WO2017168161A1 - Enzyme modifiée - Google Patents

Enzyme modifiée Download PDF

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Publication number
WO2017168161A1
WO2017168161A1 PCT/GB2017/050901 GB2017050901W WO2017168161A1 WO 2017168161 A1 WO2017168161 A1 WO 2017168161A1 GB 2017050901 W GB2017050901 W GB 2017050901W WO 2017168161 A1 WO2017168161 A1 WO 2017168161A1
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Prior art keywords
enzyme
dera
hydroxybutanal
acetaldehyde
microbial organism
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PCT/GB2017/050901
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English (en)
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Michelle GRADLEY
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Zuvasyntha Limited
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Publication of WO2017168161A1 publication Critical patent/WO2017168161A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02004—Deoxyribose-phosphate aldolase (4.1.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention provides a modified enzyme that has improved activity for catalysing the formation of 3-hydroxybutanal.
  • the modified enzyme is also capable of converting acetaldehyde to crotonaldehyde, via an aldol condensation.
  • the current invention also relates generally to microorganisms, and related materials and methods, which have been modified to express said enzyme, in order to to enhance their ability to produce commodity chemicals, for example, 1 ,3-butanediol and derivatives thereof, which can be produced in the microorganisms via the intermediates acetaldehyde and 3-hydroxybutanal.
  • 1 ,3-butanediol (1 ,3-BDO) is a four carbon diol which has a number of uses, including in the food, chemical and manufacturing industries.
  • 1 ,3-BDO has traditionally been produced from petroleum derived acetylene via its hydration. The resulting acetaldehyde is then converted to 3-hydroxybutanal which is subsequently reduced to form 1 ,3-BDO.
  • acetylene has been replaced by the less expensive ethylene as a source of acetaldehyde.
  • crude oil has become relatively more expensive than natural gas, many ethylene cracking operations are using lighter natural gas feedstocks to earn higher margins, leading to significantly lower quantities of C4 chemicals and rising prices.
  • Feedstock flexibility relies on the introduction of methods that enable access and use of a wide range of materials as primary feedstocks for chemical manufacturing.
  • the reliance on petroleum based feedstocks for either acetylene or ethylene warrants the development of renewable, or cheaper, or non-petroleum derived feedstock based routes to 1 ,3-butanediol, butadiene and other valuable chemicals such as methylethylketone.
  • the present invention relates to the engineering of organisms to imbue or enhance the ability to convert the central metabolic intermediates acetyl CoA and pyruvate to the common pathway intermediate acetaldehyde, which is then subject to an enzymatically catalysed aldol coupling, ultimately yielding 1 ,3-butanediol or other products. More specifically, in modified organisms of the invention, acetaldehyde derived from acetyl CoA or pyruvate as the primary pathway product is supplied as the substrate for an aldolase capable of the coupling of two molecules of acetaldehyde to form 3-hydroxybutanal. The 3-hydroxybutanal, which is the product of this aldol coupling, can be directed to other products or other intermediates which can then in turn enter other natural or unnatural metabolic pathways.
  • Example intermediates include 2-hydroxyisobutyryl CoA, 3-hydroxybutyryl CoA, Crotonyl CoA, Crotonaldehyde, Butyryl CoA, Butanal, Acetoacetyl CoA, and acetoacetate.
  • Desirable downstream products include 2-hydroxisobutyrate, Crotyl alcohol, Crotonic acid, Butanol, Butyrate, 3-hydroxybutyrate, 1 ,3-butanediol, 3- hydroxybutylamine, Polyhydroxybutyrate, Acetone, and Isopropanol. These products can, where desired, be recovered and used to make yet further commodities - for example butadiene, methacrylic acid, 2-methyl-1 ,4-butanediol, methyltetrahydrofuran, isoprene.
  • downstream products Any of these intermediate products, downstream products, and commodities may be referred to herein as “downstream products” or “products” herein for brevity.
  • a preferred product is 1 ,3-butanediol (1 ,3-BDO).
  • the modified organisms of the invention are typically microorganisms capable of using renewable or inexpensive feedstocks or energy sources such as sunlight, carbohydrates, methanol, synthesis gas (syngas) and ⁇ or other gaseous carbon sources such as methane to generate the appropriate metabolic intermediates.
  • renewable or inexpensive feedstocks or energy sources such as sunlight, carbohydrates, methanol, synthesis gas (syngas) and ⁇ or other gaseous carbon sources such as methane to generate the appropriate metabolic intermediates.
  • imbuing or enhancing the production of acetaldehyde from the central metabolic intermediate, or increasing its availability to the aldolase will typically involve one or more of:
  • the aldolase capable of the in vivo coupling of two molecules of acetaldehyde to 3-hydroxybutanal will itself also be the product of genetic engineering e.g. via the introduction of a heterologous aldolase as described below.
  • a genetic modification combining these changes thus serves to increase the flux of central metabolic intermediates to the 3-hydroxybutanal via the acetaldehyde intermediate.
  • this 3-hydroxybutanal is subsequently directed to a downstream product as described below.
  • a non-naturally occurring microbial organism which includes a genetic modification in its genome which enhances production of 3-hydroxybutanal by the microbial organism from at least one endogenous central metabolic intermediate via a 3-hydroxybutanal synthetic pathway in which two molecules of acetaldehyde are coupled to form 3-hydroxybutanal using an aldolase capable of accepting an aldehyde as both the acceptor and donor in an aldol coupling.
  • Preferred organisms are those in which the modification enhances production of 1 ,3-butanediol (1 ,3-BDO) via a 1 ,3-BDO synthetic pathway in which the 3- hydroxybutanal is reduced to 1 ,3-BDO.
  • the invention applies likewise to modifications and pathways in which 3-hydroxybutanal is converted to other downstream products and, unless context demands otherwise, each of the embodiments relating to 1 ,3-BDO will be understood to apply mutatis mutandis to these other products.
  • the genetic modification will be such that said modified organism produces a greater flux of or through 3-hydroxybutanal (and hence also of or through a downstream intermediate) to a product thereof such as 1 ,3-BDO) compared to a corresponding reference microbial organism not including said genetic modification, when grown on the same feedstock or energy source under the same conditions.
  • the modified organism may produce at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 times as much 3-hydroxybutanal or, more preferably, downstream product such as 1 ,3-BDO compared to the reference organism under the same conditions.
  • Non-naturally occurring in the present disclosure denotes the fact that the relevant modification, which may be any modification described herein, for example a modification that increase the flux to 3-hydroxybutanal or downstream product such as 1 ,3-BDO, is introduced to a reference organism by human intervention.
  • a microbial organism of the invention preferably includes one or more of the following modifications within its genome:
  • the invention embraces a modification which confers on the microorganism the capability to convert a feedstock described herein to 1 ,3-BDO, wherein the microorganism lacks the ability to carry out that conversion in the absence of said modification. It also embraces a modification which increases the flux of a feedstock described herein to 1 ,3-BDO, in a microorganism where that flux is initially very low or negligible.
  • the modification typically relates to an aldolase enzyme as described herein, as well as a pathway providing acetaldehyde to it.
  • said enzyme is deoxyribose phosphate aldolase, EC 4.1.2.4 (“DERA”) or a variant thereof, or other enzyme sharing the ability to accept an aldehyde (acetaldehyde) as both the acceptor and donor in an aldol coupling.
  • said enzyme is a DERA variant, as described and defined herein.
  • genetic modification can include more than one modification of the genome of the microbial organism in question.
  • Microbial organisms of the present invention may include any of the following genetic modifications in respect of the aldolase:
  • a preferred embodiment is a microbial organism wherein said modification is introduction of a heterologous nucleic acid encoding the enzyme.
  • the heterologous gene encoding the enzyme may encode a fusion protein encoding also one or more other enzymes present in a 3-hydroxybutanal pathway - for example those involved in the provision of the aldolase substrate acetaldehyde.
  • the heterologous gene encoding the enzyme may encode a fusion protein encoding also one or more other enzymes present in a downstream product pathway - for example those involved in the conversion of 3-hydroxybutanal to another product or intermediate.
  • 3-hydroxybutanal pathway or "3-hydroxybutanal synthetic pathway” in the present context refers to a series of enzymatically catalysed reactions occurring in a cell which convert one or more principle chemical starting materials or substrates (feedstocks) to central metabolic intermediates comprising one or more of: pyruvate or acetyl CoA which are in turn converted to the common pathway intermediate acetaldehyde which is condensed to form 3-hydroxybutanal.
  • a "3- hydroxybutanal (synthetic) pathway” may also include an activity involved in the conversion of 3-hydroxybutanal directly or indirectly to a downstream product derived from 3-hydroxybutanal, such as 1 ,3-BDO.
  • the term "3-hydroxybutanal pathway” enzyme should be construed accordingly i.e. an enzyme providing an activity in a "3-hydroxybutanal pathway".
  • a "3-hydroxybutanal pathway” is a "1 ,3-BDO pathway” in which a series of enzymatically catalysed reactions occurring in a cell which convert one or more principle chemical starting materials or substrates (feedstocks) to 1 ,3-BDO via central metabolic intermediates comprising one or more of: pyruvate or acetyl CoA which are in turn converted to the common pathway intermediate acetaldehyde which is condensed to form the 1 ,3-BDO precursor, 3-hydroxybutanal, which is in turn converted to 1 ,3-BDO.
  • the conversion of central metabolic intermediates to the common pathway intermediate acetaldehyde may require 1 or more steps (e.g. 2, 3, 4 steps).
  • the invention embraces the introduction of all enzymes relevant to the 3- hydroxybutanal (e.g. 1 ,3-BDO) pathway, including those relating to early substrate utilisation and generation of the central metabolic intermediates themselves, as well as those involved in conversion of the central metabolic intermediates to the common intermediate.
  • microbial organisms may include one or more other modifications within its genome:
  • said microbial organism may comprise two, three, four, five, six, seven, eight, nine, ten or more exogenous nucleic acids, each encoding a 3- hydroxybutanal (e.g, 1 ,3-BDO) pathway enzyme.
  • a 3- hydroxybutanal e.g, 1 ,3-BDO
  • the invention also embraces the knockout or other impairment of enzyme activities which would otherwise direct flux away from the pathway of choice e.g. direct flux of acetaldehyde away from the aldolase.
  • the invention provides, inter alia, a non-naturally occurring microorganism that through genetic engineering gains the ability to produce 1 ,3- BDO or other downstream product derived from 3-hydroxybutanal from acetyl-coA, or gains the ability to produce an increased flux of 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal from acetyl-coA, such that the 1 ,3-BDO or other downstream product accumulates and can be recovered or further converted enzymatically or chemically without recovery
  • acetyl CoA may optionally be utilised via acetate.
  • acetate can then be converted to acetaldehyde via carboxylic acid reductase activity, for example, EC 1.2.7.5 or EC. 1.2.99.6, ATP or ferredoxin driven or EC 1.2.1.30 or EC 1.2.1.3.
  • carboxylic acid reductase activity for example, EC 1.2.7.5 or EC. 1.2.99.6, ATP or ferredoxin driven or EC 1.2.1.30 or EC 1.2.1.3.
  • Or can be converted to acetyl CoA via EC 6.2.1.1 or EC 2.8.3.8 and subsequently converted to acetaldehyde via EC 1.2.1.10.
  • acetyl CoA may be utilised via pyruvate (see below, using enzymes such as EC 1.2.7.1 and EC 4.1.1.1) or via direct synthesis of acetaldehyde from acetyl CoA using an aldehyde dehydrogenase (acylating), for example, acetaldehyde dehydrogenase EC 1.2.1.10.
  • the invention provides, inter alia, a non-naturally occurring microorganism that through genetic engineering gains the ability to produce 1 ,3- BDO or other downstream product derived from 3-hydroxybutanal from pyruvate, or gains the ability to produce an increased flux of 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal from pyruvate, such that the 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal accumulates and can be recovered or further converted enzymatically or chemically without recovery.
  • pyruvate can be converted to acetaldehyde via acetyl CoA using enzymes such as EC 1.2.7.1 or EC 1.2.1.51 or EC 1.2.4.1 and EC 1.2.1.10.
  • pyruvate can be converted to acetaldehyde, directly via pyruvate decarboxylase (EC 4.1.1.1).
  • pyruvate may be referred to herein, depending on the pH and other conditions, it may likewise be present as pyruvic acid, and therefore all these descriptors are used interchangeably, unless context demands otherwise. This applies mutatis mutandis to other salts or acids described herein - e.g. acetic acid etc.
  • the invention further provides a method for increasing the flux of 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal produced by a microbial organism, which method comprises introducing one or more of the genetic modifications described herein into its genome.
  • the present invention relates, amongst other things, to the generation of microorganisms that are effective at producing 1 ,3-butanediol from alternative substrates to traditional petroleum-based products.
