Improved means and methods for producing isobutene from 3-methylcrotonic acid

Abstract

A method for producing isobutene from a carbon source is described, comprising: (a) culturing in a liquid culture medium a microorganism capable of producing 3-methylcrotonic acid from a carbon source, thereby producing the 3-methylcrotonic acid such that the 3-methylcrotonic acid accumulates in the liquid culture medium; (b) (i) incubating a microorganism expressing an FMN prenyltransferase and an associated FMN-dependent decarboxylase with the liquid culture medium containing the 3-methylcrotonic acid obtained in step (a); and / or (ii) incubating an FMN prenyltransferase and an associated FMN-dependent decarboxylase with the liquid culture medium containing the 3-methylcrotonic acid obtained in step (a), thereby enzymatically converting the 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene, thereby producing the isobutene; and (c) recovering the isobutene produced.

Description

Technical Field

[0001] The present invention relates to a method for producing isobutene from a carbon source, comprising: (a) culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing the 3-methylcrotonic acid such that the 3-methylcrotonic acid accumulates in the liquid culture medium; (b) (i) incubating a microorganism expressing an FMN-dependent decarboxylase associated with FMN prenyltransferase with the liquid culture medium containing the 3-methylcrotonic acid obtained in step (a); and / or (ii) incubating an FMN-dependent decarboxylase associated with FMN prenyltransferase with the liquid culture medium containing the 3-methylcrotonic acid obtained in step (a), thereby enzymatically converting the 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene; thereby producing the isobutene; and (c) recovering the produced isobutene.

Background Art

[0002] Currently, numerous chemical compounds are obtained from petrochemical products. Alkenes (e.g., ethylene, propylene, various butenes, or pentenes) are used in the plastics industry, for example, to produce polypropylene or polyethylene, as well as in other areas of the chemical and fuel industries. Butylene exists in four forms, one of which, isobutene (also called isobutylene), is part of the composition of methyl tert-butyl ether (MTBE), an anti-knock additive in automotive fuels. Isobutene can also be used to produce isooctene, which can then be reduced to isooctane (2,2,4-trimethylpentane); its extremely high octane rating makes isooctane the best fuel for so-called "gasoline" engines. Alkenes such as isobutene are currently produced by catalytic cracking of petroleum products (or, in the case of hexene, by Fischer-Tropsch derivatives from coal or gas). Production costs are therefore closely related to the price of oil. Furthermore, catalytic cracking can involve significant technical challenges that increase process complexity and production costs.

[0003] The biological pathway production of alkenes such as isobutenes is needed in the context of sustainable production processes in harmony with geochemical cycles. Given the existing fermentation and distillation processes in the food processing industry, the first generation of biofuels focused on the fermentation of ethanol. The production of second-generation biofuels, particularly long-chain alcohols (butanol and pentanol), terpenes, linear alkanes, and fatty acids, is in the experimental stage. Two recent reviews provide a summary of research in this field: Ladygina et al. (Process Biochemistry 41 (2006), 1001) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187).

[0004] The conversion of isovaleric acid to isobutene by the yeast Rhodotorula minuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583)).

[0005] Gogerty et al. (Appl. Environm. Microbial. 76 (2010), 8004-8010) and van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387) describe the production of isobutene from acetoacetyl-CoA by enzymatic conversion, the final step of which is the conversion of 3-hydroxy-3-methylbutyrate (also known as 3-hydroxyisovaleric acid (HIV)) using mevalonate diphosphate decarboxylase. This reaction for producing isobutene from 3-hydroxy-3-methylbutyrate is also described in International Publication No. 2010 / 001078. This describes, generally speaking, methods for producing alkenes through biological processes, in particular methods for producing terminal alkenes (especially propylene, ethylene, 1-butylene, isobutylene, or isoamylene) from 3-hydroxyalkanoate type molecules.

[0006] International Publication No. 2012 / 052427 also describes methods for producing alkenes through biological processes, and in particular, methods for producing alkenes (e.g., propylene, ethylene, 1-butylene, isobutylene, or isoamylene) from 3-hydroxyalkanoate type molecules. In connection with this, a reaction for producing isobutene from 3-hydroxy-3-methylbutyrate is also described in International Publication No. 2012 / 052427.

[0007] International Publication No. 2016 / 042012 describes a method for producing the aforementioned 3-hydroxy-3-methylbutyrate. In particular, International Publication No. 2016 / 042012 describes a method for producing 3-hydroxy-3-methylbutyrate comprising the steps of enzymatically converting 3-methylcrotonyl-CoA to 3-methylcrotonic acid and further enzymatically converting the 3-methylcrotonic acid thus produced to 3-hydroxy-3-methylbutyrate.

[0008] Gogerty et al. (quoted above) and van Leeuwen et al. (quoted above) propose that the production of 3-hydroxy-3-methylbutyrate is achieved by the conversion of 3-methylcrotonyl-CoA via 3-hydroxy-3-methylbutyryl-CoA. To further improve the efficiency and variability of methods for producing isobutene from reusable resources, alternative routes have been developed to provide isobutene and its precursors by providing a method for producing isobutene that involves the enzymatic conversion of 3-methylcrotonic acid (also known as 3-methyl-2-butenic acid, 3,3-dimethylacrylic acid, or senecioic acid) to isobutene.

[0009] In particular, International Publication No. 2017 / 085167 describes a method for producing isobutene, which includes the enzymatic conversion of 3-methylcrotonic acid to isobutene, achieved by using FMN prenyltransferase and associated FMN-dependent decarboxylase, wherein the FMN prenyltransferase catalyzes the prenylation of dimethylallyl phosphate (DMAP)-utilizing flavin cofactors (FMN or FAD) to flavin-derived cofactors, but these enzymes are artificially implemented in a pathway that ultimately leads to isobutene production. Furthermore, International Publication No. 2017 / 085167 describes a method that further includes the step of providing 3-methylcrotonic acid by the enzymatic conversion of 3-methylcrotonyl-CoA to 3-methylcrotonic acid, or (b) providing 3-methylcrotonic acid by the enzymatic conversion of 3-hydroxyisovaleric acid (HIV) to 3-methylcrotonic acid.

[0010] International Publication No. 2017 / 085167 also states that this method, developed to produce isobutene from 3-methylcrotonyl-CoA via 3-methylcrotonic acid, or from 3-hydroxyisovaleric acid (HIV) via 3-methylcrotonic acid, can be incorporated into the isobutene production pathway starting from acetyl-CoA, a key molecule and a central component of metabolism used in many biochemical reactions. The corresponding reaction is schematically shown in Figure 1.

[0011] International Publication No. 2018 / 206262 states that when dimethylallyl pyrophosphate (DMAPP) is used instead of DMAP, 3-methylcrotonic acid is enzymatically converted to isobutene using FMN prenyltransferase and associated FMN-dependent decarboxylase.

[0012] Furthermore, International Publication No. 2018 / 206262 describes the enzymatic conversion of 3-methylcrotonic acid to isobutene, achieved by using FMN prenyltransferase and associated FMN-dependent decarboxylase, in which the FMN prenyltransferase catalyzes the prenylation of flavin cofactors (FMN or FAD) to flavin-derived cofactors using dimethylallyl phosphate (DMAP) and / or dimethylallyl pyrophosphate (DMAPP), as a key step in the entire acetyl-CoA to isobutene metabolic pathway. In this key step, the availability of dimethylallyl phosphate (DMAP) and / or dimethylallyl pyrophosphate (DMAPP) and the availability of flavin cofactor FMN have been found to be limiting factors. However, International Publication No. 2018 / 206262 describes an improved method to ensure efficient biosynthesis of prenylated flavin cofactors (FMN or FAD) by increasing the pool / amount of dimethylallyl phosphate (DMAP) and / or dimethylallyl pyrophosphate (DMAPP).

[0013] Furthermore, International Publication No. 2020 / 188033 is based on the concept of increasing isobutene yield by providing and maintaining a high pool of acetyl-CoA in cells used for isobutene production, the acetyl-CoA pool being kept high by ensuring increased uptake of pantothenic acid by cells and / or increased conversion of pantothenic acid to CoA. Therefore, International Publication No. 2020 / 188033 describes, in particular, recombinant organisms or microorganisms capable of enzymatically converting acetyl-CoA to isobutene, (A) in said organisms or microorganisms: (i) acetyl-CoA is enzymatically converted to acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted to 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted to 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted to 3-methylcrotonyl-CoA, and (v) said 3-methylcrotonyl-CoA is converted to isobutene by: (a) enzymatically converting 3-methylcrotonyl-CoA to 3-methylcrotonic acid (b) 3-methylcrotonyl-CoA is enzymatically converted to 3-hydroxy-3-methylbutyryl-CoA, 3-hydroxy-3-methylbutyryl-CoA is then enzymatically converted to 3-hydroxy-3-methylbutyrate, 3-hydroxy-3-methylbutyrate is then enzymatically converted to 3-phosphonoxy-3-methylbutyrate, 3-phosphonoxy-3-methylbutyrate is then enzymatically converted to isobutene; (b) The recombinant organism or microorganism has an increased pool of coenzyme A (CoA) compared to the organism or microorganism from which it is derived, due to: (i) increased uptake of pantothenic acid; and / or (ii) increased conversion of pantothenic acid to CoA.

[0014] Furthermore, International Publication No. 2014 / 086780 describes a fermentation method for producing a hydrocarbon compound, preferably isobutene, comprising the step of culturing an organism in a liquid fermentation medium, wherein the organism produces a desired hydrocarbon compound by an enzymatic pathway, the enzymatic pathway comprising an intermediate that evaporates into the gas phase, the intermediate is recovered from the gas phase and reintroduced into the liquid fermentation medium.

[0015] Furthermore, International Publication No. 2014 / 086781 describes a process for fermentation production of hydrocarbons, preferably isobutene, wherein hydrocarbon-producing microorganisms are cultured in a liquid fermentation medium of a fermenter, an oxygen-containing inlet gas is supplied to the fermenter, the total pressure of the inlet gas before introduction into the fermenter is about 1.5 bar to about 15 bar (about 150 kPa to about 1500 kPa), the hydrocarbons are obtained in a gaseous state in the fermentation off-gas, and the oxygen concentration in the fermentation off-gas is controlled to be about 10 vol% or less.

[0016] As described above, various approaches have been described in the prior art to produce isobutene by enzymatic conversion in biological systems and fermentation processes / fermenters, thereby enabling the use of reusable resources as raw materials. However, there remains a need for improvements in the efficiency, effectiveness, and safety of such (fermentation) methods, in particular, to improve yield and / or safety and make them more commercially attractive. [Overview of the project]

[0017] The present invention relates to a method for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b)(i) Incubating a microorganism expressing FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and / or (ii) Incubate the FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a). The 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) is enzymatically converted to isobutene: The steps include: producing the isobutene thereafter; (c) A step of recovering the produced isobutene and This requirement is met by providing, in a first embodiment, a method for producing isobutene from a carbon source, characterized by including [a certain element].

[0018] In a second aspect, the present invention relates to a method for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b) A step of thermochemically converting the 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene, preferably at a temperature of 180°C to 400°C; (c) A step of recovering the produced isobutene and The present invention provides a method for producing isobutene from a carbon source, characterized by including [a specific component]. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows an artificial pathway for isobutene production from acetyl-CoA via 3-methylcrotonic acid. Furthermore, the enzymatic recycling of metabolites that may occur during the pathway is shown in steps Xa, Xb, XI, and XII. [Figure 2]Figure showing the main pathway of an artificial pathway for isobutene production from acetyl-CoA via 3-methylcrotonyl-CoA and a possible pathway from 3-methylcrotonyl-CoA to isobutene via 3-methylcrotonic acid. For specific steps, the corresponding enzymes are shown. [Figure 3] Figure showing the process diagram of a large-scale plant. IBN: Isobutene. [Figure 4] Figure schematically showing incubation vessels of "without gas supply" (upper figure) and "gas supply using inlet gas at <0.1 vvm (vessel volume per minute)>" (lower figure), respectively. [Figure 5] Figure showing the composition of exhaust gas over time for N2, CO2 and isobutene (IBN). [Figure 6] Figure showing the consumption rates of isobutene (IBN) and 3-methylcroton. [Figure 7] Figure showing that the total production of IBN and 100% of 3-methylcrotonic acid are converted to isobutene (IBN). [Figure 8] Figures showing that high concentrations of IBN and CO2 are produced during incubations without gas supply and with gas supply at <0.1 vvm (vessel volume per minute), respectively, compared to incubation with gas supply at 1 vvm. [Figure 9] Figure showing the correlation between the temperature and pressure at which isobutene becomes a gas in the container. Above the curve, isobutene is liquid. Below the curve, isobutene is gas.

