Wood-Ljungdahl pathway, although the general organization and abundance of these genes and proteins have been found to be the same in all species (Corke, Curr Opin Biotechnol, 22:320-325 ,2011).

Thus, in conclusion, many of the characteristics of Clostridium autoethanogenum, Clostridium coskatii, Clostridium ljungdahlii or Clostridium ragsdalei are not specific to these species, but are rather general characteristics for this cluster of C1-fixing, anaerobic, acetogenic, ethanologous and carboxydotrophic members of the genus Clostridium . However, because these species are actually different, genetic modification or manipulation in one of these species may not have an identical effect in another of these species. For example, there may be differences in growth, performance, or product production.

The microorganism of this invention can also be obtained from an isolate or mutant of Clostridium autoethanogenum, Clostridium coskatii, Clostridium ljungdahlii or Clostridium ragsdalei. Clostridium autoethanogenum isolates and mutants include JA1-1 (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200) and LZ1561 (DSM23693) (WO 2012/015 317) . Clostridium ljungdahlii isolates and mutants include ATCC 49587 (Tanner, Int J Syst Bacteriol, 43: 232236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (US 5,593,886), C-01 (ATCC 55988) (US 6,368,819), 0-52 (ATCC 55989) (US 6,368,819) and OTA-1 (Tirado-Acevedo, Production of bioethanol from synthesis gas using Clostridium ljungdahlii, PhD thesis, North Carolina State University, 2010). Clostridium ragsdalei isolates and mutants include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

Preferably, the microorganism of the invention is not phototrophic or photosynthetic. Preferably, the microorganism of the invention is not methanotrophic.

Preferably, the microorganism of the invention is not a member of the genus Alcaligenes, Azotobacter, Bacillus, Cupriavidus (Ralstonia), Rhizobium, Rhodospirillum or Pseudomonas. In particular, the microorganism of the invention is preferably not derived from Rhodospirillum rubrum, Bacillus cereus, Cupriavidus necator (formerly Ralstonia eutropha) or Pseudomonas putida. In other embodiments, the microorganism of the invention is preferably not derived from Escherichia coli.

Enzymes

Endogenous or native refers to a nucleic acid or protein that is present or expressed in the wild-type microorganism or parent microorganism from which the microorganism of this invention is obtained. For example, an endogenous gene or protein is a gene or protein that is natively present in a wild-type microorganism or the parent microorganism from which the microorganism of the present invention is derived. In one embodiment of the invention, the expression of an endogenous gene can be regulated by an exogenous regulatory element, such as an exogenous promoter.

Exogenous refers to a nucleic acid or protein that is produced outside the microorganism of the invention. For example, an exogenous gene or enzyme can be created artificially or recombinantly and introduced into or expressed in a microorganism of the present invention. An exogenous gene or enzyme can also be isolated from a heterologous microorganism and introduced into or expressed in a microorganism of the present invention. Exogenous nucleic acids can be adapted to be integrated into the genome of the microorganism of the present invention or left in an extrachromosomal state in the microorganism of the present invention, for example, in a plasmid.

Heterologous refers to a nucleic acid or protein that is not present in the wild-type microorganism or the parent microorganism from which the microorganism of this invention is derived. For example, a heterologous gene or enzyme can be obtained from a different strain or species and introduced into or expressed in a microorganism of the present invention.

Typically, at least one of the enzymes that (a) convert acetyl-CoA to acetoacetyl-CoA, (b) convert acetoacetyl-CoA to 3-hydroxybutyryl-CoA, or (c) convert 3hydroxybutyryl-CoA to RNV is heterologous (i.e. i.e. not native) in relation to the bacterium. For example, one, two, or all three of these enzymes may be heterologous (ie, not native) to the bacterium. However, if the bacterium has native enzymatic activity for one or more of these steps, then it may not be necessary to introduce heterologous

- 9 044562 enzymes for catalysis of these stages.

As used herein, the term expression refers to the process by which a polynucleotide is transcribed from a DNA template (eg, into mRNA or other RNA transcript) and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides or proteins.

Enzyme activity, or simply activity, broadly refers to enzymatic activity, including, but not limited to: the activity of the enzyme, the amount of enzyme, or the availability of enzyme required to catalyze a reaction. Accordingly, increasing enzyme activity includes increasing the activity of the enzyme, increasing the amount of enzyme, or increasing the availability of the enzyme necessary to catalyze the reaction. Likewise, a decrease in enzyme activity includes a decrease in enzyme activity, a decrease in the amount of enzyme, or a decrease in the availability of an enzyme necessary to catalyze a reaction.