  • Methods of producing such a microorganism will typically comprise the step expressing, or causing or allowing the expression of, a heterologous nucleic acid (for example, encoding at least an aldolase as described herein) within the host, following an earlier step of introducing the nucleic acid into the host or an ancestor of either.
  • a heterologous nucleic acid for example, encoding at least an aldolase as described herein
  • Suitable heterologous nucleic acids are discussed hereinafter.
  • the methods may include the step of up-regulating native enzymes using genetic engineering and ⁇ or repressing enzymes to reduce flux to competing pathways.
  • microbe utilised in the present invention will generally be based on the choice of feedstock or energy source which it is desired to use, along with the amenability of the microbe to genetic modification or introduction of a 1 ,3-BDO (or other downstream product derived from 3-hydroxybutanal) pathway.
  • Preferred processes disclosed herein involve sustainable manufacturing practices that utilise renewable feedstocks, though other feedstocks which may provide cost or environmental benefits compared to traditional petroleum products may also be used, for example natural gas derived methanol.
  • the processes disclosed herein may utilise feedstocks such as syngas, C0 2 , CO, and H 2 , methane and methanol (shale gas or biomass/ waste derived) to reduce energy intensity and cost and lower greenhouse gas emissions.
  • feedstocks such as syngas, C0 2 , CO, and H 2 , methane and methanol (shale gas or biomass/ waste derived) to reduce energy intensity and cost and lower greenhouse gas emissions.
  • feedstocks such as syngas, C0 2 , CO, and H 2 , methane and methanol (shale gas or biomass/ waste derived)
  • Syngas is a mixture of primarily H 2 and CO that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter.
  • the present invention preferably utilises microorganisms capable of utilizing syngas or other gaseous carbon sources (C0 2 , CO) with or without methanol, methane or sugar co-utilisation or by use of methanol, methane or sugars directly as sole feedstocks. Or waste streams containing acetate.
  • Photosynthetic organisms e.g. algae capable of using sunlight as an energy source are also expressly included.
  • a method for producing 1 ,3- BDO or other downstream product derived from 3-hydroxybutanal that includes culturing the aforementioned non-naturally occurring microbial organisms under conditions and for a sufficient period of time to produce 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal.
  • cultured or “culturing” on a feedstock as used herein is being used in a general sense to mean that the microbial organism utilises the feedstock in question for the production of the relevant product, and should not be taken to imply that the biomass of the microbial organism actually increases during the process.
  • a process for producing 1 ,3- BDO or other downstream product derived from 3-hydroxybutanal comprises culturing a microbial organism of the invention on a reaction feedstock as described herein so that it metabolises the feedstock to produce 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal from central metabolic intermediates.
  • the microbe may be cultured in the presence of an additional energy source e.g. a carbohydrate such as a hexose, or sunlight.
  • an additional energy source e.g. a carbohydrate such as a hexose, or sunlight.
  • the processes of the invention may further comprise recovering some or all of the 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal e.g. by one or more of electrodialysis, solvent extraction, distillation, or evaporation.
  • 1 ,3-BDO or other downstream product derived from 3- hydroxybutanal may be converted chemically or enzymatically in situ to a downstream product or products, which may in turn be recovered by similar means.
  • the processes of the invention may further comprise converting the 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal into a pharmaceutical, cosmetic, food, feed or chemical product, which may optionally be an unsaturated alcohol, alkene, carboxylic acid, ether, ester, or ketone e.g. methylethyl ketone, 1- butanol, 2-butanol, butadiene, isoprene and so on.
  • a pharmaceutical, cosmetic, food, feed or chemical product which may optionally be an unsaturated alcohol, alkene, carboxylic acid, ether, ester, or ketone e.g. methylethyl ketone, 1- butanol, 2-butanol, butadiene, isoprene and so on.
  • the invention provides non-naturally occurring microorganisms comprising one or more heterologous proteins conferring to the microorganism the capability to convert central intermediates to 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal as described herein.
  • heterologous protein may be directed at increasing the flux of reaction feedstocks such as syngas or other substrates described herein to 1 ,3- BDO or other downstream product derived from 3-hydroxybutanal, in a microorganism where that flux is initially very low or negligible under relevant industrial culture conditions.
  • the invention provides a non-naturally occurring microorganism which has been modified to up-regulate (increase expression of) a native protein, or to modify the localisation of a native protein, or to modify the activity or specificity of a native protein, thereby conferring to the microorganism the capability to convert syngas or other substrates described herein to 1 ,3-BDO or other downstream product derived from 3-hydroxybutanal, wherein the microorganism lacks the ability to carry out that conversion in the absence of said modification.
  • the heterologous protein may be directed at increasing the flux of metabolic intermediates from the feedstock being utilised in a microorganism where that flux is initially very low or negligible.
  • the invention provides a non-naturally occurring microbial organism having a genetically modified 3-hydroxybutanal or 1 ,3-BDO biosynthetic pathway and the competence to metabolise syngas or other feedstocks or energy source described herein to produce 1 ,3-BDO or other downstream product derived from 3- hydroxybutanal.
  • Pyruvate and acetyl CoA are products of a considerable range of different central metabolic pathways for assimilation of carbon. They are converted to important cellular building blocks essential for life. In the present invention they are utilised within a 1 ,3-BDO biosynthetic pathway, which pathway is at least in part the result of genetic engineering of the microbial organism.
  • a 1 ,3-BDO biosynthetic pathway which pathway is at least in part the result of genetic engineering of the microbial organism.
  • an organism is selected according to the feedstock it is desired to utilise.
  • the organism may be selected to have in its genome a particular metabolic pathway leading to acetyl CoA and ⁇ or pyruvate.
  • Example metabolic pathways include: • the Wood-Ljungdahl pathway ( Figure 5)
  • Wood-Ljungdahl pathway, reverse TCA cycle, the serine cycle, the RuMP pathway and the Calvin cycle are examples of C1 (gas and liquid) fixation pathways. In some case these pathways can be used alongside glycolysis.
  • the Wood-Ljungdahl pathway is important for redox balancing by using the reducing equivalents generated from glycolysis and pyruvate decarboxylation to acetyl CoA, to fix the released 2 C0 2 into a further molecule of acetyl CoA.
  • the serine or the RuMP pathways are generally used by methanotropic and methylotrophic organisms for assimilation of C1 feedstocks such as methanol, methane and C0 2 . These pathways are well described and well known in the art.
  • the product of the RuMP pathway is pyruvate which would normally be converted primarily to biomass.
  • Intercepting pyruvate via, for example, decarboxylation to acetaldehyde would redirect flux towards 1 ,3-butanediol synthesis using the described invention.
  • the Calvin cycle is used by photosynthetic organisms such as algae for assimilation of C0 2 using light energy.
  • the serine cycle primarily produces acetyl CoA which normally enters the ethylmalony CoA pathway for synthesis of C4 building blocks for biomass synthesis. If acetyl-CoA is required as the biosynthetic precursor of membrane fatty acids or the storage compound poly 3- hydroxybutyrate for example, then the EMC pathway is not required for oxidation of acetyl-CoA, (Anthony, C. 201 1. Science Progress, 94, 109). Hence, acetyl CoA can be tapped off to other more useful compounds such as 1 ,3-butanediol via conversion of acetyl CoA to acetaldehyde using a pathway described herein.
  • microorganisms with known pathways capable of utilising syngas, or gases such as CO, C0 2 and H 2 .
  • Microorganisms which are COteils are termed "carboxydotrophic microorganisms". Such organisms can be aerobes and anaerobes.
  • Anaerobic examples of these microorganisms fall into 3 main groups: those producing mainly acids (e.g. acetic acid, termed "acetogens"), those producing mainly methane and those producing mainly hydrogen.
  • acetogens e.g. acetic acid, termed "acetogens”
  • Carboxydotrophic acetogens are acetogenic microorganisms capable of utilising the syngas components CO and H 2 via the Wood-Ljungdahl pathway ( Figure 2) producing the key intermediate acetyl CoA.
  • the Wood-Ljungdahl pathway is well known in the art (see Figure 5) and can be separated into two branches: the methyl branch (reductive branch) and the carbonyl branch.
  • the methyl branch converts syngas (CO or C0 2 ) to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch supplies a molecule of CO which along with methyl-THF is converted to acetyl-CoA.
  • Acetogens refers to anaerobic organisms able to reduce C0 2 /CO to acetate via this pathway. Acetogens can grow on a variety of different substrates such as, for example, hexoses [glucose, fructose and xylose], C2 and C1 compounds [gas and liquid] including methanol (see Figure 2), C0 2 /H 2 and CO gases. Acetogens are also known to utilise acetate directly. Over one hundred acetogenic species, representing twenty-two genera, have been isolated so far from various habitats such as sediments, sludge, soils and the intestinal tracts of many animals, including termites and humans.
  • Acetogens are becoming a significant focus for the biotech industry, as important bulk chemicals can potentially be produced from autotrophic growth at the expense of C0 2 via syngas fermentation or via coupling with methanol, which can serve as a source of carbon and energy in the absence of syngas. in the presence of a more oxidised substrate such as C0 2 .
  • Acetogens can utilise hexoses (e.g. glucose, fructose and xylose) and other sugars as substrates.
  • hexoses e.g. glucose, fructose and xylose
  • acetogens acetate is the primary product of hexose consumption:
  • the pathway of hexose consumption starts with their oxidation via the Embden- Meyerhof-Parnas pathway to pyruvate, which is then oxidized by pyruvate:ferredoxin oxidoreductase to acetyl-CoA, reduced ferredoxin, and C0 2 .
  • the acetyl-CoA is then converted to acetate via acetyl phosphate.
  • acetogens have evolved primarily for the efficient synthesis of either biomass or acetate from the central intermediate acetyl CoA.
  • industrial products derived directly from acetyl CoA or acetate may be preferred for process development using acetogens.
  • an aldolase capable of accepting the product acetaldehyde as a substrate (both as acceptor and donor) permits the efficient generation of useful chemical products from acetate which acetogens naturally accumulate in high yield and high concentration.
  • acetyl-CoA derived acetate for generation of acetaldehyde are explained in more detail below.
  • a preferred route of acetaldehyde generation would be based on utilisation of ferredoxin driven aldehyde ferredoxin oxidoreductase. This enzymatic conversion does not require ATP so may be particularly important for bacteria growing on C1 gases such as CO, C0 2 /H 2 or syngas for the energetics reason described above. Since the natural SLP step involving the conversion of acetyl CoA to acetate can be conserved, the energetics of the Wood-Ljungdahl pathway would be unchanged.
  • the invention provides a non-naturally occurring microorganism having the Wood-Ljungdahl pathway and the capability of utilising syngas naturally and that through genetic engineering gains the ability to produce 1 ,3-BDO or gains the ability to produce an increased flux of 1 ,3-BDO.
  • Hydrogen is a major source of reducing equivalents, but equally for example, dissimilation of methanol can also generate reducing equivalents (6[H]) and ATP energy.
  • methanol utilisation confers an energetic advantage because it provides a preformed methyl group for synthesis of acetyl CoA eliminating the need for an ATP for conversion of formate to formyl-THF catalysed by formyl-THF synthetase (Bainotti, A.E and Nishio, N. 2000. J. Appl. Microbiol, 88, 191). The specific energetic requirements of the transfer of the methyl group from methanol is not clearly understood.
  • additional sources include, but are not limited to, production of C0 2 as a byproduct in ammonia and hydrogen plants, where methane is converted to C0 2 ; combustion of wood and fossil fuels; production of C0 2 as a byproduct of fermentation of sugar in the brewing of beer, whisky and other alcoholic beverages, or other fermentative processes; thermal decomposition of limestone, CaC0 3 , in the manufacture of lime, CaO; production of C0 2 as byproduct of sodium phosphate manufacture; and directly from natural carbon dioxide springs, where it is produced by the action of acidified water on limestone or dolomite.
  • acetogens to utilise methanol requires specific methyltransferases. Where such aceteogen methyltransferases are not naturally present, an acetogen can be engineered with heterologous methyltransferases and other associated proteins to allow it to utilise methanol as well as the other feedstocks discussed above. Examples of enzymes required to give an acetogen the ability to grow on methanol include:
  • Methyltetrahydrofolate:corrinoid protein methyltransferase (MtaA)
  • the invention provides a non-naturally occurring microorganism having the RUMP or serine cycle pathway encoded in its genome and the capability of utilising methanol or methane naturally and that through genetic engineering gains the ability to produce 1 ,3-BDO or gains the ability to produce an increased flux of 1 ,3-BDO.
  • Photosynthetic organisms such as microalgae or cyanobacteria are autotrophs or heterotrophs able to utilise sunlight (light energy) for C0 2 fixation via the Calvin cycle.
  • a product is glyceraldehyde-3-phosphate which can be converted to sugar or to pyruvate and acetyl CoA.