Mode for Carrying Out the Invention

[0020] As previously intended, a fermentation method for producing isobutene from 3-methylcrotonic acid (sometimes referred to herein as prenate) is based on conventional fermentation methods using microorganisms capable of producing isobutene. Isobutene, produced as a gaseous compound under normal fermentation conditions, is recovered as part of the off-gas of the culture, which is either part of the inlet gas used for ventilation of the culture during cultivation or a mixture of various gases produced during cultivation. Under such conditions, it has been found that the proportion of isobutene in the off-gas of the culture typically reaches only about 3–7 mol%. Therefore, considerable time and effort are required to purify isobutene from the off-gas to the desired purity.

[0021] The inventors have made the remarkable discovery that the amount / percentage of isobutene in the off-gas of a fermentation method involving the biological conversion from 3-methylcrotonic acid to isobutene can be dramatically increased if the enzymatic reactions resulting from the production of 3-methylcrotonic acid and its subsequent enzymatic conversion to isobutene are uncoupled, in the sense that 3-methylcrotonic acid is produced from a carbon source during fermentation and accumulates in the culture medium (and is not directly converted to isobutene), and only after the accumulation of 3-methylcrotonic acid in the culture medium is it first ensured that 3-methylcrotonic acid is subsequently converted to isobutene in a separate reaction. This second step of converting 3-methylcrotonic acid to isobutene can be achieved under simplified conditions and does not require the maintenance of fermentation conditions. In this second step, it is sufficient to contact the enzyme that converts 3-methylcrotonic acid with the accumulated 3-methylcrotonic acid or the cells of a microorganism that produces such an enzyme. It was found that simple incubation of a solution containing accumulated 3-methylcrotonic acid and enzymes / microorganisms resulted in the highly efficient conversion of 3-methylcrotonic acid to isobutene, and a very high proportion of isobutene in the off-gas of the incubation reaction.

[0022] Furthermore, the incubation step in which 3-methylcrotonic acid is converted to isobutene may be advantageous if performed in a vessel with no gas supply or a very low gas supply.

[0023] As shown in the following examples, the claimed method efficiently converts 3-methylcrotonic acid (prenate) to isobutene, and thus results in the efficient production of isobutene. Furthermore, the examples show that when the gas supply is kept low (i.e., <0.1 vvm) or no gas is supplied (0 vvm) during the incubation step used for the conversion of 3-methylcrotonic acid to isobutene, the concentration of the produced isobutene (IBN) is particularly high in the off-gas state. Therefore, it is easier to purify the isobutene to the desired purity.

[0024] As shown in the examples, using low gas supply conditions during the incubation step in which 3-methylcrotonic acid is converted to isobutene results in high concentrations of isobutene and CO2 (produced during the enzymatic decarboxylation of 3-methylcrotonic acid to isobutene). In fact, if the gas supply is kept extremely low during the incubation step, essentially only CO2 and isobutene are produced.

[0025] Whenever 3-methylcrotonic acid is mentioned, it is always referred to in its deprotonated carboxylate ion form (i.e., COO - This refers to both the form (i.e., methyl 3-crotonic acid) and its protonated acidic form (i.e., the COOH form, i.e., methylcrotonic acid). In fact, its form depends on the pH of the solution, and therefore the definition of methylcrotonic acid interchangeably refers to either form, i.e., methyl 3-crotonic acid and methylcrotonic acid.

[0026] When expressed in the form of a salt, i.e., as methyl 3-crotonate, the preferred cation is Na + Ca 2+ Mg 2+ , K + or NH4+ That is the case.

[0027] The method according to the present invention includes, in step (a), culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium.

[0028] The term "culturing," as used in this context, refers to keeping cells in a liquid medium, thereby keeping them healthy and enabling them to produce the enzymes necessary for the production of desired products. Preferably, the term "culturing" also encompasses the growth of microbial cells under conditions that allow for the propagation, proliferation, and cell division of microbial cells, thereby increasing the number of cells in the liquid culture medium. Thus, the term "culturing" refers to maintaining microorganisms under culture conditions that enable cell viability and allow for the occurrence of metabolic processes necessary for the cells, such as converting a carbon source to 3-methylcrotonic acid. Such conditions generally include providing cells with a carbon source in the culture medium, agitating the culture medium, maintaining the temperature of the culture medium at a value that allows the necessary metabolic conversions to occur (and, if necessary, the temperature for microbial growth), and supplying the cultured cells with air or a gas mixture that enables cell viability and metabolic activity.

[0029] Therefore, the term "culture," when used in this context, should be understood as "fermentation," that is, a metabolic process that brings about a chemical change in an organic substrate, preferably through the action of enzymes, and thus refers to the process by which products are synthesized from a growth substrate through the innate or genetically modified metabolism of microorganisms, and achieved by metabolic intermediates. Hence, the term culture enables the development of metabolism in cultured cells, as well as the growth and survival of cultured cells. Furthermore, the culture step requires the presence of a carbon source and an energy source (e.g., in the form of glucose and oxygen) to enable this.

[0030] In general, the culture step (a) of the method according to the present invention can be carried out using culture media and culture conditions that are suitable for the specific microorganism used in step (a), in terms of classical fermentation methods using means (such as fermenters and their equipment) and methods well known in the art.

[0031] The term "microorganisms capable of producing 3-methylcrotonic acid from a carbon source" refers to microorganisms that, when cultured under appropriate conditions, can express enzymes that enable the conversion of each carbon source to 3-methylcrotonic acid, and therefore produce 3-methylcrotonic acid from a carbon source during culture.

[0032] Methods for the enzymatic production of 3-methylcrotonic acid from a carbon source are known in the art, and (recombinant) microorganisms capable of catalyzing each enzymatic conversion have been described. In principle, any microorganism capable of converting a carbon source to 3-methylcrotonic acid can be used in step (a) of the method of the present invention.

[0033] Possible individual steps of enzymatic conversion to 3-methylcrotonic acid starting from a carbon source are described below, but not limited to, examples only. Furthermore, in preferred embodiments, and not limited to, further examples, possible individual steps of enzymatic conversion starting from a carbon source that yield the central metabolite acetyl-CoA and further 3-methylcrotonic acid are described below.

[0034] Furthermore, the term “microorganisms capable of producing 3-methylcrotonic acid from a carbon source” means, in the context of the present invention, that, under the applicable culture conditions, such microorganisms do not further metabolize or convert the produced 3-methylcrotonic acid to other compounds, particularly isobutene, or not to a considerable extent. This term specifically means that, during culture step (a), the microorganisms do not convert the produced 3-methylcrotonic acid to isobutene. This may be based on the fact that the microorganisms used in step (a) of the method according to the present invention do not contain genetic information encoding an enzyme capable of converting 3-methylcrotonic acid to isobutene, and therefore cannot produce an enzyme capable of catalyzing this reaction.

[0035] Alternatively, the microorganism used in step (a) may contain genetic information that encodes an enzyme capable of converting 3-methylcrotonic acid to isobutene, but does not express this enzyme under the culture conditions used in step (a). For example, the gene encoding the corresponding enzyme may be controlled by a promoter whose activity can be regulated and which becomes inactive under the culture conditions used in step (a).

[0036] In a preferred embodiment, the microorganism used in step (a) of the method according to the present invention is a microorganism that does not contain a gene encoding an enzyme capable of converting 3-methylcrotonic acid to isobutene.

[0037] However, it is also possible to use microorganisms that naturally contain genes that, in principle, encode enzymes capable of converting 3-methylcrotonic acid to isobutene, such as FMN prenyltransferase and associated FMN-dependent decarboxylase, but whose activity in converting 3-methylcrotonic acid to isobutene is low, so that substantial conversion does not occur during the culture step (a), in step (a) of the method of the present invention.

[0038] 3-methylcrotonic acid produced by microorganisms during the culture in step (a) of the method according to the present invention accumulates in the culture medium.

[0039] Therefore, the culture in step (a) is performed for a sufficient time to allow the conversion of the carbon source to 3-methylcrotonic acid and its accumulation in the culture medium. Preferably, the culture is performed until the carbon source provided to the culture medium is metabolized by the cells to at least 20%, 30%, 40%, 50%, or 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, or at least 95%, and particularly preferably at least 99%, (i.e., depleted from the culture medium).

[0040] "Accumulate" means that the 3-methylcrotonic acid produced by the microorganisms in step (a) is mainly generated in the culture medium, and that its concentration increases during the culturing in step (a). Preferably, the culturing is carried out until no further increase in the concentration of 3-methylcrotonic acid is observed.

[0041] Furthermore, it is preferable that the culture in step (a) be carried out until a concentration of at least 5 g / l, more preferably at least 10 g / l, even more preferably at least 20 g / l, and even more preferably at least 40 g / l of 3-methylcrotonic acid is achieved in the liquid culture medium.

[0042] In step (a) of the method of the present invention, once the desired accumulation of 3-methylcrotonic acid in the culture medium is achieved, the microbial culture is stopped. Stopping the culture means that the treatment used in the fermentation method to maintain bacterial growth is stopped.

[0043] The culture is preferably stopped when carbon source consumption is low (preferably lower than 0.03 g of carbon source / g per DCW (dry cell weight)) and / or 3-methylcotonic acid production is low (preferably lower than 0.01 g of 3-methylcotonic acid / g per DCW / hour).

[0044] Carbon source consumption and 3-methylcrotonic acid production can be measured, respectively, by methods known in the art, such as HPLC.

[0045] The culture may also be preferably stopped if undesirable byproducts (e.g., organic acids such as acetic acid or lactic acid, or alcohols such as ethanol) begin to accumulate in the culture medium. The accumulation of byproducts in the culture can be measured by methods known in the art (e.g., by HPLC or GC analysis).

[0046] Preferably, the culture can be stopped by terminating aeration and / or providing a carbon source.

[0047] Therefore, at the end of the culture step (a) of the method according to the present invention, a liquid culture medium containing accumulated 3-methylcrotonic acid and the microorganisms used to produce it is obtained. This liquid culture medium is then used in the incubation step (b) of the method according to the present invention.

[0048] According to step (b) of the method according to the present invention, 3-methylcrotonic acid produced in step (a) and contained in the liquid culture medium is then enzymatically converted to isobutene.

[0049] This step does not require microbial culture, but is characterized by the fact that it can be achieved by simply incubating the 3-methylcrotonic acid produced in step (a) with an enzyme capable of converting 3-methylcrotonic acid to isobutene (particularly FMN prenyltransferase and associated FMN-dependent decarboxylase) or a microorganism (a pre-grown culture of) that produces such an enzyme. The fact that culture is not achieved in this step (b) means, for example, that no further nutrients are generally added. Incubation simply requires contacting the substrate (3-methylcrotonic acid) with the enzyme (either in the form of an isolated enzyme or in the form of a pre-cultured microorganism that synthesized the enzyme) and incubating under controlled reaction conditions (e.g., temperature and pH values) that allow for the enzymatic conversion of 3-methylcrotonic acid to isobutene.

[0050] While the culture in step (a) may be stopped as described above, incubation may be initiated with a liquid culture medium containing 3-methylcotonic acid, together with cells expressing FMN prenyltransferase and associated FMN-dependent decarboxylase or FMN prenyltransferase and associated FMN-dependent decarboxylase.

[0051] Therefore, the term "incubating," when used in step (b) of the method of the present invention, does not necessarily require keeping the microorganisms healthy if used in this step. The term "incubating" also does not necessarily imply further proliferation / growth of the microorganisms used in step (b). In relation to the present invention, "incubation" refers to maintaining the microorganisms expressing the desired enzymes and the enzymes themselves in an active state, preferably under optimal temperature, humidity, and other conditions that enable the conversion of 3-methylcrotonic acid to isobutene. Thus, in contrast to "cultivation" / "fermentation" in step (a), "incubation" in relation to the present invention essentially refers simply to the conversion of 3-methylcrotonic acid to isobutene, but does not necessarily require the proliferation / growth of microorganisms and / or production of the respective enzymes necessary for the conversion of 3-methylcrotonic acid to isobutene. Therefore, in step (b) “Incubation” of the method of the present invention, the proliferation / growth of microorganisms and / or production of the respective enzymes necessary for the conversion of 3-methylcrotonic acid to isobutene are physically separated from the actual conversion of 3-methylcrotonic acid to isobutene, if any such processes are included or required. In contrast, the “Cultivation” described above involves the step of converting a carbon source to 3-methylcrotonic acid, as will be further described in more detail below.

[0052] The incubation step (b) of the method according to the present invention is carried out in a container containing a solution that includes 3-methylcrotonic acid produced in step (a) and a microorganism that expresses an enzyme or an enzyme for converting 3-methylcrotonic acid to isobutene.

[0053] The container is designed as a closed system that allows control over the flow of gas from the container and, in some cases, the flow of gas into the container.

[0054] The container may also include means for enabling agitation of the solution contained within the container, or means for controlling the temperature during incubation step (b).

[0055] While not bound by theory, stirring allows for the mixing of the solution containing cells / enzymes and 3-methylcrotonic acid, and therefore, the uniform dispersion of the cells and / or enzymes and 3-methylcrotonic acid in the solution. Furthermore, stirring may facilitate the release of gaseous components, such as isobutene, from the solution into the gas phase.