Codon optimization refers to the mutation of a nucleic acid, such as a gene, for optimized or improved translation of the nucleic acid in a particular strain or species. Codon optimization can lead to increased translation rates or higher translation fidelity. In a preferred embodiment, the genes of the invention are codon optimized for expression in Clostridium, in particular Clostridium autoethanogenum, Clostridium coskatii, Clostridium ljungdahlii or Clostridium ragsdalei. In the context of this document, the terms codon optimized and codon tailored may be used interchangeably.

The term variants includes nucleic acids and proteins whose sequence varies from the reference nucleic acid and protein sequence, for example, the reference nucleic acid and protein sequence disclosed in the prior art or exemplified herein. The present invention may be practiced using variant nucleic acids or proteins that perform substantially the same function as the reference nucleic acid or protein. For example, a variant protein may perform substantially the same function or catalyze substantially the same reaction as the reference protein. The variant gene may encode the same or substantially the same protein as the reference gene. The variant promoter may have substantially the same ability to stimulate expression of one or more genes as the reference promoter.

Such nucleic acids or proteins may be referred to herein as functionally equivalent variants. For example, functionally equivalent nucleic acid variants may include allelic variants, gene fragments, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These may include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii or Clostridium ljungdahlii, details of which are publicly available on websites such as GenBank or NCBI. Functionally equivalent variants also include nucleic acids that vary in sequence as a result of codon optimization for a particular microorganism. A functionally equivalent nucleic acid variant will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more nucleic acid sequence identity (percent homology) with the reference nucleic acid. A functionally equivalent protein variant will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more amino acid identity (percent homology) with the reference protein. The functional equivalence of a nucleic acid or protein variant can be assessed using any method known in the art.

The enzymes described herein are typically expressed from a nucleic acid that has been introduced into a microorganism according to the invention. Nucleic acids can be delivered to the microorganism of this invention using any method known in the art. For example, nucleic acids can be delivered as naked nucleic acids or can be formulated with one or more agents, such as liposomes. Nucleic acids may be DNA, RNA, cDNA, or combinations thereof, depending on the situation. In certain embodiments of the invention, restriction inhibitors may be used. Additional vectors may include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In a preferred embodiment of the invention, nucleic acids are delivered to the microorganism of the invention using a plasmid. For example, transformation (including transduction or transfection) can be achieved through electroporation, ultrasound, polyethylene glycol-mediated transformation, chemical or physical competence, protoplast transformation, prophage induction or conjugation. In some embodiments having active restriction enzyme systems, this may be necessary to methylate the nucleic acid before introducing the nucleic acid into the microorganism.

In addition, nucleic acids can be designed to include a regulatory element, such as a promoter, to enhance or otherwise control the expression of a particular nucleic acid. The promoter may be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate:ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or a phosphotransacetylase/acetate kinase operon promoter.

Substrates

Substrate refers to a source of carbon and/or energy for the microorganism of the present invention. Typically, the substrate is gaseous and contains a source of C1 carbon, such as CO and/or CO2. Preferably, the substrate contains a source of C1-carbon in the form of CO or CO+CO ₂ . The substrate may additionally contain other non-carbon components, for example, H ₂ , N 2 or electrons.

In general, the substrate contains at least some amount of CO, for example, about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mol%. CO. The substrate may contain CO in the range, for example, about 20-80, 30-70 or 40-60 mol%. CO. Preferably, the substrate contains about 40-70 mol%. CO (eg steel mill gas or blast furnace gas), about 20-30 mol%. CO (for example, converter furnace gas) or about 15-45 mol%. CO (for example, synthesis gas). In some embodiments, the substrate may contain a relatively low amount of CO, for example, about 1-10 or 1-20 mol%. CO. The microorganism of this invention typically converts at least a portion of the CO in the substrate into a product. In some embodiments, the substrate contains no or substantially no (<1 mol%) CO.

The substrate may contain some H2. For example, the substrate may contain about 1, 2, 5, 10, 15, 20 or 30 mol%. H2. In some embodiments, the substrate may contain a relatively high amount of H ₂ , for example, about 60, 70, 80 or 90 mol%. _H2 . In further embodiments, the substrate contains no or substantially no (<1 mol%) H ₂ .

The substrate may contain some CO ₂ . For example, the substrate may contain about 1-80 or 1-30 mol%. CO2. In some embodiments, the substrate may contain less than about 20, 15, 10 or 5 mol%. _CO2 . In another embodiment of the invention, the substrate contains no or substantially no (<1 mol%) CO2.