  • sugars as a source of carbon and energy via glycolytic pathways such as the Entner doudoroff pathway, Embden meyerhof pathway or pentose phosphate pathway. All sugar assimilation pathways are well understood in the art.
  • a product of these pathways is pyruvate which may be converted to acetyl CoA for example, for entry into the TCA cycle for supply of cellular building blocks such as malate, oxaloacetate, succinate or fumarate.
  • heterotrophs e.g. acetogens or methylotrophs
  • acetogens or methylotrophs are also capable of heterotrophic growth if sugars are supplied.
  • Photoheterotrophs are heterotrophic phototrophs - that is, they are organisms that use light for energy, but cannot use carbon dioxide as their sole carbon source, instead using carbohydrates, fatty acids, and alcohols and so on.
  • Examples of photoheterotrophic organisms include purple non-sulfur bacteria, green non-sulfur bacteria, and heliobacteria.
  • feedstocks when utilised in the present invention may where desired may include or indeed consist of sugars as all or part of the source of carbon and ⁇ or energy.
  • Enzymes suitable for converting the metabolic intermediates to acetaldehyde are discussed in more detail in the Examples below, and in Figure 3.
  • Route 1 proceeds from acetyl CoA through acetate, (a natural product of acetogenic microorganisms), to acetaldehyde via carboxylic acid reductase activity (Activity A), using for example, EC 1.2.7.5 or EC. 1.2.99.6, ATP or ferredoxin driven or EC 1.2.1.30 or EC 1.2.1.3.
  • Route 2 involves direct synthesis of acetaldehyde from acetyl CoA (Activity B) using an aldehyde dehydrogenase (acylating), for example, acetaldehyde dehydrogenase EC 1.2.1.10.
  • Route 3 involves the conversion of pyruvate to acetaldehyde via acetyl CoA (Activity C and B) using enzymes such as EC 1.2.7.1 or EC 1.2.1.51 or EC 1.2.4.1 and EC 1.2.1.10.
  • Route 4 involves the conversion of pyruvate to acetaldehyde, (Activity D) directly via for example, pyruvate decarboxylase (EC 4.1.1.1).
  • Route 5 involves the conversion of acetyl CoA to acetaldehyde via pyruvate (Activity E and D) using enzymes such as EC 1.2.7.1 and EC 4.1.1.1.
  • Route 6 involves the conversion of acetate to acetaldehyde via acetyl CoA (Activity F and B) using enzymes such as EC 6.2.1.1 or EC 2.8.3.8 and EC 1.2.1.10.
  • aldehyde ferredoxin oxidoreductase (EC 1.2.7.5) can be found in many acetogens and other organisms and has been shown to be capable of reducing unactivated carboxylic acids to the corresponding aldehyde (White, H et al. Biol. Chem Hoppe Seler 1991 , 372 (1 1) 999; White, H and Simon, H. Arch. Microbiol, 1992, 158, 81 ; Fraisse. L and Simon, H. Arch. Microbiol. 1988, 150,381 ; Basen et. al. 2014. PNAS, 11 1 (49), 17618). Kopke, M. et al.
  • PNAS,2010, 107, 15305 describes genes capable of reduction of acetate to acetaldehyde in the acetogen Clostridium ljungdahlii.
  • Alternative means of generating acetaldehyde in acetogens for supply to an aldolase which could be used separately or in conjunction with reduction of acetate are as follows:
  • Acetaldehyde dehydrogenase (EC 1.2.1.10) or any aldehyde dehydrogenase capable of converting acetyl CoA to acetaldehyde directly may be used.
  • SLP step from conversion of acetyl CoA to acetate would be lost.
  • Some compensation for this loss could be achieved from ion gradient phosphorylation from the Wood-Ljungdahl pathway when growing on gases such as CO, C0 2 /H 2 or syngas.
  • ATP may also be synthesised via NAD(P) reduction coupled to reduced ferredoxin, but growth on methanol and C0 2 or another more oxidised co substrate may be most suited due to the potential more favourable energetics and potential for supply of reducing equivalents and ATP from methanol dissimilation.
  • CAR carboxylic acid reductase
  • CAR carboxylic acid reductase
  • These enzymes catalyse reduction of carboxylic acids to the corresponding aldehyde via activation with ATP.
  • the energetics of this route would be similar to that described for acetaldehyde synthesis from acetyl CoA via acetaldehyde dehydrogenase.
  • the use of a carboxylic acid reductase in a 1 ,3- butanediol pathway for synthesis of a corresponding aldehyde is described in US8268607, albeit that the pathway for synthesis is different.
  • the modified organisms of the invention may be engineered to target (down- regulate, knockout or inhibit) the activity of enzymes which may otherwise direct the flux of intermediates in the 3-hydroxybutanal pathway to other products or biomass.
  • Methods of targeting genes in this way are known in the art, and also discussed below.
  • acetogens if the bioenergetics allow loss of ATP synthesis from acetyl CoA conversion to acetate, acetate accumulation can be reduced by targeting of phosphotransacetylase (pta) or acetate kinase (ack) genes. This can enhance the level of acetyl CoA, which can be utilised directly or via pyruvate. Thus where utilising Route 2, it may be desired to target EC 2.3.1.8 (phosphotransacetylase) or EC 2.7.2.1 (acetate kinase) or both.
  • pta phosphotransacetylase
  • ack acetate kinase
  • LDH activity EC 1.1.1.27 or 1.1.1.37; the latter is malate dehydrogenase but is known to accept pyruvate as a substrate
  • target pyruvate formate lyase EC 2.3.1.54
  • the purpose is to prevent or minimise loss of pyruvate to other products.
  • an alcohol dehydrogenase which utilises acetaldehyde as a substrate for some other purpose e.g. production of ethanol.
  • acetaldehyde a substrate for some other purpose e.g. production of ethanol.
  • Increasing the availability to the aldolase of the acetaldehyde increases production of the 3-hydroxybutanal from the aldolase.
  • any alcohol dehydrogenase with a preference for reduction of acetaldehyde to ethanol relative to reduction of 3-hydroxybutanal to 1 ,3-BDO.
  • These acetaldehyde to ethanol enzymes are generally classified in EC 1.1.1.1.
  • Acetaldehyde derived from acetyl CoA or pyruvate is used to supply the substrate for a DERA type aldolase (deoxyribose phosphate aldolase, EC 4.1.2.4, DERA or 'DERA like' enzyme) capable of the coupling of two molecules of acetaldehyde to form 3-hydroxybutanal (via "Reaction G” in Figure 3).
  • DERA type aldolase deoxyribose phosphate aldolase, EC 4.1.2.4, DERA or 'DERA like' enzyme
  • Example enzymes are given in Table 6.
  • the natural deoxyribose phosphate aldolase (DERA) reaction is:
  • the phosphorylated substrate is preferred but most wild type enzymes will catalyse the coupling of two non-phosphorylated aldehyde molecules. Primarily acetaldehyde and another aldehyde.
  • An example of a DERA which accepts phosphorylated and non-phosphorylated substrates with approximately equal preference is described by Zhong-Yu, Y. et al. (J. Ind. Microbiol Biotech. 2013, 40, 29).
  • DERAs are known to be inactivated at aldehyde concentrations above 100mM and may be sensitive to concentrations below this concentration, for both acetaldehyde and 3-hydroxybutanal and this has been a limitation for application of DERA for synthesis of statin intermediates via sequential coupling of chloroacetaldehyde and two molecules of acetaldehyde (Green Chemistry in the Pharmaceutical industry, 2010, John Wiley and sons).
  • DERA is used as part of an unnatural pathway for synthesis of 1 ,3- butanediol and other valuable chemicals where the substrate acetaldehyde is provided via de novo synthesis from a preceding pathway enzyme.
  • aldehyde concentrations in the processes of the invention will never approach 100mM, and sensitivity to this concentration of acetaldehyde is therefore immaterial.
  • Both acetaldehyde and 3-hydroxybutanal are intermediates in the pathway and accumulation of these intermediates will be avoided by ensuring adequate activity of pathway enzymes to maximise carbon flux to 1 ,3-butanediol or other target chemicals.
  • Wild type DERA aldolase has been overexpressed in E.coli and run as a high intensity process for synthesis of chiral lactol intermediates for the statin pharmaceuticals (Oslaj, M. et al Plos one, 8 (5), 1).
  • the process involves a fed batch approach involving the coupling of a 2-substituted acetaldehyde and acetaldehyde to the corresponding lactols in a tandem sequential synthesis.
  • this process was run as a whole cell system, the reactants were fed to the cells and were not generated in situ. Furthermore there were no modifications made which would have enhanced production or availability of endogenous acetaldehyde from central metabolic intermediates.
  • the processes of the present invention do not utilise batch feeding of the microbial organisms with 2-substituted acetaldehyde and/or acetaldehyde.
  • Naturally-occurring DERA enzymes are not optimised for industrial applications of coupling or condensation of two molecules of acetaldehyde. Therefore, there is a need for catalytic improvement to increase the activity of these enzymes and to fully realise their industrial potential.
  • An aspect of the present invention relates to DERA variants, or 'DERA type' enzyme variants, having improved activity for catalysing the coupling of two molecules of acetaldehyde to form 3-hydroxybutanal which is then released from the active site.
  • the invention relates to engineering a DERA enzyme, with a goal of converting two molecules of acetaldehyde to crotonaldehyde via an aldol condensation. This is achieved by the dehydration of 3-hydroxybutanal within the enzyme active site via ⁇ -elimination of a molecule of water such that crotonaldehyde rather than 3-hydroxybutanal is released.
  • the engineered aldolase may release 3-hydroxybutanal from the active site and then rebind it to carry out the dehydration step to crotonaldehyde.
  • the engineered aldolase may bind 3- hydroxybutanal produced from a separate enzyme and then carry out the dehydration to form crotonaldehyde which is released from the active site.
  • DERA enzymes are known to be capable of coupling two molecules of acetaldehyde, but there are no reported aldolase enzymes capable of crotonaldehyde synthesis from acetaldehyde directly, or via binding or rebinding and dehydration of 3-hydroxybutanal.
  • the desire is to improve the operation of the enzyme at acetaldehyde concentrations relevant for in vivo application.
  • the present invention provides a modified 2-deoxyribose phosphate aldolase (DERA) enzyme variant, comprising one or more mutations that improve the aldolase catalytic performance for synthesis of 3- hydroxybutanal or crotonaldehyde, relative to the parent DERA enzyme from which the variant originates and which does not comprise such a modification.
  • a 'DERA type' enzyme may be any enzyme capable of the coupling or condensation of acetaldehyde to 3-hydroxybutanal or crotonaldehyde respectively.
  • DERA enzymes are reported as the only type of aldolase capable of utilising aldehydes as both a donor and acceptor in an aldol coupling. For the purpose of this invention, any enzyme capable of utilising an aldehyde as both donor and acceptor in an aldol coupling is considered a DERA.
  • the parent DERA enzyme may be a wild-type enzyme or it may be a derivative of the wild-type, which has itself been modified, for example including other modifications described herein.
  • the DERA variant that is modified according to this aspect of the invention shows improved aldolase catalytic performance for synthesis of 3-hydroxybutanal or crotonaldehyde compared with the equivalent aldolase catalytic performance of the parent enzyme.
  • the present inventors have developed a consensus sequence, which can be used to facilitate appropriate engineering of any available DERA enzyme. It is desirable to be able to locate residues equivalent to those within regions of interest that may influence the catalytic activity within any DERA sequence, or to identify any additional regions of interest that may influence the catalytic activity within any DERA sequence.
  • the present invention provides a unique tool by which this can be achieved.
  • a consensus sequence is the calculated order of most frequent amino acid residues found at each position in a sequence alignment. It can be used to represent in a concise manner the "average" sequence of a population. In this way it serves as a tool against which any other amino acid sequence (e.g. that obtained from the translation of a nucleotide sequence) can be compared in order to identify homology and similarity. This is crucial when identifying residues to target for substitution by mutagenesis of the relevant position in the underlying gene when presented with a polypeptide sequence of unknown origin.
  • the method by which the consensus of DERA amino acid sequences was created for this invention is described in Example 15.
  • the new consensus sequence captures regions that are highly conserved in all DERA enzymes known to date. It can be reasonably assumed that this level of conservation is directly related to efficient processing of the natural substrates and is strongly biased towards non-preferable acceptance of a molecule of acetaldehyde in the acceptor binding site.
  • Acetaldehyde is the natural donor for the aldol coupling with the natural acceptor glyceraldehyde-3-phosphate.
  • one important aspect is to improve the ability for acetaldehyde to act as a both an efficient donor (already sufficiently present in wild type enzymes) and an efficient acceptor (required by evolution) in an aldol coupling.
  • the new consensus sequence is the following sequence, or a variant thereof:
  • Variants of the sequences disclosed herein preferably share at least 55%, 56%, 57%, 58%, 59%, 60%, 65%, or 70% identity, most preferably at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% identity. Such variants may be referred to herein as "substantially similar”.