[0056] Advantageously, in a preferred embodiment, the container includes a connection to a pH control system and / or a storage tank for an acid used to control / adjust the pH of the liquid culture medium in the container. As an example, sulfuric acid or phosphoric acid may be used to control / adjust the pH.

[0057] In another preferred embodiment, the container may be supplied during incubation step (b) with a (concentrated) solution of 3-methylcrotonic acid (or a salt thereof) produced according to step (a) of the method according to the present invention, preferably 3-methylcrotonic acid. Possible methods of supplying 3-methylcrotonic acid to the solution are described below. By supplying 3-methylcrotonic acid to the container during incubation step (b), the concentration of 3-methylcrotonic acid in the medium can be maintained at a desired level, for example, to enable efficient conversion to isobutene. Thus, in such embodiments, it is also conceivable that the concentration of 3-methylcrotonic acid in the medium is monitored (e.g., continuously or at predetermined time intervals) and additional 3-methylcrotonic acid is supplied to the medium to adjust the concentration to a desired level.

[0058] In one embodiment of the present invention, incubation step (b) is performed in a container designed as a closed system, which allows only controlled discharge of gas through an outlet but does not allow gas to enter. The corresponding container is schematically shown at the top of Figure 4. Thus, in such embodiments, there is no external gas supply, and the method is performed in a container without a gas supply. In this case, the gas (including isobutene) produced during incubation step (b) is captured as off-gas through an outlet that allows gas discharge. Thus, in such a closed system, the gas produced during incubation in step (b) of the present invention can be recovered from the container without replenishing the corresponding volume of gas with inlet gas. In other words, as gas (including isobutene) is produced during incubation, the pressure in the container increases due to gas production. To maintain a constant pressure, the gas can be recovered from the container through an outlet.

[0059] Therefore, in such a situation, the container is a closed system (away from the gas outlet), and the pressure inside the container is preferably controlled in such a way that it does not rise (i.e., by venting the gas through the outlet).

[0060] As shown in the attached examples, such incubation without any gas supply during incubation step (b) results in the production of a gas containing extremely high levels of isobutene.

[0061] In another embodiment, incubation step (b) is performed in a vessel designed as a closed system but allowing controlled gas discharge and controlled gas inflow. In such an embodiment, the inlet gas can be supplied to the system in a controlled manner and can be used to push out the gas produced by the incubation containing isobutene from the system. The corresponding vessel is schematically shown at the bottom of Figure 4.

[0062] Therefore, there is an inlet where the inlet gas (for example, nitrogen, air, etc., as outlined in more detail below) can be supplied in a controlled manner. Furthermore, there is an outlet that allows for the discharge of the gas.

[0063] In such situations, it is preferable to maintain the inlet gas flow (gas supply) at a low speed. Preferably, the flow is maintained at a speed of less than 0.1 vvm (container volume per minute).

[0064] The unit "vvm" (representing "container volume per minute") is known to those skilled in the art. The unit "vvm" represents the volume of inlet gas per minute per volume of liquid fermentation medium under standard conditions (101.325 kPa, 0°C), and can be easily adjusted in the container. Corresponding apparatus is known to those skilled in the art.

[0065] In a preferred embodiment, the gas supply is in the range of >0.0~0.1vvm, >0.0~0.09vvm, >0.0~0.08vvm, >0.0~0.07vvm, >0.0~0.06vvm, >0.0~0.05vvm, >0.0~0.04vvm, >0.0~0.03vvm, >0.0~0.02vvm, or >0.0~0.01vvm. In a more preferred embodiment, the gas supply is in the range of >0.0~0.05vvm.

[0066] In a more preferred embodiment, the gas supply is <0.09vvm, <0.08vvm, <0.07vvm, <0.06vvm, <0.05vvm, <0.04vvm, <0.03vvm, <0.02vvm, <0.01vvm, or <0.005vvm. In a more preferred embodiment, the gas supply is <0.05vvm.

[0067] In a particularly preferred embodiment, the gas supply is <0.1 vvm. In another particularly preferred embodiment, the gas supply is <0.05 vvm. In yet another particularly preferred embodiment, the gas supply is <0.02 vvm.

[0068] The gas may be supplied to the container through an inlet to the solution (for example, by blowing the gas through the solution). Alternatively, the gas may be supplied to the gas phase of the container.

[0069] Preferably, the inlet gas is supplied to the solution in the container from the bottom of the container using a sparger (for example, by "blowing" the gas through the solution).

[0070] The attached examples demonstrate that such incubation with a very low gas supply during incubation step (b) results in the production of a gas containing a very high content of isobutene. In particular, the examples show that when the incubation step is performed with no gas supply or a low gas supply, CO2 and isobutene are produced in high concentrations, and in fact, essentially only CO2 and isobutene are produced. As a result, as will be further described in more detail below, the outlet gas from the vessel consists mainly or even exclusively (with the exception of trace amounts of other gases at the end) of CO2 and isobutene, but essentially no oxygen.

[0071] The method of the present invention is therefore also advantageous in that it can eliminate, or at least significantly reduce, the risk of combustion of the outlet gas produced during isobutene production. Thus, in the absence of gas supply or when the gas supply is controlled to a low level, the oxygen in the outlet gas is less than approximately 10 vol%, and therefore lower than the minimum oxygen concentration (MOC) required for combustion of isobutene. Thus, the method of the present invention can also improve the safety of isobutene production and facilitate the subsequent treatment of the recovered outlet gas.

[0072] The inlet gas is preferably air, an inert gas, a mixture of several gases, or a mixture of air and an inert gas, the inert gas being preferably selected from nitrogen, helium, argon, neon, CO2, and mixtures of these gases.

[0073] Furthermore, if nitrogen (or another inert gas) is used in the gas supply, the nitrogen can inactivate the system (thereby avoiding / reducing the risk of combustion and / or explosion).

[0074] Incubation step (b) of the method according to the present invention is carried out under conditions that allow isobutene to be in a gaseous state and evaporate from the solution. Figure 9 shows a graph illustrating the correlation between vapor pressure and temperature for pure isobutene. Below the curve, isobutene is a gas, while above the curve, isobutene is a liquid.

[0075] Therefore, at the temperatures (typically 30°C to 40°C; generally around 37°C) and pressures applied to the enzymatic conversion of 3-methylcrotonic acid to isobutene, the isobutene produced is in gaseous form. Those skilled in the art are in an easy position to select appropriate conditions to make the produced isobutene a gaseous substance (in terms of adjusting the temperature and / or pressure in the container).

[0076] The produced isobutene, which evaporates into the gas phase, is subsequently recovered by step (c) of the method according to the present invention. Recovery involves evaporation from the solution and recovery of the gas containing isobutene. The isobutene may then be further purified according to methods well known to those skilled in the art.

[0077] As described above, the incubation step (b) of the method according to the present invention comprises incubation of a liquid culture medium containing 3-methylcrotonic acid with an enzyme capable of converting 3-methylcrotonic acid to isobutene, or a microorganism expressing such an enzyme.

[0078] After stopping the culture in step (a), the transition to step (b) can be achieved in different ways. For example, the culture medium produced at the end of step (a) can be collected as is (containing the accumulated 3-methylcrotonic acid and the cells of the microorganisms used in its production) and combined with the enzymes and / or microorganisms used in the incubation step (b). In this situation, separation of the cells used in step (a) from the culture medium is not achieved. The incubation step (b) can be carried out in the same container as the culture in step (a) by simply adding the enzymes and / or microorganisms required for step (b). In a preferred embodiment, the incubation step (b) can be carried out in the same container as the culture in step (a) by simply adding the enzymes and / or microorganisms required for step (b), and by applying the conditions optimal for incubation, i.e., the enzymatic transformation of step (b), by controlling the temperature, pH, stirring force and / or oxygen concentration by applying the corresponding routine treatment known in the art.

[0079] However, it is preferable that the culture medium obtained after stopping the culture in step (a) be transferred to a different container.

[0080] In another embodiment, the method of the present invention includes the step of separating a liquid culture medium containing 3-methylcrotonic acid, obtained after the cessation of the culture in step (a), from the microorganisms before step (b). Methods for separating microorganisms from a liquid culture medium are known to those skilled in the art. For example, centrifugation may be used to separate the microorganisms from the liquid. Once separated, the liquid culture medium containing 3-methylcrotonic acid can be subjected to incubation step (b) of the method of the present invention.

[0081] As outlined above, in a second aspect, the present invention is a method for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b) A step of thermochemically converting the 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene, preferably at a temperature of 180°C to 400°C; (c) A step of recovering the produced isobutene and The present invention provides a method for producing isobutene from a carbon source, characterized by including [a specific component].

[0082] In step (b), 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) is thermochemically converted to isobutene. Preferably, the thermochemical conversion is achieved at a temperature of 180°C to 400°C.

[0083] 3-Methylcrotonic acid can be efficiently converted to isobutene and carbon dioxide according to procedures known in the art. Preferably, 3-methylcrotonic acid is heated to a temperature of 180°C to 400°C, preferably 230°C to 350°C. In another preferred embodiment, the thermochemical conversion is achieved in a boiling reactor, a stirred tank reactor, or a tubular reactor. In another preferred embodiment, the thermochemical conversion is achieved at a pressure of 0 to 30 bar, preferably 10 to 30 bar.

[0084] In preferred embodiments, the method of the present invention includes the step of isolating or purifying the 3-methylcrotonic acid produced in step (a) of the method of the present invention from the liquid culture medium prior to step (b) of the method of the present invention. Methods for isolating, purifying, extracting or separating 3-methylcrotonic acid from the liquid culture medium are known in the art. The 3-methylcrotonic acid may be concentrated, for example (e.g., by removing water content from the liquid culture medium), or preferably (partially) purified by removing any residual sugars and other compounds that may be present. In addition or otherwise, the 3-methylcrotonic acid may be concentrated and / or purified by applying methods known in the art to a concentration of 3-methylcrotonic acid of at least 100 g / l, more preferably at least 150 g / l, even more preferably at least 200 g / l, and even more preferably at least 250 g / l. Preferably, the 3-methylcrotonic acid is recovered in the form of its sodium or potassium salt.

[0085] In another preferred embodiment, the method of the present invention includes the step of purifying the 3-methylcrotonic acid produced in step (a) of the method of the present invention from a liquid culture medium prior to step (b) of the method of the present invention.

[0086] In corresponding preferred embodiments, the 3-methylcrotonic acid produced in step (a) of the method of the present invention may be purified / separated from the liquid culture medium using batch or continuous liquid-liquid extraction prior to step (b) of the method of the present invention.

[0087] Liquid-liquid extraction of 3-methylcrotonic acid can be performed as follows: Bacteria and all other solids are preferentially removed from the fermentation broth, i.e., the liquid culture medium as defined in the present invention, by standard liquid-solid separation. Standard liquid-solid separation may preferably be centrifugation or filtration. The separated fermentation broth, i.e., the separated liquid culture medium, can then be acidified. Preferably, the acidification is to a pH below 4.2, more preferably below 4.0, and more preferably by the addition of any mineral acid. Thus, the acidified fermentation broth, i.e., the liquid culture medium as defined in the present invention, can be mixed with organic solvents, preferably alcohols (more preferably 2-octanol or 2-ethylhexanol), heavy organic acids (more preferably heptanoic acid), ketones (more preferably methyl isobutyl ketone or methyl ethyl ketone), and heavy alkanes (more preferably containing 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms or mixtures thereof). This mixing can be carried out in either a series of stirred tank reactors or a continuous liquid-liquid extraction column of any technology known in the art (e.g., by simultaneous stirring or without stirring). The organic phase can be recovered and sent to distillation. The solvent can be recovered in the distillate, and 3-methylcrotonic acid can be recovered in the residue. Alternatively, 3-methylcrotonic acid may be further distilled so that it is recovered in the distillate.

[0088] In another preferred embodiment, the liquid-liquid extraction of 3-methylcrotonic acid may be carried out directly from the liquid culture medium obtained in step (a) of the present invention, which contains the 3-methylcrotonic acid and still contains the microorganisms cultured in step (a).

[0089] This can preferably be done in a culture vessel or fermenter.

[0090] In a preferred embodiment, a solvent, preferably a heavy alkane (containing 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms or a mixture thereof), may be introduced into the culture or fermenter at the beginning of or during the culture or fermentation. The resulting mixture may be continuously drawn from the culture or fermenter. The pH may be acidified, preferably reduced to less than 4.2, to enhance the transfer of 3-methylcrotonic acid to the solvent. Both phases may be sent to a decanter. The separated organic phase may be sent to distillation, and the aqueous phase, still containing microorganisms, may be returned to the culture or fermenter.

[0091] In another preferred embodiment, the purification / extraction described above may also be performed outside the culture or fermenter. In such an embodiment, the culture or fermentation is carried out in a standard manner, and the broth (i.e., the liquid culture medium of step (a) as defined in the present invention) may be continuously drawn from the culture or fermenter. This stream (i.e., the liquid culture medium of step (a) as defined in the present invention, continuously drawn from the culture or fermenter) is preferentially acidified to a pH of preferably less than 4.2 using any mineral acid known in the art. Alternatively, it may not be acidified. The stream (i.e., the liquid culture medium of step (a) as defined in the present invention, continuously drawn from the culture or fermenter) may be mixed with a solvent (preferably the same heavy alkane) in either a series of stirred tanks as described above or in a liquid-liquid extraction column. The organic phase is recovered and sent to distillation to recover 3-methylcrotonic acid on the one hand and the solvent on the other.