In some embodiments, the growth of an RNV-producing strain is compared with the growth of a control strain (empty plasmid or EP) using two different gas mixtures containing CO and CO2, with 20% H2 similar to synthesis gas (50% CO, 20% CO2, 20% H2, 10% argon), designated as RHB20 and EP20 conditions, respectively, or 2% H2 similar to steel mill off-gas (50% CO, 20% _CO2 , 2% _H2 , 28% nitrogen), designated as conditions RHB2 and EP2, respectively.

Although typically the substrate is gaseous, the substrate may also be provided in alternative forms. For example, the substrate can be dissolved in a liquid saturated with CO-containing gas using a dispersive microbubble generator. As a further example, the substrate may be adsorbed onto a solid support.

The substrate and/or source of C1-carbon may be a waste gas obtained as a by-product of an industrial process or from some other source, for example, from automobile exhaust or from biomass gasification. In certain embodiments of the invention, the industrial process is selected from the group consisting of: ferrous metal production, such as steel production, non-ferrous metal production, petroleum refining, coal gasification, power generation, pure carbon production, ammonia production, methanol production, and coke In these embodiments, the substrate and/or source of C1-carbon can be recovered from the industrial process before being released into the atmosphere using any convenient method.

The substrate and/or source of C1-carbon may be a synthesis gas, for example, synthesis gas obtained from the gasification of coal or refinery residues, the gasification of biomass or lignocellulosic material, or from the reforming of natural gas. In another embodiment of the invention, synthesis gas can be obtained by gasification of municipal solid waste or industrial solid waste.

Substrate composition can have a significant impact on the efficiency and/or cost of the reaction. For example, the presence of oxygen (O2) can reduce the efficiency of the anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, purify or filter the substrate to remove any undesirable impurities, such as toxins, unwanted components or dust particles, and/or to increase the concentration of desired components.

In certain embodiments of the invention, fermentation or cultivation is carried out in the absence of carbohydrate substrates, for example, sugar, starch, lignin, cellulose or hemicellulose.

In the context of this document, the term PHBLowB is used to refer to experiments using a lower steady-state biomass concentration, for example, 3 times the lower steady-state biomass concentration. In the context of this document, the term PHBpH5.5 is used to refer to experiments performed at pH 5.5. See fig. 8.

Products

The microorganism of the invention can be grown to produce one or more products. In particular, the microorganism of the invention can produce RNV or its precursors, for example, acetoacetyl-CoA or 3-hydroxybutyryl-CoA.

RHB is a polymer of 3-hydroxybutyrate monomers. The PHB prepared in accordance with the invention may contain any number of 3-hydroxybutyrate monomers, for example about 10-1,000,000 monomers. As further examples, the RNV may contain about 10,100,000 monomers, 100-100,000 monomers, 100-10,000 monomers, 500-5,000 monomers, 1,000-10,000 monomers, or 5,000-20,000 monomers. In a preferred embodiment of the invention, RNV contains about 100-12,000 monomers.

The molecular weight of the RNV produced by the bacterium of the present invention may be in the range of about 1000-100000000 Da. For example, the molecular weight of RNV may be about 1000-10000 Da, 10000-1000000 Da, 10000-10000000 Da, or 10000000-100000000 Da. Preferably, the molecular weight of the RNV may be about 10,000-1000,000 Da, for example 10,000-100,000 Da, 10000500000 Da, 100,000-500,000 Da, 300,000-800,000 Da, or 500,000-1000,000 Da.

RNV production is often referred to as percentage of dry cell mass. The microorganism according to the invention can produce, for example, 0.005-0.995 wt.% RNV. Preferably, the microorganism of the invention produces about 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 3 wt%, 5 wt%, 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt% or 95 wt.% RNV.

The physical characteristics of RNV are well known in the art. As a rough approximation, RNV has a Young's modulus of 1497-3500 MPa, tensile strength of 18-43 MPa, elongation at break of 1.9-45%, crystallinity of 60-80%, melting point of 162-180°C, crystallization temperature of 45-116°C and/or glass transition temperature of -1.2-10°C.

In addition, the microorganism of the invention can also produce or can be genetically engineered to produce other products, for example ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butyrate (WO 2008/115080), 2,3butanediol (WO 2009/151342 and WO 2016/094334), lactate (WO 2011/112103), butene (WO 2012/024522), butadiene (WO 2012/024522), methyl ethyl ketone (2 -butanone) (WO 2012/024522 and WO 2013/185123), ethylene (WO 2012/026833), acetone (WO 2012/115527), isopropanol (WO 2012/115527), lipids (WO 2013/036147), 3-hydroxypropionate (3-HP) (WO 2013/180581), isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO 2013/185123), 1,2-propanediol (WO 2014/036152) , 1propanol (WO 2014/0369152) and products derived from chorismate (WO 2016/191625). In addition to one or more target products, the microorganism of the invention can also produce ethanol, acetate and/or 2,3-butanediol. In certain embodiments of the invention, the microbial biomass itself can be considered a product.