  • a DERA variant according to the invention comprises a polypeptide sequence which when aligned to a consensus sequence having at least 80% sequence identity with the sequence of SEQ I D NO: 1 shows an alignment with amino acid residues at 9 or more of 15 defined positions in the consensus sequence.
  • At least one of said one or more mutations are mutations of residues that are at equivalent positions to one or more of the amino acid residues at 9 or more of the 15 defined positions in the consensus sequence.
  • residues at equivalent positions to those of the consensus sequence are identified by alignment of the parent DERA polypeptide sequence with the consensus sequence.
  • Example 16 below provides details of how any query sequence (e.g. the parent DERA sequence) can be aligned with the consensus sequence to identify residues that are at equivalent positions within the two sequences.
  • the one or more mutations in the DERA polypeptide sequence are at residues equivalent to positions T8, L10, C35, V57, F60, D86, K147, G151 , K176, S178, G179 G180, G199, A200 and/or S201 of the consensus sequence, wherein equivalent positions are determined by alignment to the consensus sequence.
  • the one or more mutations are not at residues equivalent to positions D86, K147 and K176 of the consensus sequence. These amino acids have been demonstrated to be critical for the catalytic activity. They are essential for forming a Schiff base as a catalytic functionality, which enables binding of the substrate and performing the aldol coupling and/or condensation reactions. Mutations in these catalytic essential amino acids will ultimately lead to inactivation of the enzyme and are therefore not suitable as target for enzyme engineering.
  • the modification is substitution of one amino acid residue for another.
  • a DERA variant according to the invention may comprise one or more of these modifications: If the one or more mutations is at a residue equivalent to position 8, the modification introduces a hydrophobic residue.
  • the modification introduces a positively or negatively charged residue, or introduces a hydrophobic residue.
  • the DERA variant is used to produce 3- hydroxybutanal then a modification at a residue equivalent to position 10 introduces a hydrophobic residue.
  • a modification at a residue equivalent to position 10 introduces a positively or negatively charged residue.
  • the modification introduces a hydrophobic residue.
  • the modification introduces a hydrophobic residue.
  • the modification introduces a hydrophobic residue.
  • the modification introduces a hydrophobic or negatively charged, .
  • the modification introduces a positively or negatively charged residue, or introduces a hydrophobic residue.
  • the DERA variant is used to produce 3- hydroxybutanal then a modification at a residue equivalent to position 178 introduces a hydrophobic residue.
  • a modification at a residue equivalent to position 178 introduces a positively or negatively charged residue.
  • the modification introduces a negatively charged or a hydrophobicresidue.
  • the modification introduces a negatively charged, or a hydrophobicresidue.
  • the modification introduces a positively or negatively charged residue, or hydrophobic residue
  • the modification introduces a negatively charged, or a hydrophobic residue.
  • the modification introduces a negatively charged, or a hydrophobic residue.
  • Table 9 (also below) provides a preferred sub-set of the modifications detailed in Table 8.
  • the one or more mutations include mutations of amino acid residues that are at positions that are not equivalent to any of said 15 defined positions in the consensus sequence. In one embodiment, none of said mutations are mutations of amino acid residues that are at positions that are equivalent to any of said 15 defined positions in the consensus sequence.
  • one or more mutations improve coordination of a substrate carbonyl group. Alternatively or additionally, it is preferred that one or more mutations improve coordination of a substrate methyl group. Alternatively or additionally, it is preferred that one or more mutations reduce the coordination of a substrate phosphate group. Alternatively or additionally, it is preferred that one or more mutations increase the negative charge in the active site. Alternatively or additionally, it is preferred that one or more mutations increase the hydrophobicity in the active site.
  • DERA variants according to this aspect of the invention can be introduced into any microbial organism described herein, and can be used as a substitute for ay other aldolse enzymes described herein.
  • a separate aspect of this invention provides an isolated polypeptide comprising a polypeptide sequence which when aligned to a consensus sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 shows an alignment with amino acid residues at 9 or more of 15 defined positions in the consensus sequence.
  • said 15 defined positions in the consensus sequence are positions 8, 10, 35, 57, 60, 86, 147, 151 , 176, 178, 179 180, 199, 200 and 201.
  • the polypeptide sequence comprises one or more of the following residues at positions that are equivalent to one or more of positions 8, 10, 35, 57, 60, 151 , 178, 179 180, 199, 200 and/or 201 of the consensus sequence, wherein equivalent positions are determined by alignment to the consensus sequence:
  • hydrophobic residue selected from any of
  • hydrophobic residue selected from any of L, I, F, or A,
  • hydrophobic residue selected from any of
  • hydrophobic residue selected from any of A, , L, I, V, F, or W, or negatively charged D.
  • D a hydrophobic residue selected from any of A, L, I, V, F, or W.
  • the present invention also relates to the use of an isolated polypeptide as defined above to improve the aldolase catalytic performance for synthesis of 3- hydroxybutanal or crotonaldehyde of a 2-deoxyribose phosphate aldolase (DERA) enzyme.
  • DUA 2-deoxyribose phosphate aldolase
  • the present invention also provides an isolated polynucleotide sequence encoding said polypeptide sequences.
  • the present invention also provides an expression system comprising the isolated polynucleotide sequence of the invention, operably linked to suitable control sequences.
  • the present invention also provides a recombinant microorganism transformed with said expression system.
  • a "region of interest” is defined as any single amino acid or group of amino acids that when substituted impart an efficiency influence with respect to the coupling or condensation of two molecules of acetaldehyde to form either 3-hydroxybutanal or crotonaldehyde.
  • amino acids in these regions of interests may not promote efficient acetaldehyde acceptor binding, and hence may not promote sufficient orientation with respect to reaction with the Schiff base enzyme bound, natural acetaldehyde donor molecule.
  • acetaldehyde can be a substrate in this acceptor position, it is at lower efficiency than that required for commercial application of the enzyme, where the commercial application is for the coupling and/ or condensation of two molecules of acetaldehyde.
  • a third example region of interest ( Figure 18 - hotspot C) is known to be important for binding of the phosphate group of the natural substrate acceptor molecule glyceraldehyde-3-phosphate or the natural substrate 2-deoxyribose-5-phosphate (depending on the direction of the reaction).
  • Amino acid changes in this area may be additionally useful to suppress natural substrate binding where these natural molecules are prevalent (e.g growth on sugar based feedstocks). Changes may also promote further 3-hydroxybutanal/crotonaldehyde synthesis efficiency For example the insertion of bulky amino acids may suppress the coupling of three acetaldehyde molecules that produces the undesirable product 2,4,6 trideoxy-D- erythrohexapyranoside.
  • Some DERA enzymes are known to carry out this so called double aldol reaction, where 3-hydroxybutanal may return to the active site and may couple with another donor acetaldehyde molecule. Further, 3-hydroxybutanal may remain within the active site prior to a further acetaldehyde coupling. Contrary to this disclosure, promoting the double aldol reaction has been the target for DERA evolution over the past 20 years, for application to the synthesis of statin side chains.
  • Regions of interest within enzyme active sites may simply be regions of specific properties.
  • trypsin has a negatively charged pocket able to bind positively charged residues such as arginine.
  • the hydrophobic pocket of chymotrypsin attracts hydrophobic residues such as tyrosine, tryptophan, and phenylalanine. Therefore, creating a predominantly hydrophobic environment within the DERA active site may improve acetaldehyde binding within the acceptor site. Further, reducing the size of the active site to favour binding of a small molecule such as acetaldehyde may impart further benefit.
  • Reducing the size of the active site may also serve to reduce a DERA's potential to carry out the undesired double aldol reaction where a second molecule of acetaldehyde may react with a bound C4 aldol product such as 3-hydroxybutanal.
  • Figure 18 - Hotspot D consists of the single amino acid Leu20 in the example E. coli sequence (position 10 of SEQ ID NO: 1), which is a position in correct proximity to catalyse (if required) a deprotonation step to facilitate the ⁇ -elimination of a molecule of water from enzyme bound 3- hydroxybutanal to form crotonaldehyde.
  • Amino acid residues occupying the described example regions of interest are typically within 4A distance of bound 3-hydroxybutanal as a Schiff-base in the enzyme's catalytic site and any amino acid change introduced into these regions may be expected to influence the enzyme's ability to bind its substrates and thus influence catalytic activity. Therefore, to increase the catalytic activity of the acetaldehyde coupling reaction to 3-hydroxybutanal, whether or not further dehydration takes place, amino acids in these regions may be targeted. Targeting amino acids outside of these regions of interest, including those outside the active site may give further additional improvement in DERA activity.
  • the identified example regions of interest were selected according to their functionality in the target acetaldehyde as acceptor substrate binding (Figure. 18).
  • Amino acids residues in the first example region ( Figure 18 - Hotspot A, T18, C47, V73 and F76 in the E. coli DERA sequence) have the positional capability to interact with the methyl group of the incoming acceptor acetaldehyde.
  • Residues in the second example region ( Figure 18 - Hotspot B A203 and G236 in the E. coli DERA sequence) are positioned to interact with the carbonyl group of the acceptor acetaldehyde.
  • an aldolase capable of binding acetaldehyde efficiently in both the donor and acceptor sites.
  • orientation of the carbonyl group of acetaldehyde is facilitated by a histidine and a tyrosine residue in these natural acetaldehyde acceptor enzymes.
  • residues act in a bi-functional way by using electrostatic attractions to the carbonyl group of the acceptor acetaldehyde molecule to coordinate the acetaldehyde molecule in the catalytically active orientation and position.
  • these amino acids also act as an acid base catalyst by providing a proton for the carbonyl group during aldol coupling. The carbonyl group is thereby converted to an alcohol function.
  • Example amino acids influencing the ability of DERA to synthesise the undesired by product 2,4,6 trideoxy-D-erythrohexapyranoside and deoxyribose-5-phosphate It may be additionally desirable to avoid competition with the natural acceptor substrate glyceraldehyde-3-phosphate, which ultimately could lead to formation of the natural DERA product deoxyribose-5-phosphate particularly in processes relying on a sugar feedstock.
  • mutations in the region of interest labelled Hotspot C in Figure 18 may be desirable.
  • DERA enzymes which may significantly carry out the double aldol coupling where 3-hydroxybutanal may return to the active site or remain within it, to couple with an additional molecule of acetaldehyde, alterations in this region are expected to influence this potential and prevent the enzyme facilitated synthesis of this 2,4,6 trideoxy-D- erythrohexapyranoside by product.
  • mutations in other positions aligning with the consensus sequence may offer further advantage.
  • the present invention provides either a DERA variant which when characterized under identical conditions is capable of the coupling of two molecules of acetaldehyde to form 3-hydroxybutanai with an improved activity over the activity of the DERA enzyme from which it is derived, or a DERA variant which when characterised is capable of the condensation of two molecules of acetaldehyde to form crotonaldeyde either directly and/or via binding or rebinding of 3- hydroxybutanai. Characterisation of the enzyme may be achieved by assessment of the enzyme's performance relative to the wild type or to an enzyme from which it is derived, either within a metabolic pathway in vitro or in vivo, or as an isolated protein.
  • An improved enzyme variant or an enzyme variant capable of catalysing a reaction with increased activity is defined as an enzyme variant which differs from the wild type enzyme or an enzyme from which if is derived and which catalyses the respective coupling or condensation reaction to form 3-hydroxbutanai or crotonaldehyde so that the specific activity of the enzyme variant is higher than the specific activity of the wild type enzyme for at least one given concentration of the substrate acetaldehyde, (preferably any acetaldehyde concentration higher than 0 M and up to 1 M).
  • a specific activity may be defined as the number of moles of substrate converted to moles of product by unit of time by mole of enzyme or by weight of enzyme.
  • the power of using a rational and defined multifactorial design is two-fold. Firstly, by defining the library at the design stage, there is no need to oversample a pool of unknown mutants to ensure total library coverage - use of resources is minimised as only those mutations that are necessary are generated.
  • mutant generation in this library is based on random mutagenesis, it would be necessary to oversample and generate 1030 samples to ensure a 95% coverage of the experimental space (i.e. 1030 variants to be statistically certain of generating 95% of all variants in the library). For a defined rational library, it is possible to simply generate 343 mutants.
  • aldolase enzymes reported to be capable of crotonaldehyde synthesis from acetaldehyde directly, or via binding or rebinding and dehydration of of 3-hydroxybutanal.
  • a separate aspect of the present invention provides a modified 2-deoxyribose phosphate aldolase (DERA) enzyme variant comprising mutations that increase the ability of the enzyme to perform dehydration of 3-hydroxybutanal to form crotonaldehyde, relative to the activity of the parent DERA enzyme from which the variant originates and which does not comprise such modifications.