[0092] Preferably, in the above liquid-liquid extraction, a biosource solvent, more preferably isododecane or 2-octanol, is used.

[0093] In the method of the present invention, in incubation step (b)(i), a microorganism expressing FMN prenyltransferase and associated FMN-dependent decarboxylase is incubated with a liquid culture medium containing 3-methylcrotonic acid obtained in step (a) of the present invention, wherein the microorganism may be present as a suspension culture in the liquid culture medium (in the sense that it is freely floating). Alternatively, the microorganism may be immobilized. Immobilization may be on a suitable carrier, surface and / or support material. Suitable carriers, surfaces and / or support materials and methods of immobilization are known to those skilled in the art. Martins et al. have reviewed corresponding support materials and methods of immobilization (African Journal of Biotechnology 12(28):441-4418(2013)).

[0094] In the method of the present invention, in incubation step (b)(ii), the FMN prenyltransferase and associated FMN-dependent decarboxylase are incubated with the liquid culture medium containing 3-methylcrotonic acid obtained in step (a), wherein the enzymes may be present in the solution of the liquid culture medium (in the sense that they are freely floating). Alternatively, the enzymes may be immobilized. Immobilization may be on a suitable carrier, surface and / or support material. Suitable carriers, surfaces and / or support materials and methods for immobilizing enzymes are known to those skilled in the art (see, for example, Mohamad et al., Biotechnology & Biotechnological Equipment 29(2), 2015, 205-220).

[0095] The incubation step (b) is carried out under conditions that allow the conversion of 3-methylcrotonic acid contained in the solution to isobutene. These conditions depend on the type of organism or enzyme used for the conversion and can be adapted by those skilled in the art through routine procedures.

[0096] The incubation is carried out for a sufficient amount of time to allow for the production of isobutene. During incubation, the concentration of 3-methylcrotonic acid in the solution can be monitored. Preferably, the incubation is carried out until at least 90%, more preferably at least 95%, and even more preferably at least 98% of the 3-methylcrotonic acid has been converted to isobutene.

[0097] After incubation is stopped, the solution may be separated from the cells / enzymes and reintroduced into step (a) of the method. Preferably, the solution is sterilized before being reintroduced into step (a) of the method. The cells or enzymes used in step (b) may be reused and used in another round of step (b).

[0098] In a preferred embodiment, in the culture step (a) of the present invention, which utilizes a microorganism capable of producing 3-methylcrotonic acid from a carbon source, a microorganism capable of metabolizing the carbon source to acetyl-CoA is used prior to the enzymatic conversion of acetyl-CoA to 3-methylcrotonic acid. The corresponding enzymatic conversion and microorganism, preferably recombinant, capable of metabolizing the carbon source to acetyl-CoA, as well as the further enzymatic conversion of acetyl-CoA to 3-methylcrotonic acid, are described in more detail below.

[0099] However, the method of the present invention is not limited to the use of a corresponding microorganism capable of metabolizing the carbon source to acetyl-CoA prior to the enzymatic conversion of acetyl-CoA to 3-methylcrotonic acid. Furthermore, as will be described in more detail below, there are (recombinant) microorganisms capable of producing 3-methylcrotonic acid from a carbon source, and the metabolism in these microorganisms does not require the formation of acetyl-CoA as an intermediate.

[0100] In the culture step (a) of the present invention, which uses microorganisms capable of producing 3-methylcrotonic acid from a carbon source, it is also conceivable to use C1-fixed microorganisms or microorganisms, preferably a combination of C1-fixed microorganisms.

[0101] C1-fixed microorganisms are known in the art, for example, as described in International Publication No. 2020 / 188033, the contents of which are incorporated herein by reference.

[0102] In a preferred embodiment, the microorganism, preferably a C1-fixed microorganism as defined above, is a microorganism capable of consuming one or more sugars. Preferably, the one or more sugars include glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, and / or mannose. In a more preferred embodiment, the microorganism, preferably a C1-fixed microorganism, is a microorganism capable of consuming two or more sugars selected from the group consisting of glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, and mannose. Organisms and / or microorganisms capable of consuming glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, and / or mannose exist in nature and are known in the art.

[0103] In a preferred embodiment, the microorganism, preferably a C1-fixing microorganism, is an organism capable of consuming CO and / or synthesis gas. In another preferred embodiment, the microorganism, preferably a C1-fixing microorganism, is an organism capable of consuming a mixture of CO and / or CO2 and H2.

[0104] In another embodiment, the microorganism is genetically modified to be able to consume glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, mannose and / or CO (or synthesis gas), and / or genetically modified to enhance the ability of the microorganism to consume glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, mannose and / or CO (or synthesis gas). The corresponding genetic modifications are known in the art.

[0105] In another preferred embodiment, the microorganism, preferably a C1-fixed microorganism, is a microorganism capable of consuming sugar through a phosphotransferase transport system (PTS).

[0106] In a preferred embodiment, the microorganism, preferably a C1-fixed microorganism, is an organism capable of consuming sugar through a non-phosphotransferase transport system (non-PTS).

[0107] In a preferred embodiment, the culture step (a) of the present invention utilizes a microorganism capable of producing 3-methylcrotonic acid from a carbon source, wherein the carbon source is selected from the group consisting of glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, and mixtures thereof.

[0108] In preferred embodiments, the microorganisms used in the culture step (a) of the present invention are recombinant microorganisms. The corresponding recombinant microorganisms are described in more detail below.

[0109] In a preferred embodiment, the microorganism used in the culture step (a) of the present invention is a microorganism that, when cultured in step (a), exhibits reduced expression and / or decreased activity of FMN prenyltransferase and associated FMN-dependent decarboxylase, or does not express FMN prenyltransferase and associated FMN-dependent decarboxylase, preferably a recombinant microorganism.

[0110] As outlined above, in preferred embodiments, the method of the present invention includes a step in which the liquid culture medium containing 3-methylcrotonic acid in step (a) is isolated from the microorganisms before step (b). Therefore, the isolated microorganisms do not necessarily have to be discarded. In fact, in preferred embodiments, the microorganisms isolated from the liquid culture medium can be reused and reintroduced into step (a) of the method of the present invention. The corresponding reuse is schematically shown in Figure 3.

[0111] In another preferred embodiment, the microorganisms used in step (b)(i) are pre-cultured in a suitable liquid culture medium under suitable conditions prior to the conversion step (b)(i) of the method of the present invention. Although not bound by theory, the corresponding pre-culture step has the effect of growing and concentrating the microorganisms to a certain degree that increases the number and / or density of the microorganisms (and correspondingly the desired enzymes for the desired enzymatic conversion). Suitable liquid culture media and suitable conditions are known to those skilled in the art and are described in more detail below.

[0112] The microorganisms used in step (b)(i) are preferably added to a solution containing 3-methylcrotonic acid to achieve a cell density expressed as a dry cell weight per liter of at least 1 g / l, more preferably at least 5 g / l, and most preferably at least 10 g / l. As outlined above, incubation step (b)(i) is preferably carried out under conditions that do not support further cell growth. Preferably, incubation step (b)(i) is carried out under conditions that do not support further cell growth such that the cell density remains essentially constant or shows only a slight increase (preferably not exceeding 20%, more preferably not exceeding 10%) during incubation. In another preferred embodiment, incubation step (b)(i) is carried out under conditions that do not support further cell growth such that the cell density remains essentially constant or shows only a slight decrease (preferably not exceeding 20%, more preferably not exceeding 10%) during incubation. A decrease in cell density can occur, for example, when adjusting the pH of a liquid culture medium, by adding an (acidic) solution, which causes some cells to lyse and increases the volume of the liquid culture medium during incubation.

[0113] A decrease in cell density may also occur by the addition of 3-methylcrotonic acid to the container, for example, when supplying 3-methylcrotonic acid to the container during incubation step (b) as described herein above and below, thereby diluting the biomass / cells in solution.

[0114] As outlined above, the method of the present invention includes, in incubation step (b), a step in which a microorganism expressing FMN prenyltransferase and associated FMN-dependent decarboxylase is incubated with the liquid culture medium containing 3-methylcrotonic acid obtained in step (a) of the method of the present invention. Alternatively, the method of the present invention includes, in incubation step (b), a step in which FMN prenyltransferase and associated FMN-dependent decarboxylase is incubated with the liquid culture medium containing 3-methylcrotonic acid obtained in step (a) of the method of the present invention. In preferred embodiments, in both cases, the liquid culture medium is recovered after the completion of step (b) of the method of the present invention, may be sterilized, and may be reintroduced into step (a) of the method of the present invention. The corresponding reuse is schematically shown in Figure 3.

[0115] In another preferred embodiment described above, the microorganisms present in the recovered liquid culture medium (i.e., microorganisms expressing FMN prenyltransferase and associated FMN-dependent decarboxylase) are removed from the liquid culture medium and reintroduced into the bioconversion step (b)(i) of the method of the present invention. The corresponding reuse is schematically shown in Figure 3.

[0116] In another preferred embodiment, the recovered isobutene is subsequently purified and / or concentrated.

[0117] In this invention, purification or partial purification preferably means the (partial or complete) removal of other compounds (components other than isobutene) that may be present or remain.

[0118] In this invention, concentration means an increase in the concentration of isobutene in the gas phase or liquid phase.

[0119] Methods for purifying and / or concentrating isobutene are known in the art. Isobutene can be recovered or isolated from the off-gas of incubation step (b) of the method of the present invention using methods known in the art, such as physical absorption, reactive absorption, adsorption, aggregation, cryogenic techniques, and / or membrane-based separation.

[0120] In preferred embodiments, the microorganism used in step (a) and / or step (b) of the method of the present invention, preferably a recombinant microorganism, is a bacterium, yeast, fungus, or algae. In particularly preferred embodiments, the microorganism is a bacterium, for example, Escherichia coli (E. coli). Corresponding suitable microorganisms, preferably recombinant microorganisms, are described in more detail below.

[0121] Enzymatic production of 3-methylcrotonic acid from a carbon source by step (a) As described above, methods for the enzymatic production of 3-methylcrotonic acid from carbon sources are known in the art, and (recombinant) organisms capable of catalyzing each enzymatic conversion have been described; see, for example, International Publication Nos. 2017 / 085167, International Publication Nos. 2018 / 206262, and International Publication Nos. 2020 / 188033, the contents of which are incorporated herein by reference.

[0122] The enzymatic transformations beginning with a carbon source that yields the central metabolite acetyl-CoA, and the possible individual steps of its further conversion to 3-methylcrotonic acid, are described only as examples, and are not limited to them.

[0123] Acetyl-CoA (acetyl coenzyme A) is a central metabolite found in all living organisms and is involved in many biochemical reactions in protein, carbohydrate, and lipid metabolism. Acetyl-CoA is produced, for example, when CoA is acetylated to acetyl-CoA through the breakdown of carbon sources by glycolysis and the breakdown of fatty acids by β-oxidation.

[0124] Therefore, since acetyl-CoA produced from fatty acids, and especially from carbon sources, is a central metabolite present in all living organisms, its production is not described herein.

[0125] Without limitation, the following summarizes the individual steps of possible enzymatic conversion of acetyl-CoA to 3-methylcrotonic acid, and microorganisms capable of producing 3-methylcrotonic acid from, for example, acetyl-CoA. Corresponding enzymatic conversions and microorganisms are described, for example, in International Publication No. 2017 / 085167, International Publication No. 2018 / 206262, and International Publication No. 2020 / 188033, the contents of which are incorporated herein by reference.

[0126] Methods for producing 3-methylcrotonic acid via different possible pathways are described (see Figures 1 and 2 for an overview). Methods utilizing these pathways and enzymatic conversions, as well as recombinant organisms and microorganisms, are described in particular in International Publication Nos. 2010 / 001078, 2012 / 052427, 2017 / 085167, 2018 / 206262, and 2020 / 188033 (incorporated herein by reference).

[0127] However, in step (a) of the method of the present invention, not only may these reactions be used, but in principle, any other route for converting acetyl-CoA to 3-methylcrotonic acid described in the prior art publications International Publication Nos. 2017 / 085167, 2018 / 206262, 2010 / 001078, 2012 / 052427 and 2016 / 042012 may also be used.

[0128] Furthermore, in step (a) of the method of the present invention, the 3-methylcrotonic acid may also be produced from a carbon or nitrogen source via pyruvate and leucine as a thioester, i.e., 3-methylcrotonyl-CoA. The corresponding biosynthetic pathway and microorganisms capable of producing 3-methylcrotonyl-CoA are described in Li et al. (Angew. Chem. Int. Ed. (2013) 52: 1304); see in particular Figure 1.