Preferably, the microorganism of the invention is not capable of degrading RNV. Organisms that natively produce RHB and other polyhydroxyalkanoates typically synthesize these polymers as carbon storage material when other nutrients (eg, nitrogen and phosphorus) are limited and carbon is abundant. These organisms can then depolymerize/degrade the polymers when limited nutrients are replenished to access the stored carbon. In order to maximize RNV production, non-native producers, such as the microorganisms of this invention, often have the advantage of not being able to enzymatically degrade polymers once they are produced. This essentially locks the carbon in the polymers permanently and can improve yield.

Selectivity refers to the ratio of the production of the target product to the production of all fermentation products produced by the microorganism. The microorganism of the invention can be engineered to produce products with a certain selectivity or with minimal selectivity. In one embodiment of the invention, the target product represents at least about 5%, 10%, 15%, 20%, 30%, 50%, or 75% of the total fermentation products produced by the microorganism of the invention. In one embodiment of the invention, the target product constitutes at least 10% of the total fermentation products produced by the microorganism of the invention, such that the microorganism of the invention has a selectivity for the target product of at least 10%. In another embodiment of the invention, the target product constitutes at least 30% of the total fermentation products produced by the microorganism of the invention, such that the microorganism of the invention has a selectivity for the target product of at least 30%.

Fermentation

The present invention also provides a method for producing RNV, comprising culturing the microorganism of the invention in the presence of a gaseous substrate through which the microorganism produces RNV. The gaseous substrate generally contains one or more of CO, CO2 and _H2 .

Typically, cultivation is carried out in a bioreactor. The term bioreactor includes a culture/fermentation apparatus consisting of one or more vessels, columns or piping, such as a continuous stirred reactor (CSTR), an immobilized cell reactor (ICR), a trickle bed reactor ( TBR), bubble column, gas lift fermenter, static mixer or other vessel or other device suitable for bringing gas into liquid contact. In some embodiments, the bioreactor may comprise a first growth reactor and a second culture/fermentation reactor. One or both of such reactors may be provided with a substrate. In the context of this document, the terms cultivation and fermentation are used interchangeably. These terms cover both the growth phase and the product biosynthesis phase of the culture/fermentation process.

The culture is typically maintained in an aqueous culture medium containing nutrients, vitamins and/or minerals in sufficient quantities to support growth of the microorganism. Preferably, the aqueous culture medium is an anaerobic microbial growth medium, for example, a minimal anaerobic microbial growth medium. Suitable media are well known in the art.

To obtain the target product, cultivation should preferably be carried out under appropriate conditions. As a rule, cultivation is carried out under anaerobic conditions. Reaction conditions to consider include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, pH of the medium, redox potential of the medium, stirring speed (if using a continuous mechanically stirred reactor), level inoculum, maximum gas substrate concentrations to ensure that gas in the liquid phase does not become a limiting factor, and maximum product concentrations to avoid product inhibition.

Operating a bioreactor at elevated pressures makes it possible to increase the rate of gas mass transfer from the gas phase to the liquid phase. Accordingly, it may be preferable to carry out fermentation at pressures above atmospheric pressure. In addition, since the target gas conversion rate depends in part on the residence time of the substrate in the reactor, with the residence time in the reactor determining the required bioreactor volume, the use of pressurized systems can significantly reduce the required bioreactor volume and therefore the capital cost of fermentation equipment. This in turn means that by maintaining bioreactors at elevated pressure rather than at atmospheric pressure, residence time, defined as the volume of liquid in the bioreactor divided by the flow rate of the injected gas, can be reduced. Optimal reaction conditions will depend in part on the specific microorganism used. However, in general, it is preferable to carry out fermentation at pressures above atmospheric pressure. In addition, since the target gas conversion rate depends in part on the residence time of the substrate in the reactor, and achieving the required residence time in the reactor, in turn, determines the required bioreactor volume, the use of pressurized systems can significantly reduce the required bioreactor volume and, therefore, capital costs for fermentation equipment.

In certain embodiments of the invention, fermentation is carried out in the absence of light or in the presence of an amount of light insufficient to meet the energy needs of photosynthetic or phototrophic microorganisms.