  • DERA 2-deoxyribose phosphate aldolase
  • a DERA variant according to this aspect of the invention comprises a polypeptide sequence which, when aligned to a consensus sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 1 , shows an alignment with amino acid residues at 9 or more of 15 defined positions in the consensus sequence, and wherein said mutations are substitutions of residues at a position equivalent to positions 10, 178 and 199 in the consensus sequence, wherein at positions 10, 178 and 199 there is a H or a negatively charged residue selected from D or E.
  • Hotspot B in Figure 18 may be mutated to facilitate the aldol condensation reaction to crotonaldehyde.
  • these residues may be mutated to a combination of Histidine and Glutamate or Aspartate.
  • Glutamate or Aspartate are able to act as acid catalysts to protonate the hydroxyl group of the bound substrate, thus converting it to a good leaving group in the ⁇ -elimination reaction.
  • Histidine as previously described for natural acetaldehyde acceptor aldolases, is able to orientate the acetaldehyde acceptor molecule to the catalytic active position.
  • Example region of interest contains Leu20 in the E. coli sequence ( Figure 18 - Hotspot D). This amino acid is in close proximity to the ⁇ -hydrogen in the 3- hydroxybutanal Schiff-base complex. By introducing an amino acid side chain in the position of Leu20 that is capable of acting as a base, abstraction of the ⁇ -hydrogen would be expected which would assist the dehydration and formation of crotonaldehyde bound to the Schiff-base complex. Crotonaldehyde would then be released from the active site.
  • the present invention also provides a novel consensus sequence (SEQ ID NO: 1) which captures regions that are highly conserved in all DERA enzymes known to date.
  • the novel consensus sequence serves as a tool against which any other amino acid sequence can be compared in order to identify homology and similarity.
  • the consensus sequence therefore enables the identification of the example regions of interest as described above in any query sequence which aligns at any position in the consensus sequence (single amino acid or group of amino acids) and which imparts an efficiency influence with respect to the coupling or condensation of two molecules of acetaldehyde to form either 3- hydroxybutanal or crotonaldehyde.
  • the consensus sequence may be used to identify other regions of interest outside of the given examples which when mutated enhance the coupling of two molecules of acetaldehyde or the dehydration of the product thereof relative to the parent DERA enzyme that does not contain the modifications.
  • a further aspect of the invention provides a non-naturally occurring polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO. 1.
  • said has at least 90%, prefereably 95% identity to SEQ ID NO: 1.
  • the non-naturally occurring polypeptide may preferably comprise the amino acid sequence of SEQ ID NO. 1 , or alternatively, it may consist of the amino acid sequence of SEQ ID NO. 1.
  • the present invention also relates to the use of the non-naturally occurring polypeptide of the invention to identifying regions within a DERA enzyme that can be mutated to influence the catalytic activity of the DERA enzyme, wherein said catalytic activity is the coupling or condensation of two molecules of acetaldehyde to form 3-hydroxybutanal and/or crotonaldehyde. Examples of such use are provided herein.
  • a separate aspect of the invention provides a method for identifying one or more residues within a DERA polypeptide sequence that can influence catalytic activity of the DERA enzyme, comprising:
  • said catalytic activity is: coupling of two aldehyde molecules to produce 3-hydroxybutanal; and/ or dehydration of 3- hydroxybutanal to crotonaldehyde.
  • step (b) comprises identifying residues within the DERA sequence that are in alignment with residues at 9 or more of the 15 positions within the consensus sequence which include: 8, 10, 35, 57, 60, 86, 147, 151 , 176, 178, 179 180, 199, 200 and 201. If the DERA sequence comprises at least 9 residues at positions equivalent to 8, 10, 35, 57, 60, 86, 147, 151 , 176, 178, 179 180, 199, 200 and/or 201 of the consensus sequence, it can be concluded that the DERA sequence can be modified to improve the catalytic activity of the enzyme, relative to the parent DERA enzyme.
  • a further aspect of the invention provides a method of identifying regions within a DERA enzyme that can be mutated to influence the coupling or condensation of two molecules of acetaldehyde to form 3-hydroxybutanal and/or crotonaldehyde, comprising aligning the polypeptide sequence of the DERA enzyme with a consensus sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 , and identifying regions in the DERA enzyme sequence that align with known regions within the consensus sequence.
  • the known regions within the consensus sequence comprise one or more of the following residues: 10, 35, 57, 60, 86, 147, 151 , 176, 178, 179 180, 199, 200 and/or 201.
  • a further aspect of the invention provides a method of increasing the catalytic activity of a DERA enzyme, comprising:
  • said regions are single amino acids or groups of amino acids that can be substituted to influence the coupling or condensation of two molecules of acetaldehyde to form either 3-hydroxybutanal or crotonaldehyde.
  • said mutation of one more or amino acid resides results in increased synthesis of 3-hydroxybutanal and/or synthesis of crotonaldehyde.
  • a further related aspect of the invention is directed to the use of a polypeptide having at least 80% sequence identity with the sequence of SEQ ID NO: 1 in the method for identifying regions a DERA enzyme that can be mutated to influence the coupling or condensation of two molecules of acetaldehyde to form 3- hydroxybutanal and/or crotonaldehyde or in the method for increasing the catalytic activity of a DERA enzyme according to the present invention.
  • a further aspect of the invention provides a method of increasing the catalytic activity of a DERA enzyme, comprising:
  • a further aspect of the invention provides a method of increasing the catalytic activity of a DERA enzyme, comprising: (i) aligning the polypeptide sequence of the DERA enzyme with a consensus sequence having at least 80% identity to the amino acid sequence of SEQ ID NO. 1 ,
  • mutations are substitutions of residues at a position equivalent to positions 10, 178 and 199 in the consensus sequence, wherein at positions 10, 178 and 199 there is a H or a negatively charged residue selected from D or E, and
  • a further aspect of the invention provides an assay for screening for DERA variants having improved aldolase catalytic performance for the synthesis of 3- hydroxybutanal or downstream products thereof, comprising determining an increase in the rate of H 2 O 2 formation from the oxidation of 3-hydroxybutanal or downstream products thereof in the presence of an alcohol oxidase.
  • the rate of H 2 O 2 formation is compared to a control value.
  • the downstream product is 1 ,3-butanediol formed by the selective reduction of 3-hydroxybutanal.
  • selective reduction is performed by introducing a heterologous enzyme with alcohol dehydrogenase or aldehyde reductase activity which shows a preference for reduction of 3-hydroxybutanal over acetaldehyde.
  • the heterologous enzyme is selected from Table 7, or is a variant of an enzyme from Table 7, preferably wherein the heterologous enzyme is GOX 1615, GRE2, or BdhB, or a variant thereof.
  • the assay is performed in a microbial host, preferably wherein the host is E.coli.
  • the assay may be performed in a cell lysate or may be performed in vitro using recombinant proteins. Fusions
  • the aldolase such as DERA may be provided as a fusion protein encoding also one or more other enzymes involved in the provision of the aldolase substrate acetaldehyde, or linked to such other enzymes using chemical or other means (e.g. scaffoldins or dockerins).
  • Examples include fusions of DERA with an acetaldehyde dehydrogenase or pyruvate decarboxylase or a carboxylic acid reductase such as AOR which catalyse reactions B, D and A described herein (see Tables 2, 4, 1).
  • Example 11 demonstrates the production of a DERA-EutE fusion.
  • the aldolase such as DERA may be provided as a fusion protein encoding also one or more other enzymes involved in a downstream product pathway, or linked to such other enzymes using chemical or other menas (e.g. scaffoldins or dockerins).
  • the enzyme may, for example, be one involved in the conversion of 3-hydroxybutanal to another product or intermediate. Examples include enzymes listed in Table 7.
  • This reaction is preferably catalysed by a medium chain alcohol dehydrogenase or aldehyde reductase, ideally which shows preference for alcohols of C4 or greater, for example see Appl. Environ. Microbiol, 2000, 66, 5231. More specifically the enzyme preferably shows a preference for reduction of 3-hydroxybutanal to 1 ,3- BDO relative to reduction of acetaldehyde to ethanol (Example 9). An alcohol dehydrogenase described by Wales, M and Fewson, C. Microbiol 1994, 140, 173 again shows preference for longer chain alcohols. Although measured in the oxidative direction, the dehydrogenase also accepts 1 ,4-butanediol as a substrate.
  • 2,3- butanediol is not a substrate, clearly demonstrating the desired primary alcohol as opposed to secondary alcohol specificity for application to 3-hydroxybutanal reduction.
  • Other enzymes which it may be desired to utilise for conversion of 3- Hydroxybutanal to 1 ,3-butanediol are described in Example 3 and Table 7 hereinafter.
  • 1 ,3-BDO has numerous utilities in industry.
  • 1 ,3-BDO is commonly used as an organic solvent for food flavoring agents. It is also used as a co- monomer for polyurethane and polyester resins and is widely employed as a hypoglycaemic agent.
  • Optically active 1 ,3-BDO is a useful starting material for the synthesis of biologically active compounds and liquid crystals.
  • Another use of 1 ,3- butanediol is that its dehydration affords 1 ,3-butadiene and other important chemicals such as methylethyl ketone (lchikawa et al., J.
  • 1 ,3-butadiene is an important chemical used to manufacture synthetic rubbers (e.g. tyres), latex, and resins.
  • synthetic rubbers e.g. tyres
  • latex e.g. tyres
  • resins e.g. tyres
  • 1 ,3-Butadiene and further examples of products produced by chemical conversion of 1 ,3-butanediol are shown in Figure 1.
  • 3-hydroxybutanal can also be directed to products other than 1 ,3-BDO.
  • 3-hydroxybutanal can be considered a branch point for a number of possible unnatural DERA-based pathways leading to a variety of immediate or further downstream products.
  • 3-Hydroxybutanal can be converted (e.g. oxidised, reduced) to:
  • 3-hydroxybutyrate has utility as a biodegradable plastics monomer.
  • 3-hydroxybutanal can be also be converted to metabolic intermediates such as 3-hydroxybutyryl CoA using for example butanal dehydrogenase (EC 1.2.1.57) or another aldehyde dehydrogenase such as EC1.2.1.10 which can allow metabolic access to a range of other products.
  • Aldehyde dehydrogenases have been mutated to improve their preference for C4 aldehydes relative to C2 aldehydes (e.g. acetaldehyde).
  • Baker et al. describe a mutant with a preference for butanal relative to acetaldehyde, Biochemistry. 2012 Jun 5; 51 (22):4558-67. Epub 2012 May 21.
  • This enzyme may have utility in the conversion of 3-hydroxybutanal to 3-hydroxybutytyl CoA.
  • Several other enzymes have a natural preference for a C4 aldehyde. Yan, R.T and Chen, J. S. 1990 Appl Environ Microbiol 56, (9) 2591. Any of these enzymes could, if desired, be further engineered to optimise their activity in generating 3-hydroxybutyryl CoA in the context of the present invention.
  • Downstream products from 3-hydroxybutyryl CoA include:
  • Crotyl alcohol (which can be converted enzymatically or chemically to 1 ,3- butadiene).
  • Butanol (which can be converted enzymatically or chemically to 1 ,3-butadiene)Other downstream products include Crotonic acid, butyrate, 3-hydroxybutyrate, 3- hydroxybutylamine, Polyhydroxybutyrate, Acetone, and isopropanol.
  • a route to 3-hydroxybutyryl CoA via acetate in acetogens allows for generation of this intermediate without sacrificing the ATP energy which would otherwise be lost if 3-hydroxybutyryl CoA was provided via, for example, acetyl CoA to acetoacetyl CoA. This is because preventing acetate formation from acetyl CoA loses the molecule of ATP generated from the acetate kinase reaction. Generation of acetoacetyl CoA is energetically unfavourable under most conditions.
  • the pathway to 3-hydroxybutyryl CoA via acetate through the DERA pathway described herein retains the energetics of acetogenesis. Hence, the same product is reached through a more energetically favourable route.
  • the key intermediate branch point is the DERA product 3-hydroxybutanal.
  • a non-naturally occurring microorganism of the invention can have one, two, three, or more, up to all nucleic acids encoding the enzymes or proteins constituting a 3-hydroxybutanal or downstream product derived therefrom (e.g.1 ,3-BDO) pathway revealed herein.
  • the non-naturally occurring microorganisms can also include other genetic modifications that facilitate or optimise 1 ,3-BDO (or other downstream product derived from 3-hydroxybutanal) biosynthesis or that confer other useful functions onto the host microorganism.
  • yeasts such as Saccharomyces cerevisiae, Kluveromyces lactis Candida boidinii, Pichia angusta, Ogataea polymorpha, Komagataella pastoris.