[0129] The thioester of 3-methylcrotonic acid produced, i.e., 3-methylcrotonyl-CoA, can then be further converted to 3-methylcrotonic acid by enzymatic conversion as described in the prior art.

[0130] The disclosures of these documents, particularly with respect to preferred embodiments of enzymes for the individual transformations of the pathways described therein, are incorporated herein by reference in their entirety. Therefore, in preferred embodiments, it is preferable to use enzymes selected from the preferred embodiments described in these prior art documents with respect to each enzymatic transformation. Accordingly, the same as described in International Publication No. 2017 / 085167, International Publication No. 2018 / 206262, International Publication No. 2010 / 001078, International Publication No. 2012 / 052427, International Publication No. 2016 / 042012 and Li et al. (Angew. Chem. Int. Ed. (2013) 52: 1304) applies to the enzymatic transformation of step (a) of the method of the present invention.

[0131] In a preferred embodiment, in step (a) of the method of the present invention, the 3-methylcrotonic acid is produced by a pathway that starts with acetyl-CoA, then enzymatically converts acetyl-CoA to acetoacetyl-CoA, then enzymatically converts acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, then enzymatically converts 3-hydroxy-3-methylglutaryl-CoA to 3-methylglutaconyl-CoA, then enzymatically converts 3-methylglutaconyl-CoA to 3-methylcrotonyl-CoA, and then enzymatically converts 3-methylcrotonyl-CoA to the 3-methylcrotonic acid (see, for example, Figure 2 for an overview). Corresponding methods, enzymatic conversions, and recombinant organisms and microorganisms utilizing these pathways and enzymatic conversions are described in the above-mentioned prior art literature. The disclosures in these documents, particularly with regard to preferred embodiments of enzymes for the above-mentioned preferred pathway (i.e., starting from acetyl-CoA, via acetoacetyl-CoA, then via 3-hydroxy-3-methylglutaryl-CoA, then via 3-methylglutaconyl-CoA, then via 3-methylcrotonyl-CoA, and then converted to the aforementioned 3-methylcrotonic acid), are incorporated herein by reference in their entirety. Accordingly, in preferred embodiments relating to this possible preferred pathway, it is preferable to use enzymes selected from the preferred embodiments described in these prior art documents with respect to each enzymatic conversion. Thus, the same as described in the above-mentioned prior art documents applies to the enzymatic conversion of step (a) of the method of the present invention.

[0132] As described above, in preferred embodiments, in the culture step (a) of the present invention, the microorganism used in step (a) is a microorganism having reduced activity of FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene), preferably a recombinant microorganism. Using a corresponding microorganism having reduced activity of FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene) is beneficial in avoiding the metabolism of the 3-methylcrotonic acid thus produced and therefore allowing the accumulation of 3-methylcrotonic acid in the liquid culture medium.

[0133] Therefore, microorganisms in which the activity of FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene) is reduced are either microorganisms that do not naturally express FMN prenyltransferase and associated FMN-dependent decarboxylase, or microorganisms that have been modified, particularly genetically modified, so that the activity of each enzyme is completely abolished or reduced compared to the corresponding unmodified microorganism.

[0134] Corresponding microorganisms that do not naturally express FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene) are known in the art.

[0135] In preferred embodiments, microorganisms in which the activity of FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene) is reduced / decreased compared to unmodified organisms or microorganisms preferably refer to microorganisms in which the reduction / decreased enzymatic activity compared to unmodified microorganisms is achieved by genetic modification of the microorganism that results in the inactivation or reduction.

[0136] In preferred embodiments, the recombinant microorganisms of the present invention are recombinant microorganisms in which the activity of FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene) is reduced compared to non-modified microorganisms. Preferably, this reduction is achieved by genetic modification of the microorganism. This can be achieved, for example, by random mutagenesis or site-directed mutagenesis of the promoter and / or enzyme, followed by selection of a promoter and / or enzyme with desired properties, or by complementary nucleotide sequences or RNAi activity as described above.

[0137] In the context of the present invention, "decreased activity" means that the activity of enzymes in genetically modified microorganisms, particularly FMN prenyltransferase and associated FMN-dependent decarboxylase, is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80%, and particularly preferably at least 90% or 100% lower than that of the corresponding unmodified microorganisms. Assays for measuring the decrease in enzymatic activity of FMN prenyltransferase and associated FMN-dependent decarboxylase are known in the art.

[0138] In another embodiment, the microorganism according to the present invention is a microorganism that lacks the activity of FMN prenyltransferase and associated FMN-dependent decarboxylase (preferably an FMN-dependent decarboxylase capable of enzymatically converting 3-methylcrotonic acid to isobutene). This preferably means that such a microorganism does not naturally possess the activity of FMN prenyltransferase and associated FMN-dependent decarboxylase. This means that such a microorganism does not naturally contain in its genome a nucleotide sequence encoding an enzyme having the activity of FMN prenyltransferase and associated FMN-dependent decarboxylase.

[0139] In another preferred embodiment, the microorganism of the present invention is an organism genetically modified to avoid the leakage of acetyl-CoA, thereby increasing the intracellular concentration of acetyl-CoA, which is ultimately converted to 3-methylcrotonic acid. Genetic modifications resulting in an increase in the intracellular concentration of acetyl-CoA are known in the art. Such microorganisms may preferably be genetically modified by deleting or inactivating the following genes: ΔackA (acetate kinase), Δldh (lactate dehydrogenase), ΔadhE (alcohol dehydrogenase), ΔfrdB and / or ΔfrdC (fumarate reductase and fumarate dehydrogenase).

[0140] In preferred embodiments, methods are used to increase the yield, pool, and / or flow of acetyl-CoA. Corresponding methods, as well as recombinant organisms and microorganisms with increased acetyl-CoA pools, are described in the prior art, for example, in International Publication No. 2013 / 007786, International Publication No. 2020 / 021051, and International Publication No. 2020 / 188033, the contents of which are incorporated herein by reference.

[0141] In preferred embodiments, the yield, pool, and / or flow of acetyl-CoA is increased by utilizing recombinant organisms or microorganisms having phosphoketolase (PKT) activity, as described, for example, in International Publication No. 2013 / 007786, International Publication No. 2020 / 021051, and International Publication No. 2020 / 188033, the contents of which are incorporated herein by reference.

[0142] Enzymatic conversion of 3-methylcrotonic acid to isobutene by step (b) As outlined above, the incubation step (b)(i) or (b)(ii) of the method of the present invention uses FMN prenyltransferase and associated FMN-dependent decarboxylase or a microorganism expressing such enzymes, which is incubated with a liquid culture medium containing 3-methylcrotonic acid obtained in step (a) of the present invention.

[0143] In principle, any FMN-dependent decarboxylase (or a microorganism expressing it) associated with FMN prenyltransferase may be used in step (b) of the method according to the present invention. The use of such enzymes for the conversion of 3-methylcrotonic acid to isobutene is described in the prior art, for example, in International Publication No. 2017 / 085167, International Publication No. 2018 / 206262 and International Publication No. 2020 / 188033 (incorporated herein by reference).

[0144] The following describes the enzymatic conversion of 3-methylcrotonic acid to isobutene using an FMN prenyltransferase and associated FMN-dependent decarboxylase, which can enzymatically convert 3-methylcrotonic acid to isobutene. Whenever the FMN prenyltransferase and associated FMN-dependent decarboxylase as used in this invention are referred to, for simplicity of reference, they may also be more accurately referred to as "prenylated FMN-dependent decarboxylase."

[0145] The enzymatic conversion of 3-methylcrotonic acid to isobutene is schematically shown in step I of Figure 1. This conversion can be achieved by decarboxylation using FMN prenyltransferase and associated FMN-dependent decarboxylase. Decarboxylation is generally a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2).

[0146] The enzymatic conversion of 3-methylcrotonic acid to isobutene using FMN prenyltransferase and associated FMN-dependent decarboxylase relies on a two-step reaction catalyzed by two enzymes: FMN prenyltransferase, which provides a modified flavin cofactor, and associated FMN-dependent decarboxylase (which catalyzes the actual decarboxylation of 3-methylcrotonic acid to isobutene).

[0147] The flavin cofactor may preferably be FMN or FAD. FMN (flavin mononucleotide; also called riboflavin-5'-phosphate) is a biomolecule produced from riboflavin (vitamin B2) by the enzyme riboflavin kinase and functions as a prosthetic group in various reactions. FAD (flavin adenine dinucleotide) is a redox cofactor, more specifically a prosthetic group, involved in several important metabolic reactions.

[0148] Therefore, in the conversion of 3-methylcrotonic acid to isobutene, the first step involves the modification of the flavin cofactor (FMN or FAD) into a (modified) flavin-derived cofactor. This modification is catalyzed by the FMN prenyltransferase. The FMN prenyltransferase prenylates the flavin ring of the flavin cofactor (FMN or FAD) into a (modified) prenylated flavin cofactor. More specifically, the FMN prenyltransferase catalyzes the prenylation of the flavin cofactor (FMN or FAD) to a flavin-derived cofactor using dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP).

[0149] In the second step, the actual conversion of 3-methylcrotonic acid to isobutene is catalyzed by the FMN-dependent decarboxylase via a 1,3-dipolar cycloaddition-based mechanism, which in turn utilizes a prenylated flavin cofactor (FMN or FAD) provided by an associated FMN prenyltransferase.

[0150] In a preferred embodiment, the flavin cofactor (FMN or FAD) is modified to a (modified) flavin-derived cofactor (using dimethylallyl phosphate (DMAP) or dimethylallyl pyrophosphate (DMAPP)). The FMN prenyltransferase is a phenylacrylate decarboxylase (PAD) type protein, or a closely related prokaryotic enzyme UbiX, which is an enzyme involved in ubiquinone biosynthesis in prokaryotes.

[0151] In Escherichia coli, the protein UbiX (also known as 3-octaprenyl-4-hydroxybenzoate carboxylyase) has been shown to be involved in the third step of ubiquinone biosynthesis.

[0152] In preferred embodiments, the modification of a flavin cofactor (FMN or FAD) to a corresponding (modified) flavin-derived cofactor is catalyzed by FMN-containing protein phenylacrylate decarboxylase (PAD). The enzymes involved in the modification of a flavin cofactor (FMN or FAD) to a corresponding modified flavin-derived cofactor were initially annotated as decarboxylases (EC4.1.1.~). Some phenylacrylate decarboxylases (PADs) are now annotated as flavin prenyltransferases (EC2.5.1.~). The enzymes capable of catalyzing the enzymatic reactions of flavin prenyltransferases described herein have recently also been annotated as flavin prenyltransferases (EC2.5.1.129).

[0153] In a more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene is performed using a phenylacrylate decarboxylase (PAD) type protein as an FMN prenyltransferase that modifies a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor. The phenylacrylate decarboxylase (PAD) type protein is derived from Candida albicans (Uniprot accession number Q5A8L8), Aspergillus niger (Uniprot accession number A3F715), Saccharomyces cerevisiae (Uniprot accession number P33751), or Cryptococcus gattii (Uniprot accession number E6R9Z0).

[0154] In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) to a corresponding (modified) flavin-derived cofactor is catalyzed by FMN-containing protein 3-octaprenyl-4-hydroxybenzoate carboxylyase (originally annotated as EC 4.1.1.~), also known as UbiX. As described above, the enzymes involved in the modification of a flavin cofactor (FMN or FAD) to a corresponding modified flavin-derived cofactor were originally annotated as decarboxylases. Some phenylacrylate decarboxylases (PADs) are now annotated as flavinprenyltransferases as EC 2.5.1.~.

[0155] As described above, the enzyme capable of catalyzing the enzymatic reactions described herein, flavinprenyltransferase, has recently been annotated as flavinprenyltransferase, EC 2.5.1.129.

[0156] In a more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene is performed using 3-octaprenyl-4-hydroxybenzoate carboxylyase (also known as UbiX) as an FMN prenyltransferase that modifies a flavin cofactor (FMN or FAD) into the corresponding (modified) flavin-derived cofactor. The 3-octaprenyl-4-hydroxybenzoate carboxylyase (also known as UbiX) is derived from Escherichia coli (Uniprot accession number P0AG03), Bacillus subtilis (Uniprot accession number A0A086WXG4), Pseudomonas aeruginosa (Uniprot accession number A0A072ZCW8), or Enterobacter sp. DC4 (Uniprot accession number W7P6B1).

[0157] In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) to a corresponding (modified) flavin-derived cofactor is catalyzed by Ubx-like flavin prenyltransferases derived from Escherichia coli (E. coli) encoded by kpdB and ecdB (UniProt accession numbers A0A023LDW3 and P69772, respectively), and by UbiX-like flavin prenyltransferases derived from Klebsiella pneumoniae (UniProt accession number Q462H4) encoded by kpdB.

[0158] In another preferred embodiment, the modification of a flavin cofactor (FMN or FAD) to a corresponding (modified) flavin-derived cofactor is catalyzed by a flavin prenyltransferase.