The methods of the invention may further include separating or purifying the RNV. RNV can be separated or purified using any method known in the art. For example, cells can be collected by sedimentation (Chen, Appl Microbiol Biotechnol, 57: 50-55, 2001) or continuous separation (Elbahloul, Appl Environ Microbiol, 75: 643-651, 2009; Heinrich, AMB Express, 2: 59, 2012 ) followed by lyophilization. After freeze-drying, the polymer can be removed from the cells along with substances such as ethyl acetate (Chen, Appl Microbiol Biotechnol, 57: 5055, 2001), acetone (Elbahloul, Appl Environ Microbiol, 75: 643-651, 2009) or sodium hypochlorite ( Heinrich, AMB Express, 2:59, 2012). The Polymer can then be removed from the residual/solubilized cell mass. A large number of alternative methods for the purification of polyhydroxyalkanoates have been published and developed, but have not yet been validated for purification on a large scale (Kunasundari, Express Polym Lett, 5, 620-634, 2011).

- 13 044562

Examples

The following examples further illustrate the present invention, but, of course, should not be construed as limiting its scope in any way.

Example 1

This example demonstrates the design of a Wood-Ljungdahl microorganism capable of synthesizing RNV.

RNV pathway genes (phaC, phaA and phaB) from C. necator (SEQ ID NOs: 1, 4 and 7) were introduced into C. autoethanogenum, a Wood-Ljungdahl microorganism that does not natively produce RNV. It should be noted that these species have significant differences in chromosomal GC content. In particular, C. necator has 66% GC content (Pohlmann, Nat Biotechnol, 24:1257-1262, 2006), and C. autoethanogenum has only 31% GC content (Brown, Biotechnol Biofuels, 7: 40, 2014) . Anticipating problems of gene expression based on codon usage, the RNV gene sequences from C. necator were codon-tailored to better match the higher expression profile for proteins in C. autoethanogenum. Genes with new sequences (SEQ ID NO: 3, 6 and 9), encoding identical proteins as in C. necator, were synthesized and assembled into the expression vector pMTL83157 (SEQ ID NO: 10). This plasmid is similar to the pMTL8000 series (Heap, J Microbiol Methods, 78: 79-85, 2009) with a native Wood-Ljungdahl promoter taken from C. autoethanogenum to drive gene transcription. These genes were placed downstream of the promoter in the same order in which they appear in the C. necator genome: phaC, phaA, and phaB. An antibiotic selection marker, catP, was also used. The resulting plasmid was named pPHB_01 (SEQ ID NO: 11) (Fig. 2).

pPHB_01 was inserted into C. autoethanogenum by bacterial conjugation using E. coli HB101 as described elsewhere (Mock, J Bacteriol, 197: 2965-2980, 2015). In addition, empty plasmid pMTL83157 was inserted into C. autoethanogenum as a negative control. These strains were then used to test the production of RNV from gaseous substrates.

In a preferred embodiment of the invention, the microorganism contains an enzyme that converts acetyl-CoA to acetoacetyl-CoA having at least 80% sequence identity with the amino acid sequence specified in SEQ ID NO: 2, an enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl -CoA having at least 80% sequence identity with the amino acid sequence specified in SEQ ID NO: 5, and/or an enzyme that converts 3-hydroxybutyryl-CoA to polyhydroxybutyrate having at least 80% sequence identity with the amino acid sequence, specified in SEQ ID NO: 8.

Example 2

This example demonstrates the production of RNV from gaseous substrates in Schott bottles.

The strains constructed in Example 1 were grown in small batches to test for RNV production. All work was carried out under strictly anaerobic conditions (Hungate, Methods in microbiology, pages 117-132, Academic Press, New York, NY, 1969). High-pressure Schott bottles containing modified PETS medium (Corke, Appl Environ Microbiol, 11: 54675475, 2011) with thiamphenicol for plasmid retention and 2-(N-morpholino)ethanesulfonic acid for buffering were inoculated with strains, and as the sole source carbon, gas containing CO, CO ₂ , H ₂ and N2 (at 50, 18, 3 and 29%, respectively) was added to the bottles to 21 psi (0.1448 MPa). Cell cultures were grown at 37°C with rotational shaking.

Cell growth was periodically monitored until the cultures entered the stationary phase. Once growth was complete, cells were no longer treated under anaerobic conditions. Cells were collected by centrifugation, their supernatants were discarded, frozen at -20°C and freeze-dried.