  • bacteria such as Moorella thermoacetica, Moorella thermoautotrophica, Thermoacetogenium phaeum, Thermoanaerobacter kivu, Acetobacterium woodii, Clostridium carboxidivorans, Clostridium drakei, Clostridium formicoaceticum, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium aceticum, Clostridium autoethanogenum, Clostridium scatologenes, Acetitomaculum ruminis, Acetogenium kivui, Eubacterium limosum, Oxobacter pfennigii, Acetobacterium tundrae, Acetobacterium noterae, Acetobacterium carbinolicum, Acetobacterium dehalogenans, Acetobacterium fimetarium, Acetobacterium
  • Hydrogenibacillus schlegelii Lactococcus. sp. Lactobacillus.sp., Bacillus sp. Geobacillus sp. Corynebacterium. sp. Klebsiella, oxytoca, Ralstonia. sp., Alcaligenes. sp. Cupriavidus. sp.
  • the host is not E. coli.
  • Sources of encoding nucleic acids for use in the present invention can include any species where the encoded gene product is capable of catalysing the referenced reaction.
  • Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal and mammal, including human.
  • Exemplary sources of nucleic acids are described herein. However, with the large number of complete genome sequences available, the identification of genes encoding the requisite 1 ,3-BDO biosynthetic activity (e.g.
  • the aldolase-type enzymes described herein for one or more genes in related or distant species, including for example, homologs, orthologs, paralogs and non-orthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known to those skilled in the art, and can be carried out in the present context in the light of the teaching herein. Consequently, in the light of the present disclosure, the metabolic modifications enabling biosynthesis of 3-hydroxybutanal or downstream product derived therefrom (e.g.1 ,3-BDO) described herein with reference to a particular organism such as Moorella thermoacetica can be readily applied to other microorganisms. Those skilled in the art will know that a metabolic modification exemplified in one organism can be applied equally to other organisms.
  • Non-limiting examples of variants include the following:
  • Novel, naturally occurring, nucleic acids, isolatable using the recited or referred to sequence may include alleles (which will include polymorphisms or mutations at one or more bases), paralogues, isogenes, or other homologous genes belonging to the same families as the relevant enzymes. Also included are orthologues or homologues from different microbial or other species.
  • nucleic acid molecules which encode amino acid sequences which are homologues of the genes referred to herein. Homology may be at the nucleotide sequence and/or amino acid sequence level, as discussed below. A homologue from a different species or strain encodes a product which causes a phenotype similar to that caused by the recited sequence.
  • Artificial nucleic acids which can be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence referred to herein.
  • Changes may be desirable for a number of reasons. For instance they may introduce or remove restriction endonuclease sites or alter codon usage. Alternatively changes to a sequence may produce a derivative by way of one or more (e.g. several) of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more (e.g. several) amino acids in the encoded polypeptide. Other desirable mutations may be random or site directed mutagenesis in order to alter or evolve the activity (e.g. specificity) or stability of the encoded polypeptide. Changes may be by way of conservative variation, i.e.
  • altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.
  • variants having non-conservative substitutions are also included.
  • variant' nucleic acid as used herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expression product of the variant nucleic acid.
  • Sequence identity may be assessed as using BLASTp (proteins) or Megablast (nucleic acids) from NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) using default settings, as used in the Examples.
  • Variants of the sequences disclosed herein preferably share at least 55%, 56%, 57%, 58%, 59%, 60%, 65%, or 70%, or 80% identity, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identity. Such variants may be referred to herein as “substantially homologous”.
  • Nucleic acid fragments may encode particular functional parts of the enzyme (i.e. encoding a biological activity of it).
  • the present invention provides for the production and use of fragments of the full-length polypeptides disclosed herein, especially active portions thereof.
  • An "active portion" of a polypeptide means a peptide which is less than said full length polypeptide, but which retains its essential biological activity.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • a "vector” as used herein need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce nucleic acid into cells for recombination into the genome.
  • the nucleic acid in the vector will typically be under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a microbial host cell. It may include a native promoter. In the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA).
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • the promoter is an inducible promoter.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is “switched on” or increased in response to an applied stimulus.
  • the nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.
  • the present disclosure teaches how pathways may be engineered into an organism by selection of the appropriate enzymes, cloning their corresponding genes into a production host, optimising the stability and expression of these genes, attenuation or functional deletion of the competitive pathways, optimising fermentation conditions for the genetically engineered strain to produce the desired product, and assaying for product formation following fermentation.
  • heterologous is used broadly herein to indicate that the gene/sequence of nucleotides in question (e.g. encoding an aldolase) has been introduced into a host cell or an ancestor thereof, using genetic engineering, i.e. by human intervention. Nucleic acid heterologous to a host cell will be non-naturally occurring in cells of that type, variety or species.
  • heterologous nucleic acid may comprise a coding sequence of or derived from a microorganism, placed within a different microorganism.
  • a nucleic acid sequence may be placed within a cell in which it or a homologue is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
  • Transformed in this context means that the nucleotide sequences of the heterologous nucleic acid alter one or more of the cell's characteristics and hence phenotype e.g. with respect to 3-hydroxybutanal or downstream product derived therefrom (e.g.1 ,3-BDO).
  • Nucleic acid when used in the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed. Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin, and double or single stranded. Where used herein, the term “isolated” encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic.
  • nucleic acids may comprise, consist, or consist essentially of, any of the sequences discussed hereinafter.
  • any shuttle vectors available for Gram-positive bacteria that carry at least one nucleotide sequence homologous to one gene encoding the desired enzyme can be employed for transformation of M. thermoacetica or other microorganism of interest.
  • An expression plasmid is obtained by inserting at least a gene responsible for replication of the plasmid in Gram-positive and more specifically in Clostridia species or acetogens.
  • the plasmid capable of introducing the desired gene into an acetogen is not particularly limited as long as it contains at least a gene responsible for replication and amplification in acetogenic bacteria. Specific examples thereof include pAK201 (Kim, A. and Blashek, H. P., Appl. Environ. Microbiol. 55 (2): 360- 365 (1988), pHB101 (Blaschek H. P. et. al, J. Bacterial.
  • any of the series modular plasmids pMTL8000 Heap, J.T. et al., J. Microbiol. Methods 78:79-85 (2009), pMS1 , pMS2, pMS3, pMS4, pKV12 (Staetz, M. et al, Appl. Environ. Microbiol. 1033-1037 (1994), pUB110 (McKenzie et al., 1984), plMP1 (Mermelstein, L et al. 1992), pITF (Dong, H. et al. 2010).
  • pMTL80000 Kopke, M. et al., Appl. Environ. Microbiol. 3394-3403, 2014
  • pMTL80000 Kopke, M. et al., Appl. Environ. Microbiol. 3394-3403, 2014
  • Novel shuttle vectors which are chimeras of pUB1 10 or any of the above mentioned plasmids and a general E. coli cloning vectors such as pUC19 (Yanisch-Perron, C. et al., Gene 33:103-1 19 (1985)) or pBluescript II SK (+/-) can be easily generated and tested.
  • E. coli cloning vectors such as pUC19 (Yanisch-Perron, C. et al., Gene 33:103-1 19 (1985)) or pBluescript II SK (+/-) can be easily generated and tested.
  • These chimera plasmids are propagated in E. coli for plasmid isolation and employed for the genetic engineering work of M. thermoacetica or another acetogen or Gram-positive bacteria which is naturally sensitive towards the antibiotic gene expressed by the plasmid.
  • sub-cloning can be employed to replace the antibiotic resistance cassettes on the existing plasmids with suitable ones based on the antibiotic sensitivity of the target organism.
  • Standard techniques for DNA amplification using a high-fidelity DNA polymerase and molecular sub- cloning, including restriction enzyme digestion, ligation and E. coli transformation can be used for engineering of the plasmids (Sambrook, 1989).
  • kanamycin and chloramphenicol may be utilised as antibiotic markers for selection of the genetic engineered M. thermoacetica strains.
  • the operon or one gene of the operon encoding the required activity can be ligated into the multiple cloning site between two convenient restriction sites.
  • heterologous genes can be codon optimised for the target organism with techniques well known to those skilled in the art.
  • an N-or C-terminus tag sequence can be added to the gene sequences cloned as understood by those skilled in the art.
  • methylation of the transformable DNA protects it from being degraded by the host.
  • In vivo methylation of the transformable DNA is achieved by its propagation in methylation E. coli strains such as Top10 (pAN2) (Kuit et al., Appl. Microbiol. Biotechnol. 94:729-741 (2012)).
  • Heterologous (or exogenous, the terms are used interchangeably) gene(s) can be introduced into the chosen host cell, exemplified herein by M.
  • thermoacetica and Acetobacterium woodii using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection and electrofusion.
  • electroporation and conjugation published protocols of Clostridium perfringens, Clostridum. acetobutylicum, Clostridium, cellulolyticum and Acetobacterium woodii may be used.
  • target metabolic intermediates and ⁇ or 1 ,3-BDO or divert metabolic pathways away from biomass generation.
  • An example is to minimise loss of pyruvate away from a 3-hydroxybutanal pathway.
  • a plasmid can be constructed for gene deletion by integrational mutagenesis or gene replacement techniques well known in the art. Integrational mutagenesis and gene replacement can selectively inactivate undesired genes from host genomes. Such methods have been developed and successfully used to create metabolically engineered mutants of Clostridial strains (Green et al., 1996). In this technique, a fragment of the target gene is cloned into a non-replicative vector with a selection marker, resulting in the non-replicative integrational plasmid.
  • the partial gene in the non-replicative plasmid can recombine with the internal homologous region of the original target gene in the parental chromosome (double crossover), which results in the insertional inactivation of the target gene, Idh locus in this particular example.
  • double crossover results in the insertional inactivation of the target gene, Idh locus in this particular example.
  • the use of gene replacement is preferred to insertional inactivation (single recombination) since it permits the generation of more stable engineered strains, without the need to maintain selection of vectors.
  • An example describing a double crossover in an acetogen is shown in Example 5. Using this technique, in the same manner non-natural microorganisms can be generated having complete or partial deletion of one, two, three, four, five, or more genes in order to remove competitive pathways.
  • Reduction of expression of the target genes can also be used as an alternative to gene disruption. This may be achieved using expression of antisense RNA for the target gene, which will inhibit but not completely abolish gene expression.
  • the antisense RNA system serves as a convenient approach of gene knock-down of a desired gene with the advantage that it can reduce expression of genes for which complete inactivation could be damaging or lethal to the organism.
  • a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et a/,(1988) Nature 334, 724-726.
  • the complete sequence corresponding to the coding sequence need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence.
  • the sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14- 23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.
  • sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence in the terms described above.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable.
  • Clostridium acetobutylicim (Desai R. et al. Appl. Environ & Eviron Microbiol. 65(3):936-945 (1999)) Fierro-Monti IP et. ai, J Bacteriol. 174(23) :7642-7647 (1992)) and Clostridium cellulolyticum (Perret S, et ai, Mol. Microbiol. 51 (2):599-607 (2004)) as well as for termophiles such as Thermus thermophilus (Moreno, R. et ai., J. Bacteriol., 7804-7806(2004) and may be applied herein.
  • Clostridium acetobutylicim (Desai R. et al. Appl. Environ & Eviron Microbiol. 65(3):936-945 (1999)) Fierro-Monti IP et. ai, J Bacteriol.
  • An attractive approach for down-regulation expression of a target gene is to replace the native promoter with a less active promoter for example one from another gene. This can be achieved by double-recombination/gene replacement techniques well known in the art. Alternatively, expression can be reduced by altering the ribosome binding site or the spacing between the RBS and the translation initiation start codon, or using a less efficient start codon.
  • Figures Figure 1 Example of chemical transformation of 1 ,3-butanediol into industrially important chemicals including butadiene and methylethyl ketone. Ichikawa et al., J. Molecular Catalysis A- Chemical, 256: 106-112 (2006) Figure 2. Shows the Wood Ljungdahl pathway for synthesis of 3 acetyl CoA (3 acetate), from gaseous carbon sources with or without methanol, showing the entry point for methanol. Associated equations are: 4CH 3 OH + 2C0 2 ⁇ 3CH 3 COOH; 12CO + 6H 2 0 3CH 3 COOH + 6C0 2 ; 12H 2 + 6C0 2 ⁇ 3CH 3 COOH + 6H 2 0.
  • the Wood Ljungdahl pathway can also fix C0 2 derived from the glycolytic pathway (pyruvate decarboxylation) using reducing equivalents derived from glycolysis and pyruvate decarboxylation.
  • Figure 3 Shows metabolic pathways (route 1 , 2, 3, 4, 5 and 6) for the synthesis of 1 ,3-butanediol from the central metabolic intermediates acetyl CoA or pyruvate, via the common intermediate acetaldehyde. Enzyme activities required to catalyse these steps are listed as Activity A, B, C, D, E, F, G, and H. Example gene sequences coding for these activities can be found in Tables 1 , 2, 3, 4, 5, 6, and 7.