[0159] As described above, the actual decarboxylation, i.e., the conversion of 3-methylcrotonic acid to isobutene, is catalyzed by FMN-dependent decarboxylases via a 1,3-dipolar cycloaddition-based mechanism, which utilizes prenylated flavin cofactors (FMN or FAD) provided by one of the associated FMN prenyltransferases.

[0160] In a preferred embodiment, the FMN-dependent decarboxylase that catalyzes the decarboxylation of 3-methylcrotonic acid to isobutene is catalyzed by ferulic acid decarboxylase (FDC). Ferulic acid decarboxylase (FDC) belongs to enzyme class EC 4.1.1.~.

[0161] In a more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene is performed using ferulic acid decarboxylase (FDC) derived from budding yeast (Saccharomyces cerevisiae) (Uniprot accession number Q03034), Enterobacter sp. (Uniprot accession number V3P7U0), Bacillus pumilus (Uniprot accession number Q45361), Aspergillus niger (Uniprot accession number A2R0P7), or Candida dubliniensis (Uniprot accession number B9WJ66).

[0162] In another, more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene is performed using protocatechuic acid decarboxylase (EC 4.1.1.63).

[0163] In a preferred embodiment of the present invention, the PCA decarboxylase used in the method of the present invention is a PCA decarboxylase derived from Klebsiella pneumoniae (Uniprot accession number B9AM6), Leptolyngbya sp. (Uniprot accession number A0A0S3U6D8), or Phascolarctobacterium sp. (Uniprot accession number R6IIV6).

[0164] In another preferred embodiment, the FMN-dependent decarboxylase that catalyzes the decarboxylation of 3-methylcrotonic acid to isobutene is an enzyme closely related to the ferulate decarboxylase (FDC) described above, namely 3-polyprenyl-4-hydroxybenzoate decarboxylase (also known as UbiD). 3-polyprenyl-4-hydroxybenzoate decarboxylase belongs to the UbiD decarboxylase family, which is classified as EC 4.1.1.~.

[0165] In a more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene is carried out by Hypocrea atroviridis (UniProt accession number G9NLP8), Sphaerulina musiva (UniProt accession number M3DF95), Penicillium requeforti (UniProt accession number W6QKP7), Fusarium oxysporum f. sp. lycopersici (UniProt accession number W9LTH3), Saccharomyces kudriavzevii (UniProt accession number J8TRN5), Saccaromyces cerevisiae, and Aspergillus paradicicus. 3-polyprenyl-4-hydroxybenzoic acid decarboxylase (UbiD) derived from *Candida parasiticus*, *Candida albicans*, *Grosmannia clavigera*, *Escherichia coli* (Uniprot accession number P0AAB4), *Bacillus megaterium* (Uniprot accession number D5DTL4), *Methanothermobacter sp.* CaT2 (Uniprot accession number T2GKK5), *Mycobacterium chelonae* 1518 (Uniprot accession number X8EX86), or *Enterobacter cloacae* (Uniprot accession number V3DX94) is used.

[0166] In another, more preferred embodiment, the conversion of 3-methylcrotonic acid to isobutene is performed using a UbiD-like decarboxylase derived from a species of the genus Streptomyces (Streptomyces sp) (UniProt accession number A0A0A8EV26).

[0167] In a preferred embodiment, in incubation step (b)(i) of the method of the present invention, a microorganism expressing an FMN prenyltransferase and / or an FMN-dependent decarboxylase that overexpresses an enzyme exhibiting improved properties, such as higher enzyme activity or higher substrate specificity, is used.

[0168] In another preferred embodiment, in incubation step (b)(ii) of the method of the present invention, an FMN-dependent decarboxylase accompanied by an FMN prenyltransferase is used, which is produced using a microorganism that overexpresses and / or uses an enzyme exhibiting improved properties, such as higher enzyme activity or higher substrate specificity. Corresponding recombinant microorganisms and enzymes exhibiting such improved properties are described in the prior art, for example, in International Publication No. 2018 / 206262.

[0169] The disclosures in this document, particularly those relating to the overexpression of enzymes exhibiting improved characteristics, such as higher enzyme activity or higher substrate content, and / or enzymes exhibiting improved characteristics, such as higher enzyme activity or higher substrate content, are incorporated herein by reference in their entirety.

[0170] The present invention also aims to provide an alternative method for producing isobutene from 3-methylcrotonic acid or a compound closely related to 3-methylcrotonic acid.

[0171] For example, in one embodiment, the culture step (a) of the method according to the present invention may be omitted, and 3-methylcrotonic acid (regardless of how it was produced) may be used in the incubation step (b) above, in which the gas supply is controlled to enable the efficient conversion of 3-methylcrotonic acid to isobutene.

[0172] Therefore, in one embodiment, the present invention relates to a method as described in the present invention, wherein the culture step (a) is omitted and 3-methylcrotonic acid is (directly) supplied to a container used in the incubation step (b). Thus, in one embodiment, the present invention relates to a method for producing isobutene from 3-methylcrotonic acid: (a) The step of supplying 3-methylcrotonic acid to the liquid culture medium in the container; (b)(i) Incubating a microorganism expressing FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and / or (ii) Incubate the FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a). The 3-methylcrotonic acid contained in the liquid culture medium is enzymatically converted to isobutene; The steps include: producing the isobutene thereafter; (c) A step of recovering the produced isobutene and It is characterized by including, The incubation in step (b) is (a) The container without a gas supply; or (b) A method for producing isobutene from 3-methylcrotonic acid, performed in a vessel having a gas supply of <0.1 vvm (vessel volume per minute) using an inlet gas.

[0173] The term "supplying 3-methylcrotonic acid to the liquid medium in the container" means that 3-methylcrotonic acid is supplied to the liquid medium in the container in which incubation step (b) is to be performed. This can be achieved by first supplying the liquid medium in which incubation is to be performed to the container, and then adding 3-methylcrotonic acid to the medium at the desired concentration, or by supplying the container with a medium that already contains 3-methylcrotonic acid. The microorganisms / enzymes used to achieve the conversion of 3-methylcrotonic acid can be added before supplying the 3-methylcrotonic acid, after supplying the 3-methylcrotonic acid, or simultaneously. Furthermore, 3-methylcrotonic acid may be supplied to the medium in the container during incubation step (b). This can be done continuously or in batches. In this way, the concentration of 3-methylcrotonic acid in the medium can be maintained at a desired level, for example, to enable efficient conversion to isobutene. Therefore, in such embodiments, it is conceivable that the concentration of 3-methylcrotonic acid in the culture medium is monitored (for example, continuously or at predetermined time intervals), and that additional 3-methylcrotonic acid is supplied to the culture medium to adjust the concentration to a desired level.

[0174] For more preferred embodiments of incubation step (b), the same applies as described above with respect to the first aspect of the present invention.

[0175] Alternatively, the present invention also relates to the method described above, but in step (a) the hydrated form of 3-methylcrotonic acid (i.e., 3-hydroxy-3-methylbutyrate (also known as 3-hydroxyisovaleric acid, HIV)) is produced rather than 3-methylcrotonic acid.

[0176] Methods for producing 3-hydroxy-3-methylbutyrate via various possible pathways are described (see Figure 2 for an overview). Methods utilizing these pathways and enzymatic conversions, as well as recombinant organisms and microorganisms, are described, for example, in International Publication No. 2012 / 052427, International Publication No. 2017 / 085167, and International Publication No. 2016 / 042012.

[0177] The disclosures of these documents are incorporated herein by reference in their entirety, particularly with regard to preferred embodiments of enzymes for the individual transformations of the pathway yielding 3-hydroxy-3-methylbutyrate as described therein. Therefore, in preferred embodiments, it is preferable to use enzymes selected from the preferred embodiments described in these prior art documents with respect to each enzymatic transformation. In such alternative methods, once 3-hydroxy-3-methylbutyrate is produced in step (a), the incubation step (b) then includes the production of 3-methylcrotonic acid from the 3-hydroxy-3-methylbutyrate thus produced, preferably by thermochemical conversion, by dehydrating it to 3-methylcrotonic acid (see Figure 2 for illustration). In the same step (b), the 3-methylcrotonic acid thus produced is then converted to isobutene as described above.

[0178] In another preferred embodiment, as outlined above with respect to the thermochemical conversion of 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene, the above alternative method is used in which 3-hydroxy-3-methylbutyric acid is used instead of 3-methylcrotonic acid, with the necessary modifications made. In this alternative method, the 3-hydroxy-3-methylbutyric acid contained in the liquid culture medium obtained in step (a) is thermochemically converted to isobutene, preferably at a temperature of 180°C to 400°C.

[0179] Therefore, in the corresponding step (b), the 3-hydroxy-3-methylbutyrate contained in the liquid culture medium obtained in step (a) is thermochemically converted to isobutene. Preferably, the thermochemical conversion is achieved at a temperature of 180°C to 400°C.

[0180] 3-Hydroxy-3-methylbutyric acid can be efficiently converted to isobutene and carbon dioxide according to procedures known in the art. Preferably, 3-Hydroxy-3-methylbutyric acid is heated to a temperature of 180°C to 400°C, preferably 230°C to 350°C. In another preferred embodiment, the thermochemical conversion is achieved in a boiling reactor, a stirred tank reactor, or a tubular reactor. In another preferred embodiment, the thermochemical conversion is achieved at a pressure of 0 to 30 bar, preferably 10 to 30 bar.

[0181] Preferably, 3-hydroxy-3-methylbutyrate is converted to isobutene in gaseous form, water, and carbon dioxide.

[0182] In another alternative embodiment, the present invention relates to the method described above, but wherein step (a) produces a phosphorylated form of 3-hydroxy-3-methylbutyrate (i.e., 3-phosphonoxy-3-methylbutyrate; also known as 3-phosphonoxyisovaleric acid (PIV)) rather than 3-methylcrotonic acid.

[0183] Methods for producing 3-phosphonoxy-3-methylbutyrate via various possible pathways are described (see Figure 2 for an overview). Methods utilizing these pathways and enzymatic conversions, as well as recombinant organisms and microorganisms, are described, for example, in International Publication No. 2012 / 052427, International Publication No. 2017 / 085167, and International Publication No. 2016 / 042012.

[0184] The disclosures in these documents are incorporated herein by reference in their entirety, particularly with regard to preferred embodiments of enzymes for the individual transformations of the pathway yielding 3-phosphonoxy-3-methylbutyrate as described therein. Therefore, in preferred embodiments, it is preferable to use enzymes selected from the preferred embodiments described in these prior art documents with respect to each enzymatic transformation.

[0185] In such alternative methods, once 3-phosphonoxy-3-methylbutyrate is produced in step (a), the incubation step (b) then includes the production of 3-hydroxy-3-methylbutyrate from the 3-phosphonoxy-3-methylbutyrate thus produced, preferably by dephosphorylation of 3-phosphonoxy-3-methylbutyrate (hydrolysis resulting in the formation of an -OH bond) (see Figure 2 for illustration), and the step (b) then further includes the production of 3-methylcrotonic acid from the 3-hydroxy-3-methylbutyrate thus produced, preferably by thermochemical conversion, by dehydrating it to 3-methylcrotonic acid.

[0186] In the same step (b), the 3-methylcrotonic acid thus produced is then converted to isobutene as described above.

[0187] Alternatively, in such an alternative method, once 3-phosphonoxy-3-methylbutyrate is produced in step (a), the incubation step (b) then includes the direct production of 3-methylcrotonic acid from the 3-phosphonoxy-3-methylbutyrate thus produced, preferably by dephosphorylation and simultaneous double bond formation.

[0188] In the same step (b), the 3-methylcrotonic acid thus produced is then converted to isobutene as described above.

[0189] In further alternative embodiments, the present invention relates to the method described above, but wherein, as described above, in step (a), the hydrated form of 3-methylcrotonic acid (i.e., also known as 3-hydroxy-3-methylbutyrate (3-hydroxyisovaleric acid, HIV)) and the phosphorylated form of 3-hydroxy-3-methylbutyrate (i.e., 3-phosphonoxy-3-methylbutyrate; also known as 3-phosphonoxyisovaleric acid (PIV)) are produced rather than 3-methylcrotonic acid.

[0190] In such an alternative method, once 3-hydroxy-3-methylbutyrate is produced in step (a), the incubation step (b) then includes the production of 3-methylcrotonic acid from the 3-hydroxy-3-methylbutyrate thus produced, as described above. In the same step (b), the 3-methylcrotonic acid thus produced is then converted to isobutene as described above.

[0191] Furthermore, in such alternative methods, once 3-phosphonoxy-3-methylbutyrate is produced in step (a), the incubation step (b) then includes the production of 3-hydroxy-3-methylbutyrate from the 3-phosphonoxy-3-methylbutyrate thus produced, as well as the production of the 3-methylcrotonic acid described above from the 3-phosphonoxy-3-methylbutyrate thus produced and / or the direct production of 3-methylcrotonic acid. In the same step (b), the 3-methylcrotonic acid thus produced is then converted to isobutene as described above.