The yield of RNV was assessed by high performance liquid chromatography (HPLC) in the same manner as described elsewhere (Karr, Appl Environ Microbiol, 46, 1339-1344, 1983). Briefly, dried cells were treated with concentrated sulfuric acid and heated to convert PHB to crotonic acid. The samples were cooled, diluted, filtered and analyzed by HPLC with a UV/Vis detector to quantify crotonic acid. The results of initial RNV production are summarized in FIG. 3, which illustrates the successful production of ~1.15 wt.% RNV in the Wood-Ljungdahl microorganism.

However, given the low yields compared to native producers such as Cupriavidus and Pseudomonas, which are capable of synthesizing polymers such as RHV such that they constitute over 90% of their weight, it appears that the synthesis of RHV from gas in WoudLjungdahl microorganisms is not so simple , as in native producers growing on non-gaseous substrates. Without wishing to be bound by any particular theory, the inventors postulate that the production of RNV in Wood-Ljungdahl microorganisms may require codon adaptation to overcome differences in pH preference, oxygen demand, substrate utilization pathways, etc. between Wood-Ljungdahl microorganisms and native RNV producers.

After achieving the formation of RNV in C. autoethanogenum from gaseous substrates, the work described above was repeated with changes in growth conditions in order to study the conditions that may promote/improve the yield of RNV. In particular, experiments were carried out to replicate the conditions

- 14 044562 described above (condition 1, Fig. 4A) to change the gas compositions to 50/30/10/10 CO/CO2/H2/N2 (condition 2, Fig. 4B) to extend the culture incubation to the stationary phase ( condition 3, Fig. 4C) and periodically refresh the bottled gas (condition 4, Fig. 4D). As illustrated in FIG. 4A-4D, growth was similar for both the engineered strain and the control strain under all conditions tested.

Cells were collected and analyzed for RNV production as described above. The results are illustrated in Fig. 5, which shows the production of ~1.65 wt.% RNW in condition 1, ~1.50 wt.% RNW in condition 2, ~1.50 wt.% RNW in condition 3 and ~0.85 wt.% RNW in condition 4.

Example 3

This example demonstrates the production of RHB from gaseous substrates during continuous fermentation.

The strain constructed in Example 1 was tested in continuous fermentation using gas as the main carbon source under conditions similar to those described in Valgepea, Cell Syst, 4: 505-515, 2017. Similar to the experiments performed in Schott bottles, continuous cell cultures grown and processed anaerobically. Unlike Schott bottles, cell cultures were grown continuously for approximately 20 days with a constant supply of nutrient medium. To grow and obtain RNV, two different gas compositions were used: 50/20/20/10 CO/ _CO2 / _H2 /Ar and 50/20/2/28 CO/CO2/H2/N2. Gas uptake was monitored by mass spectrometry (MS) and samples were taken periodically for quantification of liquid metabolites by HPLC.

RNV was not quantified until the end of the continuous fermentation. Similar to the Schott bottle experiments, cells were collected by centrifugation, frozen, and freeze-dried. The dried cells were subsequently analyzed for RHB by treatment with sulfuric acid and heating to convert RHB to crotonic acid. Then the quantitative determination of RNV was carried out by HPLC. The results of RNV production during continuous fermentation are illustrated in FIG. 6. Specifically, microorganisms grown on 20% hydrogen gas produced ~0.45 wt.% RHB, and microorganisms grown on 2% hydrogen gas produced ~0.25 wt.% RHB.

Example 4

This example demonstrates the optimization of a fermenter to increase RHB production.

Various conditions were tested within a continuous fermentation to increase the RNV content in the cells. Acetyl-CoA and NADPH pools increase at lower biomass concentrations (Valgepea, Cell Syst., 4: 505-515, 2017). Therefore, it was tested whether lower steady-state biomass levels would lead to higher RBI by increasing the levels of acetyl-CoA and NADPH pools. It has been shown that reducing the rate of CO uptake and, in general, the concentration of biomass in the fermenter increases the flow of cellular resources to the RNV (Fig. 7).

Another factor found to increase RHB was pH. At higher pH, less acetic acid will diffuse and uncouple the proton motive force (PMF) (Valgepea, Cell Syst., 4: 505-515, 2017). Thus, it was tested whether less energy would be expended to maintain PMF by increasing the pH from 5 to 5.5 or 6. The additional energy available would provide additional ATP to support PPH production by reducing the production of acetate required to produce ATP. Changing the pH from 5.0 to 5.5 or 6.0 resulted in an increase in RHB production (~12.5-fold at pH 5.5). However, a pH value of 6.0 is difficult to maintain because C. autoethanogenum grows optimally at a more acidic pH.