  • Route 1 proceeds from acetyl CoA through acetate (a natural product of acetogenic microorganisms) to acetaldehyde via carboxylic acid reductase activity, for example, EC 1.2.7.5 or EC. 1.2.99.6, ATP or ferredoxin driven or EC 1.2.1.30 or EC 1.2.1.3.
  • carboxylic acid reductase activity for example, EC 1.2.7.5 or EC. 1.2.99.6, ATP or ferredoxin driven or EC 1.2.1.30 or EC 1.2.1.3.
  • Route 2 involves direct synthesis of acetaldehyde from acetyl CoA using an aldehyde dehydrogenase (acylating), for example, acetaldehyde dehydrogenase EC 1.2.1.10.
  • aldehyde dehydrogenase acylating
  • Route 3 involves the conversion of pyruvate to acetaldehyde via acetyl CoA using enzymes such as EC 1.2.7.1 or EC 1.2.1.51 or EC 1.2.4.1 and EC 1.2.1.10.
  • Route 4 involves the conversion of pyruvate to acetaldehyde, directly via pyruvate decarboxylase (EC 4.1.1.1).
  • Route 5 involves the conversion of acetyl CoA to acetaldehyde via pyruvate using enzymes such as EC 1.2.7.1 and EC 4.1.1.1.
  • Route 6 involves the conversion of acetate to acetaldehyde via acetyl CoA using enzymes such as EC 6.2.1.1 or EC 2.8.3.8 and EC 1.2.1.10.
  • Two molecules of acetaldehyde are coupled to form 3-hydroxybutanal using an aldolase capable of accepting an aldehyde as both the acceptor and donor in an aldol coupling, for example, deoxyribose phosphate aldolase (DERA, EC 4.1.2.4).
  • DEA deoxyribose phosphate aldolase
  • 3- Hydroxybutanal is reduced to 1 ,3-butanediol by an alcohol dehydrogenase or aldehyde reductase, for example, using enzymes categorised in EC 1.1.1.1 , EC 1.1.1.2, EC 1.1.1.72 or EC 1.1.1.265 or EC 1.1.1.283.
  • FIG 4. Shows the RuMP pathway and its association with the TCA cycle (modified from Appl. Environ Microbiol. 2003 69, 3986).
  • Pyruvate is the primary product of the RuMP pathway which is converted to acetyl CoA prior to entry to the TCA cycle.
  • Either pyruvate or acetyl CoA can be converted directly to the common intermediate acetaldehyde thereby supplying substrate for a DERA type aldolase capable of accepting acetaldehyde as both the donor and acceptor in an aldol coupling for synthesis of 1 ,3-butanediol.
  • Figure 5. Shows the Wood Ljungdahl pathway.
  • Either pyruvate or acetyl CoA can be converted directly to the common intermediate acetaldehyde thereby supplying substrate for a DERA type aldolase capable of accepting acetaldehyde as both the donor and acceptor in an aldol coupling for synthesis of 1 ,3-butanediol. Modified from Fung Min Liew, Michael Kopke and Sean Dennis Simpson (2013). Gas Fermentation for Commercial Biofuels Production, Liquid, Gaseous and Solid Biofuels - Conversion Techniques, Prof. Zhen Fang (Ed.), ISBN: 978-953-51-1050- 7, InTech, DOI: 10.5772/52164.
  • Acetate derived from acetyl CoA can also be directly reduced to acetaldehyde for supply to the aldolase.
  • Figure 6. Shows the reverse TCA cycle. Either pyruvate or acetyl CoA can be converted directly to the common intermediate acetaldehyde thereby supplying substrate for a DERA type aldolase capable of accepting acetaldehyde as both the donor and acceptor in an aldol coupling for synthesis of 1 ,3-butanediol. Modified from Mar. Drugs. 201 1 , 9, 719.
  • Figure 7. Shows the serine cycle.
  • Acetyl CoA can be converted directly to the common intermediate acetaldehyde supplying substrate for a DERA type aldolase capable of accepting acetaldehyde as both the donor and acceptor in an aldol coupling for synthesis of 1 ,3-butanediol.
  • Central metabolism also converts PEP (phosphoenol pyruvate) into pyruvate which can be decarboxylated to acetaldehyde as described previously.
  • FIG. 8 Shows the coupling of acetaldehyde catalysed by deoxyribose phosphate aldolase (DERA).
  • DEA deoxyribose phosphate aldolase
  • Figure 9 Shows the Cavin cycle linked to sugar synthesis (or utilisation) and or conversion to pyruvate or acetyl CoA directly. Either pyruvate or acetyl CoA can be converted directly to the common intermediate acetaldehyde thereby supplying substrate for a DERA type aldolase capable of accepting acetaldehyde as both the donor and acceptor in an aldol coupling for synthesis of 1 ,3-butanediol.
  • Figure 10 Shows the Cavin cycle linked to sugar synthesis (or utilisation) and or conversion to pyruvate or acetyl CoA directly. Either pyruvate or acetyl CoA can be converted directly to the common intermediate acetaldehyde thereby supplying substrate for a DERA type aldolase capable of accepting acetaldehyde as both the donor and acceptor in an aldol coupling for synthesis of 1 ,3-butanediol.
  • Figure 10
  • A1 Colony 1 of A woodii carrying plasmid pEP55
  • A2 Colony 2 of A woodii carrying plasmid pEP55
  • Figure 1 Cloning strategy to construct an A. woodii LDH knockout mutant by replacing the LDH gene with an Erythromycin resistance marker.
  • Figure 12. Cloning strategy to construct an A. woodii LDH knockout mutant by disrupting the LDH gene via single cross-over recombination event and integration of the complete plasmid.
  • FIG. 13 Growth of A. woodii wildtype and A. woodii mutants in the presence of 20 mM Fructose and 40 mM DL-Lactate.
  • Aw A. woodii wildtype
  • Plasmid A. woodii transformant harboring plasmid pUC19-Ery-pAIV ⁇ 1
  • dLDH double crossover LDH knockout.
  • SR Single cross-over LDH knockout.
  • FIG. 14 Utilization of Fructose and Acetate production by A. woodii wildtype and A. woodii mutants.
  • Aw A. woodii wild type
  • P A. woodii transformant harboring plasmid pUC19-Ery- ⁇
  • dLDH double cross-over LDH knockout
  • SR Single cross-over LDH knockout.
  • Figure 15 Utilization of Lactate and Acetate production A. woodii wildtype and A. woodii mutants.
  • Aw A. woodii wild type
  • P A. woodii transformant harboring plasmid pUC19-Ery- ⁇
  • dLDH double cross-over LDH knockout
  • SR Single cross-over LDH knockout.
  • FIG. 18 E.coli DERA with amino acids within 4A of the lysine 137 3- hydroxybutanal Schiff-base complex. Residues in positions particularly influencing binding of the acetaldehyde as acceptor aldehyde are clustered in example hotspots A and B.
  • Example hotspot C is involved with binding the phosphate group of the natural substrate.
  • Example hotspots B and D are regions able to influence formation of crotonaldehyde by the dehydration of 3-hydroxybutanal.
  • Figure 19 Alignment of the consensus sequence (SEQ ID N01) with amino acid sequence of E. coli DERA (SEQ ID NO.2) as a query.
  • FIG. 20 Alignment of the consensus sequence (SEQ ID N01) with amino acid sequence of Homo sapiens DERA (SEQ ID NO.3) as a query. 12 Amino acid residues that are found in Hotspots A, B, C and D and serve as target sites for mutation are highlighted in green on the consensus sequence (top). By performing a pairwise alignment, all those amino acids in the H. sapiens DERA sequence (bottom) aligning to those highlighted in green on the consensus (i.e. T57, L59, C100, V125, F129, G179, A213, G214, G215, G246, A247 and S248 of the H. sapiens DERA) are those which should be targeted in order to improve the H. sapiens DERA's ability to perform the aldol coupling and condensation. Amino acid positions (e.g. T57) are counted from the N terminal ignoring gaps (-).
  • FIG. 21 Alignment of the consensus sequence (SEQ ID N01) with amino acid sequence of Plasmodium falciparum DERA (SEQ ID NO.4) as a query. 12 Amino acid residues that are found in hotspots A, B, C and D and serve as target sites for mutation are highlighted in green on the consensus sequence (top). By performing a pairwise alignment, all those amino acids in the P. falciparum DERA sequence (bottom) aligning to those highlighted in green on the consensus (i.e. T20, L22, C49, V76, F79, G175, A212, G213, G214, G245, A246 and S247 of the P. falciparum DERA) are those which should be targeted in order to improve the P.
  • T20, L22, C49, V76, F79, G175, A212, G213, G214, G245, A246 and S247 of the P. falciparum DERA are those which should be targeted in order to improve the P.
  • A8, L10, C35, V56, F59, G150, A190, G191 , G192, G218, T219 and S220 of the P. aerophilum DERA) are those which should be targeted in order to improve the P. aerophilum DERA's ability to perform the aldol coupling or condensation.
  • Amino acid positions e.g. T20 are counted from the N terminal ignoring gaps (-).
  • FIG. 23 Alignment of the consensus sequence (SEQ ID N0.1) with amino acid sequence of Geobacillus thermoglucosidasius.
  • DERA SEQ ID NO.6 as a query. 12 Amino acid residues that are found in hotspots A, B, C and D and serve as target sites for mutation are highlighted in green on the consensus sequence (top). By performing a pairwise alignment, all those amino acids in the Geobacillus. DERA sequence (bottom) aligning to those highlighted in green on the consensus (i.e.
  • T12, L14, C39, V62, F65, G158, S185, G186, G187, G206, T207 and S208 of the Geobacillus DERA are those which should be targeted in order to improve the Geobacillus DERA's ability to perform the aldol coupling or condensation.
  • Amino acid positions e.g. T20 are counted from the N terminal ignoring gaps (-).
  • FIG. 24 Alignment of the consensus sequence (SEQ ID N0.1) with amino acid sequence of Acetobacterium woodii DERA (SEQ ID NO.7) as a query. 12 Amino acid residues that are found in hotspots A, B, C and D and serve as target sites for mutation are highlighted in green on the consensus sequence (top). By performing a pairwise alignment, all those amino acids in the A. woodii DERA sequence (bottom) aligning to those highlighted in green on the consensus (i.e. T13, L15, C40, V62, F65, G157, A184, G185, G186, G205, T206 and S207 of the A. woodii DERA) are those which should be targeted in order to improve the A. woodii DERA's ability to perform the aldol coupling or condensation. Amino acid positions (e.g. T20) are counted from the N terminal ignoring gaps (-).
  • Figure 25 shows the DERA consensus sequence.
  • Figure 26 shows a schematic of the screen for identifying DERA variants with improved production of 3-hydroxybutanal.
  • Metabolic engineering steps required to generate a 1 ,3-butanediol production strain will depend on whether pyruvate or acetyl CoA or both are selected as the source of acetaldehyde. Subsequent conversion of acetaldehyde is common to all routes. For example, for Route 1 , acetaldehyde is derived from acetyl CoA via acetate. Acetate is a natural acetogen product which can accumulate to 10s grams per litre. For example 44g/l was obtained from the acetogen Acetobacterium woodii growing on C0 2 and H 2 (Demlar, M. et al. Biotech. Bioeng. 201 1 , 108, 470).
  • Direct conversion of acetyl CoA to acetaldehyde using acetaldehyde dehydrogenase can operate in the absence of acetate accumulation (Route 2) or alongside acetate accumulation where flux is directed to acetaldehyde directly or via acetate.
  • the route chosen may be influenced by the energetics requirement of organism which can be related to the feedstock provided. It is most preferable to convert a primary central metabolic intermediate to acetaldehyde directly.
  • acetate accumulation can be prevented in an acetogen by knockout of one or more phosphotransacetylase (pta) or acetate kinase (ack) genes (Example 6 and 8). Furthermore, acetate accumulation may be prevented by natural regulation, or by mutation which directs flux away from acetate synthesis while maintaining Wood Ljungdahl pathway activity. For example growth of the acetogen Moorella thermoacetica (renamed from C.
  • thermoaceticum on CO and methanol in the presence of nitrate led to no acetate accumulation due to repression of key Wood Ljungdahl related gene expression (Seifritz, C. et al. J. Bacteriol. 1993, 175, 8008). In that example, sufficient ATP appeared to be provided from nitrate respiration.
  • Acetyl CoA can also be converted to acetaldehyde via pyruvate (Route 5) using pyruvate synthase (EC 1.2.7.1 , Table 3).
  • pyruvate synthase EC 1.2.7.1 , Table 3
  • pyruvate is the primary central metabolic intermediate
  • Route 4 the natural metabolic route prior to entry to the TCA cycle
  • Table 3 the gene sequence examples shown in Table 3.
  • acetaldehyde it is desirable that the maximum amount of acetaldehyde be converted to 3- hydroxybutanal via an overexpressed endogenous or heterologous DERA (example sequences are shown in Table 6).