[0192] As described above, the methods according to the present invention are particularly useful for the large-scale in vivo production of isobutene, especially for commercial production. The present invention describes novel means and methods for the commercial and cost-effective production of large quantities of isobutene that have not been previously obtainable. The large quantities of isobutene produced can then be further converted in a commercial environment to produce large quantities of, for example, drop-in gasoline (e.g., isooctane, ETBE, MTBE), jet fuel, cosmetics, chemicals, for example, methacrylic acid, polyisobutene, or butyl rubber. As used herein, “large-scale production,” “commercial production,” and “bioprocessing” of isobutene in a fermentation vessel or in vitro are carried out at a capacity of at least 100 liters, preferably at least 400 liters, more preferably 1,000 liters or more, and even more preferably 5,000 liters or more. As used herein, “large quantities” specifically excludes trace amounts that can be essentially produced by microorganisms.

[0193] This specification references numerous documents, including patent applications. While the disclosures of these documents are not deemed to relate to the patentability of the present invention, their entirety is incorporated herein by reference. More specifically, all references are incorporated by reference to the same extent that each individual document is specifically and individually indicated as being incorporated by reference. The present invention may include the following embodiments. [1] A method for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b)(i) Incubate a microorganism expressing FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and / or (ii) Incubate the FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a). The 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) is enzymatically converted to isobutene; The steps include: producing the isobutene thereafter; (c) A step of recovering the produced isobutene and Characterized by including; or Methods for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b) A step of thermochemically converting the 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene, preferably at a temperature of 180°C to 400°C; (c) A step of recovering the produced isobutene and A method for producing isobutene from a carbon source, characterized by including [a specific component]. [2] The incubation in step (b) is (a) A container without a gas supply; or (b) A container with an inlet gas supply at <0.1 vvm (container volume per minute) The method according to claim 1, which is performed by [the specified method]. [3] The method according to claim 1 or 2, wherein the liquid culture medium containing 3-methylcrotonic acid in step (a) is isolated from the microorganisms before step (b). [4] The method according to any one of claims 1 to 3, wherein the 3-methylcrotonic acid is isolated or purified from the liquid culture medium before step (b) of claim 1. [5] The inlet gas is air, an inert gas, or a mixture of air and an inert gas, and the inert gas is preferably nitrogen, helium, argon, neon, or CO2. 2 The method according to any one of claims 1 to 4, selected from a mixture of these gases. [6] The method according to any one of claims 1 to 5, wherein the carbon source is metabolized to acetyl-CoA before its enzymatic conversion to 3-methylcrotonic acid. [7] The method according to any one of claims 1 to 6, wherein the carbon source is selected from the group consisting of glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, and mixtures thereof. [8] The method according to any one of claims 1 to 7, wherein the microorganism used in claim 1(b)(i) is pre-cultured in a suitable liquid culture medium under suitable conditions prior to the conversion step (b)(i) of claim 1. [9] The method according to any one of claims 1 to 8, further comprising the step of purifying / concentrating the recovered isobutene.

[10] The method according to any one of claims 1 to 9, wherein the microorganism is a bacterium, yeast, fungus, or algae.

[0194] The present invention is described herein by reference to the following examples, which are merely illustrative and should not be construed as limitations on the scope of the invention. [Examples]

[0195] [Example 1] Isobutene production by a two-stage process in a 15L reactor

[0196] Step 1: In vivo production of 3-methylcrotonic acid from acetyl-CoA This example demonstrates the production of 3-methylcrotonic acid by a recombinant Escherichia coli (E. coli) strain expressing an exogenous gene, thereby forming the 3-methylcrotonic acid pathway.

[0197] Like most microorganisms, Escherichia coli converts glucose to acetyl-CoA. The enzymes used in this experiment to convert acetyl-CoA to 3-methylcrotonic acid (Figure 2) are summarized below.

[0198] Expression of the 3-methylcrotonic acid biosynthesis pathway in Escherichia coli (E. coli) The following genes were codon-optimized for expression in Escherichia coli (E. coli) and synthesized by GeneArt (Life Technologies): - thl (Uniprot accession number Q6LD78) derived from Clostridium acetobutylicum - Ech (enoyl-CoA hydratase) derived from Pseudomonas sp. (Uniprot accession number K9NHK2) - mvaS (Uniprot accession number P54874) derived from fission yeast (Schizosaccharomyces pombe) - aibA and aibB (Uniprot accession numbers AKQ65711.1 and AKQ65710.1) encoding two subunits of glutaconic acid-CoA transferase derived from Myxococcus hansupus. - MenI derived from Escherichia coli (strain K12) (Uniprot accession number P77781).

[0199] An expression vector containing the origin of replication for pSC101 (see: http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC320470 / ) was used to express the genes mvaS, Ech, aibA, aibB, and ydiI according to the procedure described in Example 12 of International Publication No. 2017 / 085167 (except for the incorporation of the FDC1 gene). The recombinant pGBE13786 plasmid was validated by sequencing.

[0200] Strain MG1655 was modified by incorporating the thl gene from Clostridium acetobutylicum into the ssrS locus. The resulting strain (GBI19077) was electrocompetent and transformed with pGBE13786.

[0201] Transformed cells, strain SB1429, were then placed in LB plates and supplied with tetracycline. The plates were incubated overnight at 30°C. The isolated colonies were used to prepare the pre-cultures described below.

[0202] Production of 3-methylcrotonic acid A 15L container was filled with 6L of culture medium containing 15g / L yeast extract, 50mM monosodium glutamate, 4mM magnesium sulfate, 5mM sodium sulfate, 10mM ammonium sulfate, 25mM potassium dihydrogen phosphate, and 25mM disodium hydrogen phosphate, and sterilized at 121°C for 20 minutes. After cooling, filter-sterilized vitamins were added at final concentrations of 0.6mM for thiamine and 5mM for calcium panthotenate. Filter-sterilized trace metals were also added at final concentrations of 10μM iron(III) chloride, 4μM calcium chloride, 2μM manganese chloride, 2μM zinc sulfate, 0.4μM copper chloride, and 0.4μM sodium molybdate. Then, filter-sterilized glucose was added at a final concentration of 1g / L.

[0203] In addition to the batch culture medium, two types of Fed-Batch solutions were prepared. The first Fed-Batch solution was a 300 g / L yeast extract solution that had been filtered and sterilized. The second Fed-Batch solution was a 700 g / L glucose solution containing 50 μM iron(III) chloride, 20 μM calcium chloride, 10 μM manganese chloride, 10 μM zinc sulfate, 2 μM copper chloride, and 2 μM sodium molybdate at a final concentration of 5 g / L magnesium sulfate heptahydrate, 10 mM sodium glutamate, and trace metals.

[0204] 500 mL of pre-culture of strain (SB1429), which had been previously grown in LB medium containing 50 mM sodium glutamate and tetracycline at 30°C, was inoculated into the culture medium. The temperature was maintained at 32°C for 30 hours, and then raised to 34°C. Aeration was set to 0.77 vvm, and stirring was controlled to maintain dissolved oxygen at a saturation of 5%.

[0205] Six hours after the start of culture, 400 mL of yeast extract was continuously added over 18 hours. In parallel, a glucose-fed batch was started eight hours after the start of culture, and a specific supply rate of 0.1 g glucose per gram of dry cell weight per hour was maintained for 22 hours.

[0206] Next, the specific supply rate was initially increased to 0.25 g / g / h, and then adjusted to maintain low levels of glucose and acetic acid in the culture medium. 3-methylcrotonic acid production was monitored by HPLC, and fermentation was stopped when acetic acid began to accumulate instead of the desired product.

[0207] When fermentation was stopped, more than 20 g / L of 3-methylcrotonic acid was produced at that point. The culture medium was then clarified by centrifugation and used in the second step and in Example 2.

[0208] Step 2: Production of isobutene from 3-methylcrotonic acid pSC101 induction vector Using (reference: http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC320470 / ), we expressed mutants of prenylated FMN-dependent 3-methylcrotonic acid decarboxylase (FDC) from Streptomyces sp. 769 (UniProt accession number A0A0A8EV26) and Ubx-like flavin prenyltransferase from Klebsiella pneumoniae (kpdB; UniProt accession number Q462H4). We transformed E. coli (MG1655) cells with a constructed plasmid, and the newly obtained strain SB1505 cells were grown to a cell density of approximately 35 g / L in a nutrient-rich medium containing yeast extract and inorganic salts, along with glucose as a carbon source. The cells were collected by centrifugation, resuspended in the supernatant at a concentration of 250 g / L, and kept at 4°C for up to 3 weeks before use.

[0209] A 15L reactor was filled with 12L of culture medium containing methyl 3-crotonate and stirred at 800 RPM. The temperature was set to 37°C and the pH was controlled to 6.3 with 20% phosphoric acid. Air was circulated from the reactor headspace (approximately 3L) by venting the container with nitrogen through a sparger, and the pressure was controlled to 0.5 bar. The outlet gas was analyzed, and when oxygen was no longer detectable, the gas supply nitrogen was set to 0.017 vvm. 1L of concentrated cells of strain MB106 was added to the container to start isobutene production.

[0210] At that point, the concentration of 3-methylcrotonic acid was 231 mM. Figure 5 shows the composition of exhaust gas over time. Production of isobutene was stopped once 3-methylcrotonic acid was no longer detected in the culture medium.

[0211] [Example 2] Production of isobutene from 3-methylcrotonic acid in a 1L reactor

[0212] The incubation step was performed in a 1L container under the following conditions:

[0213] [Table 1] The incubation medium consisted of the following:

[0214] [Table 2]

[0215] The outlet gases (IBN, CO2) and 3-methylcrotonic acid consumption were monitored over time.

[0216] The results are shown in Figure 6 (showing the consumption rates of isobutene (IBN) and 3-methylcroton), Figure 7 (showing the total IBN production and the conversion of 100% of 3-methylcrotonic acid to isobutene (IBN)), and Figure 8 (showing that high concentrations of IBN and CO2 are produced during incubations without gas supply and with gas supply at <0.1 vvm, respectively, compared to incubation with gas supply at 1 vvm).

[0217] summary: If the gas supply is low (0.05 vvm) or there is no gas supply (0 vvm): • High IBN concentration (and therefore easy to purify) • No risk of combustion / explosion This was shown.

[0218] [Example 3] 3-Methylcrotonic acid purification, process 1

[0219] A 15-liter fermentation tank was treated according to the conditions described in Example 1. The biomass was removed by centrifugation to obtain 10.8 L of supernatant of 29 g / L 3-methylcrotonate. The supernatant was then acidified by adding 270 g of 98% sulfuric acid until the pH was adjusted to 3.5 before the evaporation step.

[0220] Evaporation was carried out using a RotaVapor R300 (Buchi) at a heating temperature of 80°C, a cooling temperature of 10°C, and a pressure of 150 mbar. 3-methylcrotonic acid crystals were recovered in a condenser. The crystals were washed off with water and mixed with the distillate. Evaporation was continued until the residue became viscous. 11.7 kg of distillate containing 24.5 g / L of 3-methylcrotonic acid was recovered.

[0221] Next, 600 g of 3M sodium hydroxide was added to the distillate to adjust the pH to 9.1. Evaporation was carried out at a heating temperature of 80°C, a cooling temperature of 10°C, and a pressure of 150 mbar until a solid appeared in the residue. 900 g of 35 w% sodium 3-methylcrotonate was recovered.

[0222] 488 g of 20% sulfuric acid and 11 g of 80% sulfuric acid were added to precipitate 3-methylcrotonic acid. The slurry was filtered through a Buchner funnel to obtain 488 g of a wet solid of 57 w% 3-methylcrotonic acid.

[0223] [Example 4] 3-Methylcrotonic acid purification, process 2

[0224] A 15-liter fermentation tank was treated according to the conditions described in Example 1. Biomass was removed by centrifugation to obtain 7.1 L of supernatant of 27.3 g / L 3-methylcrotonate. Next, 204 g of 3M sodium hydroxide was added to alkalize the mixture and adjust the pH to pH 9.0.

[0225] Evaporation was carried out using a RotaVapor R300 (Buchi) at a heating temperature of 80°C, a cooling temperature of 10°C, and a pressure of 150 mbar. Evaporation was continued until a solid appeared in the residue. 1.4 kg of distillate containing 117 g / L of 3-methylcrotonic acid was recovered.

[0226] The distillate was cooled to 10°C and filtered through a Buchner funnel.

[0227] Next, 165 g of 98% sulfuric acid was added to the residue to adjust the pH to 3.78. Evaporation was carried out at a heating temperature of 80°C, a cooling temperature of 10°C, and a pressure of 150 mbar until a solid appeared in the residue. 900 g of 35 w% sodium 3-methylcrotonate was recovered, yielding 146 g of wet solid 52 w% 3-methylcrotonic acid.