Example 5

This example demonstrates changes in the level of transcription and metabolism during the production of RNV compared to the control strain (empty plasmid).

Analysis of transcriptome data from RNA sequencing was based on a previously published R script (Valgepea, Cell Syst., 4: 505–515, 2017) with the following modifications: use of the C. autoethanogenum reference sequence CP006763.1 and its annotated genome described in Brown, Biotechnol . Biofuels, 7: 40, 2014; addition of nucleotide sequence for three RNV genes (SEQ ID NO: 3, 6, 9.)

The metabolomics package available in R (Livera and Bowne, R package, 2014) was used to perform statistical analysis of intracellular metabolomics data. This script normalizes and integrates metabolomics data into a linear model fit (De Livera, Anal. Chem., 84: 1076810776, 2012). Intracellular metabolite concentrations were normalized to biomass (μmol/gSCM) before importing data into the script. Linear model similarity using conventional statistics (i.e. non-Bayesian) was used to statistically analyze metabolome data (De Livera, Anal. Chem., 84: 10768-10776, 2012; De Livera, Metabolomics Tools for Natural Product Discovery, 2013) .

Although arginine was not supplied, increased activity was observed for the arginine deiminase pathway, and an alternative pathway was discovered to provide ATP in acetogens (Valgepea, Metab. Eng. 41: 202-211, 2017)

- 15 044562 (q-value<0.01): arginine deiminase (CAETHG_3021, ~7 times); ornithine carbamoyltransferase (CAETHG_3022, ~6 times); carbamate kinase (CAETHG_3025, ~3.3-fold). In addition, three genes encoding the Rnf complex, which is part of the energy-saving complex in acetogens (Schuchmann and Muller, Nat. Rev. Microbiol. 12: 809-821, 2014), showed a ~2-fold increase in the RNV strain: (CAETHG_3231, q-value=0.02; CAETHG_3228, q-value=0.04 and CAETHG_3230, q-value=0.03). These observations highlight changes in energy metabolism due to heterologous production. In addition, the expression of two Wood-Ljungdahl pathway (WLP) genes encoding CO-dehydrogenase/acetyl-CoAsynthase (CAETHG_1610, ~1.4-fold; CAETHG_1611, ~1.2-fold) and a gene encoding (FeFe)hydrogenase ( CAETHG_1691 (~2.5-fold) were upregulated in the RNV strain.These changes may reflect the increase required for acetyl-CoA and NADPH production for RNV production (Fig. 1).

At the metabolome level, strain RNV had a higher intracellular NADH/NAD+ ratio compared to EP. This suggests potential changes in the redox state following RNV expression. Production of acetate, the main native metabolic byproduct of C. autoethanogenum (Abrini, Arch. Microbiol., 161: 345-351, 1994; Marcellin, Green Chem., 18: 3020-3028, 2016), is reduced compared to the synthesis strain EP -gas (p value <0.01; two-tailed equal variance t-test). No changes were observed in the steel mill waste gas.

Example 6

This example shows the results of genome-scale metabolic model (GEM) reconstructions. The genome-scale metabolic model GEM iCLAU786 (Valgepea, Cell Syst., 4: 505–515, 2017) was used with the addition of the RNV pathway. The simulation was carried out for the RNV strain grown on synthesis gas under all the conditions listed above.

Flux modeling confirmed that less CO ₂ was dissipated under conditions with higher RPH (i.e. low biomass and pH 5.5). In addition, as noted previously (Valgepea, Cell Syst, 4: 505-515, 2017), these simulations also showed that CO2 was directly reduced to formate by _H2 through the formate- _H2 -lyase activity of the electron bifurcation hydrogenase-formate enzyme complex -dehydrogenase (HytA-E/FdhA) (Wang, J. Bacteriol, 195: 4373-4386, 2013). This provides an advantage over CO2 reduction by formate dehydrogenase redox consumption because redox potential is not consumed in the process of _CO2 reduction in WLP using the former enzyme complex. It was also noted that in low biomass and pH 5.5 experiments, equilibration of the total amount of reduced ferredoxin was achieved by increasing or decreasing the flux for some key reactions, e.g., AOR (aldehyde ferredoxin oxidoreductase), Nfn complex, or methylene-THF reductase bifurcation reaction , compared to control (РНВ20).

Surprisingly, the control condition (PHB20), in silico, had a lower ATP maintenance cost (mmol/gSCM/h) and ATP maintenance cost of total ATP production (%mATP) compared to the PHBpH5.5 condition.