  • loss to oxidation or reduction products should be avoided by knockout of undesired genes, for example, short chain alcohol dehydrogenases highly active on acetaldehyde, or non-acetylating acetaldehyde dehydrogenase (e.g. EC 1.2.1.5).
  • Reduction of 3- hydroxybutanal is achieved by overexpression of an endogenous, or introduction of a heterologous alcohol dehydrogenase or aldehyde reductase which shows preference for C4 aldehydes (3-hydroxybutanal) relative to C2 aldehydes (acetaldehyde) e.g Example 9.
  • a heterologous alcohol dehydrogenase or aldehyde reductase which shows preference for C4 aldehydes (3-hydroxybutanal) relative to C2 aldehydes (acetaldehyde) e.g Example 9.
  • Example 7 The introduction of a heterologous gene into an acetogen is described in Example 7, this method can be cross applied to the introduction of any heterologous gene, for example, a gene within a 1 ,3-butanediol pathway.
  • the overall conversion of pyruvate to 1 ,3-butanediol is accomplished in 3 or 4 steps depending on the route taken ( Figure 3) and in 1 or 2 steps to the common pathway intermediate acetaldehyde.
  • the two steps from acetaldehyde to 1 ,3-butanediol are common to all 1 ,3,- butanediol synthetic routes.
  • Acetogens naturally produce acetate in high yield from sugars, or C1 feedstocks (syngas, C0 2 /H 2 , C0 2 and methanol) via conversion of acetyl CoA derived from the Wood Ljungdahl pathway. Yields are typically approximately 80% of theoretical or greater, for example, A.E. Bainotti et al., 1988. Journal of fermentation and bioengineering, 85(2), 223-229. Although it is anticipated that even higher yields may be achievable, for example, via modification of the Wood Ljungdahl pathway which converts C0 2 , H 2 , CO, or methanol to acetyl CoA or via optimisation of the growth medium.
  • Acetate can be reduced to acetaldehyde using a carboxylic acid reductase enzyme.
  • carboxylic acid reductase enzyme mainly uses either reduced ferredoxin (aldehyde ferredoxin oxidoreductase) or ATP to drive the thermodynamically unfavourable reduction of a carboxylic acid moiety and tend to be classified in EC 1.2.7.5, EC 1.2.1.30, EC 1.2.99.6. or EC 1.2.1.3.
  • carboxylic acid reductase and aldehyde oxidoreductase are used interchangeably in the literature.
  • Aldehyde dehydrogenase is also used to describe enzymes capable of carboxylic acid reduction.
  • the npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme.
  • PPTase phosphopantetheine transferase
  • the natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates as small as lactic acid (Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical and Biotechnology Industries, ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, Fla. (2006)). Activity towards acetate was not discussed. However, high activity towards lactate suggests that the enzyme is capable of accepting molecules containing as few as three carbons.
  • this enzyme may potentially be used for acetate reduction in either its native form or as an evolved enzyme.
  • a further well studied enzyme is the example from Mycobacterium marinum which has a wild type substrate preference for C6 to C18 acids (Kalim Akhtar, M. et al. PNAS, 2013, 110, 87).
  • Enzymes capable of carboxylic acid reduction may be evolved or mutated as described above to increase activity towards acetate using enzyme evolution techniques common in the art.
  • the griC and griD genes from Streptomyces also code for a carboxylic acid reductase with diverse capability for acid reduction Suzuki et al. 2007. J. Antibiot. 60 (6) 380.
  • Aldehyde ferredoxin oxidoreductase enzymes use ferredoxin not ATP to drive the carboxylate reduction and are present in many acetogens and other organisms (White, H et al. Biol. Chem Hoppe Seler 1991 , 372 (11) 999; White, H and Simon, H. Arch. Microbiol, 1992, 158, 81 ; Fraisse. L and Simon, H. Arch. Microbiol. 1988, 150,381 ; (Basen et. al. 2014. PNAS, 11 1 (49), 17618 ).
  • the carboxylic acid reducing enzyme from Moorella thermoacetica has been purified and characterised .White, H. et al. Eur.
  • Example genes for acetate reduction are shown in Table 1.
  • the aldehyde oxidoreductase (AOR) genes CLJU_20110 and CLJU_20210 from Clostridium ljungdahlii are reported to reduce acetate to acetaldehyde, Kopke, M. et al. PNAS, 2010, 107, 15305. Hence, demonstrating the activity of a wild type enzyme towards the target reduction.
  • Various authors have also described conditions under which AOR enzymes are induced in ethanologenic acetogens for synthesis of ethanol from acetate via acetaldehyde, (Mock et al.
  • aldehyde ferredoxin oxidoreductase A further source of aldehyde ferredoxin oxidoreductase are the hyperthermophiles, Thermococcus sp. (Kesen, J.H. J. Bacteriol. 1995, 177, 4757 and Pyrococcus sp. (Basen et. al. 2014. PNAS, 111 (49), 17618 where this enzyme has been used to effectively synthesise ethanol from acetate via acetataldehyde driven by carbon monoxide . Although described mainly for oxidation of aldehydes to the corresponding acids, reduction of acetate is also mentioned.
  • aldehyde ferredoxin oxidoreductase in the aldehyde oxidation direction is further described by Kletzin, A., et al. J. Bacteriol. 1995,177, 4817.
  • An aldehyde dehydrogenase (aldH) from E.coli has been shown to reduce 3- hydroxypropionic acid to the corresponding aldehyde as well as the preferred oxidation of 3-hydroxpropionaldehyde, Ji-Eun, J. et al., Appl. Microbiol. Biotechnol 2008. 81 , 51.
  • This enzyme was also shown to oxidise acetaldehyde to acetate. Hence, as these authors have shown the enzyme to be reversible, activity towards reduction of acetate would be expected.
  • aldehyde ljungdahlii ferredoxin strain ATCC oxidoreductase 55383 / DSM
  • ferredoxin strain ATCC oxidoreductase (EC 51850 / DSM 1.2.7.5) 5473 / JCM
  • Acetaldehyde can be synthesised from acetyl CoA via the reversible enzyme acetaldehyde dehydrogenase EC 1.2.1.10.
  • the gene coding for this enzyme can be found in a wide range of different organisms such as: Acinetobacter sp.; Burkholderia xenovorans; E. coli; Clostridium beijerinckii, (Run-Tao, Y and Jiann-Shin, C. 1990, Appl. Environ. Microbiol. 56, 2591 ; Appl. Environ Microbiol, 1999, 65 (11) 4973); Clostridium kluyveri; Pseudomonas sp.
  • acetogens also have annotated acetaldehyde dehydrogenase genes e.g. Moorella thermoacetica (Moth_1776). Acetobacteri urn wood ii (Arch. Microbiol, 1992, 158, 132). Clostridium ljungdahlii CLJU_c11960.
  • the eutE gene from the eut operon also encodes for an acetaldehyde dehydrogenase.
  • the eutE gene from Salmonella enterica has been cloned into E.coli and shown to efficiently produce acetaldehyde from growth on glucose via acetyl CoA reduction (Huilin, Z. et al. 201 1. Appl. Environ. Microbiol. 77, 6441).
  • 1 ,3-Butanediol production using eutE to deliver acetaldehyde to DERA from acetyl CoA in a 1 ,3-BDO pathway is shown in Example 10.
  • H6LJM8 1 1871 155 1.1.1.1 ; adhE Bifunctional Acetobacterium
  • acetaldehyde (Activity B) can be identified based on sequence homology to those
  • the conversion of pyruvate to acetyl CoA can be carried out using an enzyme such
  • ferredoxin linked enzymes are particularly common in anaerobes such as the
  • acetogens but are also present in other aerobic or facultatively anaerobic
  • the pyruvate dehydrogenase complex is also a central metabolic enzyme well understood in the art which is responsible for conversion of pyruvate (for example, generated from glycolysis) to acetyl CoA for entry into the TCA cycle.
  • pyruvate for example, generated from glycolysis
  • acetyl CoA for entry into the TCA cycle.
  • the subsequent conversion of acetyl CoA to acetaldehyde is described in Route 2.
  • CSIM 742 (EC 1.2.7.1) jejuni subsp. jejuni
  • thermophilus 8773721 oxidoreductase thermophilus
  • thermophilus 8773666 oxidoreductase thermophilus
  • MTBMA_c031 subunit PorA (EC er marburgensis 40 1.2.7.1) (Pyruvate (strain DSM 2133 / oxidoreductase alpha 14651 / NBRC chain) (POR) 100331 / OCM 82 / (Pyruvic-ferredoxin Marburg) oxidoreductase (Methanobacterium subunit alpha) thermoautotrophicu m)
  • MTBMA_c031 subunit PorB (EC er marburgensis 30 1.2.7.1) (Pyruvate (strain DSM 2133 / oxidoreductase beta 14651 / NBRC chain) (POR) 100331 / OCM 82 / (Pyruvic-ferredoxin Marburg) oxidoreductase (Methanobacterium subunit beta) thermoautotrophicu m)
  • MTBMA_c031 subunit PorC (EC er marburgensis 60 1.2.7.1) (Pyruvate (strain DSM 2133 / oxidoreductase 14651 / NBRC gamma chain) (POR) 100331 / OCM 82 / (Pyruvic-ferredoxin Marburg) oxidoreductase (Methanobacterium subunit gamma) thermoautotrophicu m)
  • thermoacetica gamma subunit (strain ATCC 1.2.7.1) 39073)
  • thermoacetica gamma subunit (strain ATCC 1.2.7.1) 39073)
  • TM_0015 subunit PorC (EC maritima (strain
  • Additional genes coding for enzymes capable of the conversion of pyruvate to acetyl CoA can be identified based on sequence homology to those examples in Table 3, or to common sequences for the pyruvate dehydrogenase complex.
  • Pyruvate decarboxylase is a homotetrameric enzyme (EC 4.1.1.1) that catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide in the cytoplasm of prokaryotes, and in the mitochondria of eukaryotes. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase. Under anaerobic conditions, this enzyme is part of the fermentation process that occurs in yeast, especially of the Saccharomyces genus, to produce ethanol by fermentation.
  • Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide.
  • Examples 12, 13 and 14 show the production of 1 ,3-butanediol using pyruvate decarboxylase to deliver acetaldehyde to DERA from pyruvate, in a novel, unnatural
  • PDC11 PDC1 Candida albicans (strain CaO19.10395 Pyruvate SC5314 / ATCC MYA-
  • Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 /
  • Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 /
  • Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-
  • Neosartorya fumigata strain ATCC MYA-4609 / Af293 / CBS 101355 / pdcA Pyruvate FGSC A1100
  • OSJNBa0052E20 decarboxylase 1 Oryza sativa subsp.
  • PDC1 PDC (PDC) Zea mays (Maize)
  • the conversion of acetate to acetyl CoA can be achieved using acetyl CoA synthetase or a CoA transferase for example, EC 6.2.1.1 or EC 2.8.3.8 and subsequently converted to acetaldehyde via EC 1.2.1.10 (Route 2.).
  • acetyl CoA synthetase or a CoA transferase for example, EC 6.2.1.1 or EC 2.8.3.8 and subsequently converted to acetaldehyde via EC 1.2.1.10 (Route 2.).
  • Examples of gene sequences coding for enzymes capable of the conversion of acetate to acetyl CoA are shown in Table 5.
  • ASA_09 synthetase (AcCoA salmonicida
  • CT1652 synthetase (AcCoA tepidum
  • PAE286 synthetase (AcCoA aerophilum
  • CoA Iigase 1 (Acyl- (Sinorhizobiu activating enzyme 1) m meliloti)
  • V5MSC 1770359 U712_10 Putative coenzyme A Bacillus 1 8 415 transferase subunit beta subtilis PY79
  • V5MSQ 1770359 U712_10 Putative coenzyme A Bacillus 3 9 420 transferase subunit subtilis PY79 alpha (EC 2.8.3.8)
  • M7PTY6 G000_0 Acetate CoA- Klebsiella 9783 transferase YdiF (EC pneumoniae
  • RVA1- (Acetate coa- asymbiotica
  • the NIH Genbank® database of publicly available nucleotide sequences may be used to identify genes encoding proteins classified as EC 4.1.2.4.
  • Bacterial genes annotated with EC 4.1.2.4 number 1 137 as of 6th July 2014; by phylum, there are 394 examples in the firmicutes, 387 in proteobacteria, 153 in actinobacteria, 50 in cyanobacteria and 153 in others.

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Abstract

La présente invention concerne un variant d'enzyme 2-désoxyribose phosphate aldolase (DERA) modifié comprenant une ou plusieurs mutations qui améliorent les performances catalytiques de l'aldolase pour la synthèse de 3-hydroxybutanal ou de crotonaldéhyde, par rapport à l'enzyme DERA parente à partir de laquelle la variante provient et qui ne comprend pas une telle modification.
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