Simulations were also run to determine whether ATP, NADH, NADPH, or reduced ferredoxin (Fd _red ) were limiting for RNV production. The simulations showed that upon ATP delivery, RPH production (mmol/gSCM/h) reached its maximum value among the limiting candidates in all tested conditions (i.e., RHB20, RHB low biomass, and PHBpH5.5). This observation is consistent with the understanding that acetogen metabolism is limited by ATP (Schuchmann and Muller, Nat. Rev. Microbiol. 12: 809–821, 2014). The model also showed that, after being limited by ATP, PHB production is limited by the presence of Fd _red , NADPH and then NADH (Fig. 8).

This result confirms the importance of ATP and Fd _red as high-energy carriers in acetogens. Since ATP primarily supports anabolism and cell performance, Fd _red is essential for the energy-saving Rnf complex (Biegel, Cell. Mol. Life Sci. 68: 613-634, 2011) and only Fd _red is known to provide electrons for the reduction of CO ₂ to CO in the carbonyl branch of WLP (Schuchmann and Muller, Nat. Rev. Microbiol. 12: 809-821, 2014).

All references, including publications, patent applications and patents cited herein, are incorporated herein by reference to the same extent as if each reference had been separately and specifically indicated for inclusion by reference and set forth herein in its entirety. volume. In this specification, reference to any prior art does not constitute, and should not be construed as, an admission that such prior art is part of the public knowledge of the art in any country.

The use of singular terms and similar references in the context of the description of this invention (especially in the context of the claims below) should be considered to include both the singular and plural, unless otherwise stated herein or otherwise clearly inconsistent context. Terms consisting of, having, including

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Claims (5)

including and containing should be considered non-limiting terms (i.e., including but not limited to) unless otherwise noted. The term essentially consisting of limits the scope of the composition, process or method to specified materials or steps, or that does not significantly affect the essential and novel characteristics of the composition, process or method. The use of an alternative (eg, or) should be understood to mean one, both, or any combination of the above alternatives. As used herein, the term about means ±20% of the stated range, value, or structure unless otherwise noted. The listing of ranges of values herein is simply intended to serve as a shorthand way of referring individually to each individual value falling within the specified range, unless otherwise indicated herein, each individual value being included in this description as if has been provided separately in this document. For example, any concentration range, percentage range, ratio range, integer range, size range, or thickness range should be understood to include the value of any integer within the range and, where appropriate, fractions thereof (e.g., one tenth and one hundredth whole number) unless otherwise noted. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use of any and all examples or exemplary language (eg, for example) proposed herein is intended merely to better illuminate the present invention and does not constitute a limitation on the scope of the present invention unless otherwise stated. Nothing contained in this specification should be construed as indicating any unclaimed element as being necessary for the practice of this invention. This document describes preferred embodiments of the present invention. Variations on such preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will use such variations as necessary, and the inventors expect that the invention will be practiced differently than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter set forth in the appended claims, as provided by applicable law. In addition, any combination of the elements described above, in all possible variations thereof, is included in this invention, unless otherwise indicated herein or otherwise clearly inconsistent with the context. CLAIM 1. A non-naturally occurring Wood-Ljungdahl microorganism Closthdium autoethanogenum for the production of polyhydroxybutyrate, containing: a. a nucleic acid encoding a heterologous enzyme that converts acetylCoA to acetoacetyl-CoA and is acetyl-CoA-C acetyltransferase (EC 2.3.1.9), b. a nucleic acid encoding a heterologous enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA and is acetoacetyl-CoA reductase (EC 1.1.1.36) or 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157), and c. a nucleic acid encoding a heterologous enzyme that converts 3hydroxybutyryl-CoA into polyhydroxybutyrate and is a polyhydroxyalkanoate synthase (EC 2.3.1.-), where the specified microorganism consumes gaseous substrates containing H ₂ and at least one of CO and CO ₂ . 2. The microorganism according to claim 1, characterized in that the acetyl-CoA-C-acetyltransferase is obtained from: Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Clostridium acetobutylicum, Cupriavidus necator, Escherichia coli, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida or Streptomyces coelicolor. 3. Microorganism according to claim 1, characterized in that the acetoacetyl-CoA reductase is obtained from: Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescen s, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida or Streptomyces coelicolor. 4. Microorganism according to claim 1, characterized in that the 3-hydroxybutyryl-CoA dehydrogenase is obtained from: Clostridium beijerinckii, Clostridium acetobutylicum or Clostridium kluyveri. 5. Microorganism according to claim 1, characterized in that the polyhydroxyalkanoate synthase is obtained from: Acinetobacter baumannii, Aeromonas caviae, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, -