[0228] [Example 5] 3-Methylcrotonic acid purification, process 3

[0229] 40 mL of clarified broth containing 21 g / L 3-methylcrotonate (obtained according to Example 1) was introduced into a 200 mL glass jacketed stirring cell. 98% sulfuric acid was then added to acidify the broth to pH=2. Next, 40 mL of solvent was added to the cells in a 1 / 1 vol% / vol% ratio. The following solvents were tested in this experiment: 2-Octanol (CAS number: 123-96-6) Isododecane (IDD, CAS number: 31807-55-3) Heptanoic acid (CAS number: 111-14-8) 4-Methyl-2-pentanone (CAS number: 108-10-1)

[0230] The cell temperature was set to 20°C, and the mixture was vigorously stirred for 2 hours. Then, stirring was stopped for 16 hours to allow the two liquid phases to form. Finally, each phase was collected separately and weighed. Next, each phase was analyzed as follows: The water phase analyzed the situation as follows: • LC-RID: Quantification of 3-methylcrotonic acid • Dry mass at 200°C The organic phase was analyzed as follows: • Karl Fisher method: Quantification of water • Dry mass at 200°C

[0231] The measured distribution coefficient (K) is shown in the following table:

[0232]

Table 3

[0233] This shows that the four solvents tested can be used efficiently for the liquid - liquid extraction of 3 - methylcrotonic acid from the fermentation broth.

[0234] 3 - methylcrotonic acid was extracted from 1 kg of a clarified fermentation broth containing 2.5 w% 3 - methylcrotonic acid and 1.1 w% acetic acid. 1 kg of 2 - octanol was added and stirred for 16 hours. After decantation, the mass of the organic phase was 1.03 kg. The composition was 97.1 w% 2 - octanol, 2.3 w% prenic acid, 0.5 w% acetic acid, and 0.02 w% water. This mixture was distilled in a 21 - theoretical - stage batch column at a pressure of 100 mbar and a reflux ratio of 2. The first fraction contained 2 - octanol, acetic acid, and water: their compositions changed over time until only 2 - octanol was recovered at the top of the distillation column. When the temperature rose in the middle of the distillation column (indicating that prenic acid also evaporated), the reflux ratio was increased to 5. An intermediate fraction containing 35% 2 - octanol and 65% prenic acid was recovered. The next fraction consisted of 4.9 g of 99.9% prenic acid.

[0235] [Example 6] Thermal conversion of 3 - methylcrotonic acid to isobutene

[0236] The 3 - methylcrotonic acid obtained in Examples 3 - 5 above was dissolved to a temperature of 70 °C and fed into a stirred - tank reactor, tubular reactor. The reactor was operated at a temperature of at least 220 °C and a pressure of 10 - 30 bar. 3 - methylcrotonic acid was converted to gaseous isobutene and carbon dioxide.

[0237] [Example 7] Thermal conversion of 3 - methylcrotonic acid to isobutene

[0238] 3-Methylcrotonic acid was dissolved at 100 °C and pumped continuously. This was preheated to 85 °C and fed into the reactor at a flow rate of 22 g / h. The reactor is a tubular reactor containing 2 mm glass beads (280 mL - 1 inch diameter). The reactor pressure is 15 bar and the temperature is 290 °C. After the reactor, two types of liquid traps (made continuously of water and ethanol and water) were added so that the gas had to pass through the liquid. After running for 115 minutes, the sample was analyzed. The isobutene ratio compared to CO2, which indicates that the yield from 3-methylcrotonic acid to isobutene is 95%, is measured to be 65 (area ratio in GC). The collected liquid in the trap analyzed by GC-MS shows only minor impurities. Some 3-methylcrotonic acid remains in the reactor. This explains that the conversion rate to isobutene is less than 100%.

[0239] [Example 8] Thermal conversion of 3-methylcrotonic acid to isobutene

[0240] 3-Methylcrotonic acid was dissolved at 100 °C and pumped continuously. This was preheated to 85 °C and fed into the reactor at a flow rate of 22 g / h. The reactor is a tubular reactor containing 2 mm glass beads (280 mL - 1 inch diameter). The reactor pressure is 25 bar and the temperature is 290 °C. After the reactor, two types of liquid traps (made continuously of water and ethanol and water) were added so that the gas had to pass through the liquid. After running for 70 minutes, the sample was analyzed. The yield from 3-methylcrotonic acid to isobutene is measured to be 101%. The collected liquid in the trap analyzed by GC-MS shows only minor impurities. Some 3-methylcrotonic acid is recovered in the reactor.

[0241] [Example 9] Production of 3-hydroxy-3-methylbutyric acid from acetyl CoA

[0242] This example demonstrates the production of 3-hydroxy-3-methylbutyrate by a recombinant Escherichia coli (E. coli) strain expressing an exogenous gene, thereby forming the 3-hydroxy-3-methylbutyrate pathway.

[0243] Like most microorganisms, Escherichia coli (E. coli) converts glucose to acetyl-CoA. The enzymes used in this experiment to convert acetyl-CoA to 3-hydroxy-3-methylbutyrate (Figure 2) are summarized below.

[0244] 9.1 Expression of the 3-hydroxy-3-methylbutyrate biosynthesis pathway in Escherichia coli (E. coli) The following genes were codon-optimized for expression in Escherichia coli (E. coli) and synthesized using GeneArt (Life Technologies): - thl (Uniprot accession number P45359) derived from Clostridium acetobutylicum - mvaS (Uniprot accession number P54874) derived from Enterococcus faecalis aibA and aibB (Uniprot accession numbers A0A0H4WQB1 and A0A0H4WWJ4) encode two subunits of glutaconic acid-CoA transferase derived from Myxococcus hansupus. - tesB derived from Escherichia coli (strain K12) (Uniprot accession number P0AGG2) - liuC (Uniprot accession number Q1D5Y4) derived from Myxococcus xanthus.

[0245] An expression vector containing the origin of replication for pSC101 (see: http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC320470 / ) was used to express the genes mvaS, Ech, aibA, aibB, menI, and liuC, following the procedure described in Example 12 of International Publication No. 2017 / 085167 (with the exception of integration of the FDC1 gene and substitution of the ech gene by the liuC gene). The recombinant pGB 5550 plasmid was validated by sequencing (SEQ ID NO: 1).

[0246] Strain MG1655 was modified by incorporating the thl gene from Clostridium acetobutylicum into the ssrS locus.

[0247] To reduce acetyl-CoA conversion to acetate, deletions were made in the ackA, pta, and poxB genes. Lactate production was also reduced by deleting the ldhA gene. The resulting strain (GBI19706) was electrocompetent and transformed with plasmid pGB 5550.

[0248] Transformed cells, strain SB1653, were then placed in an LB plate and supplied with spectinomycin. The plate was incubated overnight at 30°C. The isolated colonies were used to prepare the pre-cultures described below.

[0249] 9.2 Production of 3-hydroxy-3-methylbutyrate in Fed-Batch Mode A 1L container was filled with 0.5L of culture medium containing 5g / L yeast extract, 10g / L tryptone, 50mM monosodium glutamate, 4mM magnesium sulfate, 5mM sodium sulfate, 10mM ammonium sulfate, 25mM potassium dihydrogen phosphate, and 25mM disodium hydrogen phosphate, and sterilized at 121°C for 20 minutes. After cooling, filter-sterilized vitamins were added at final concentrations of 0.6mM for thiamine and 5mM for calcium panthotenate, and filter-sterilized spectinomycin 50mg / L was also introduced into the culture medium. Filter-sterilized trace metals were also added at final concentrations of 10μM iron(III) chloride, 4μM calcium chloride, 2μM manganese chloride, 2μM zinc sulfate, 0.4μM copper chloride, and 0.4μM sodium molybdate. Then, filter-sterilized glucose was added at a final concentration of 1g / L.

[0250] In addition to the batch culture medium, two types of Fed-Batch solutions were prepared. The first Fed-Batch solution was a 250 g / L yeast extract solution that had been filtered and sterilized. The second Fed-Batch solution was a 600 g / L glucose solution containing 50 μM iron(III) chloride, 20 μM calcium chloride, 10 μM manganese chloride, 10 μM zinc sulfate, 2 μM copper chloride, and 2 μM sodium molybdate at final concentrations of 5 g / L magnesium sulfate heptahydrate, 20 g / L monosodium glutamate, and trace metals.

[0251] 500 mL of a pre-culture of strain SB1653, which had been previously grown in LB medium containing 50 mM sodium glutamate and 50 mg / L spectinomycin at 30°C, was inoculated into the culture medium. The temperature was maintained at 32°C for 30 hours, and then raised to 34°C. Aeration was set to 2 vvm, and stirring was controlled to maintain dissolved oxygen at a saturation of 5%. The pH was controlled to 6.5.

[0252] 10 mL of yeast extract was added at 8, 12, and 16 hours after the start of culture. In parallel, a glucose-fed batch was started 8 hours after the start of culture, and a specific supply rate of 0.08 g glucose per gram of dry cell weight per hour was maintained for 22 hours.

[0253] Next, the supply rate was increased to deliver 4 g / l / h of glucose, and later adjusted to maintain low levels of glucose and acetic acid in the culture medium. 3-hydroxy-3-methylbutyrate production was monitored by HPLC, and fermentation was stopped when acetic acid began to accumulate instead of the desired product.

[0254] When fermentation was stopped, more than 80 g / L of 3-hydroxy-3-methylbutyric acid had been produced at that point.

[0255] 9.3 Production of 3-hydroxy-3-methylbutyrate in a quasi-continuous mode A 1L container was filled with 0.5L of culture medium containing 15g / L yeast extract, 13.4g / L monosodium glutamate, 2.2g / L magnesium sulfate, 0.85g / L potassium dihydrogen phosphate, and 1.1g / L disodium hydrogen phosphate, and sterilized at 121°C for 20 minutes. After cooling, sterilized by filtration were added to the culture medium at final concentrations of 0.6mM thiamine and 5mM calcium panthotenate, and sterilized by filtration 50mg / L spectinomycin was also introduced. Sterilized by filtration trace metals were also added at final concentrations of 10μM iron(III) chloride, 4μM calcium chloride, 2μM manganese chloride, 2μM zinc sulfate, and 0.4μM copper chloride. Then, sterilized by filtration glucose was added at a final concentration of 5g / L.

[0256] In addition to the batch culture medium, two types of Fed-Batch solutions were prepared. The first Fed-Batch solution was a filter-sterilized 600 g / L glucose solution. The second Fed-Batch solution was physiological saline containing 0.85 g / L potassium dihydrogen phosphate, 1.1 g / L disodium hydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 50 μM iron(III) chloride, 20 μM calcium chloride, 10 μM manganese chloride, 10 μM zinc sulfate, and 2 μM copper chloride. The cells were reused while maintaining the culture volume at approximately 0.5 liters, and a 1 L container was placed in a Vivaflow 200 PES (200 cm³) to obtain a permeate containing 3-hydroxy-3-methylbutyrate. 2, it was connected to a 0.2 μ module (Sartorius).

[0257] 500 mL of a preculture of strain (SB1653) that had been pre-grown in LB medium containing 50 mM sodium glutamate and spectinomycin at 30 °C was inoculated into the culture medium. The temperature was maintained at 32 °C. Aeration was set to 0.5 vvm, and stirring was controlled to maintain dissolved oxygen at 5% saturation. The pH was controlled to 7.5 using 30% ammonia solution and 5 M phosphoric acid.

[0258] Glucose 5 g / l was added 7.5 hours after the start of the culture. Then, a supply of glucose at 3 g / l / h was applied for 7 hours 9 hours after the start of the culture. 16 hours after the start of the culture, a specific supply of 0.3 g glucose per gram of dry cell weight per hour was applied.

[0259] The production of 3-hydroxy-3-methylbutyric acid was monitored by HPLC, and when the fermentation was stopped, more than 12 L of 3-hydroxy-3-methylbutyric acid solution was produced.

[0260] [Example 10][[ID=​​​​​​​​​​​​​​As described in Example 9.3, 11.9 L of permeate containing 177 g 3-hydroxy-3-methylbutyrate (HMB) and approximately 153 g acetic acid, pH 7.6, was concentrated to 1.7 L using a Buchi 300 evaporator at 80°C and a pressure of 180 mbar. The concentrate was diluted to pH 3.84 with concentrated sulfuric acid and then extracted twice, first with 1.3 L and then with 1.1 L of MIBK (methyl isobutyl ketone). The two MIBK layers were combined and evaporated using a Buchi 300 evaporator at 85°C, initially at a pressure of 150 mbar and reduced to 10 mbar at the end of the operation. 125 ml of liquid phase (132 g) containing 93 g HMB and 10 g acetic acid was recovered.

Claims

1. A method for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b) (i) Incubating a microorganism expressing FMN prenyltransferase and associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and / or (ii) Incubate the FMN prenyltransferase and the associated FMN-dependent decarboxylase in the liquid culture medium containing 3-methylcrotonic acid obtained in step (a). The 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) is enzymatically converted to isobutene; The steps include: producing the isobutene thereafter; (c) A step of recovering the produced isobutene and A method characterized by including, Here, the liquid culture medium containing 3-methylcrotonic acid in step (a) is isolated from the microorganisms before step (b); or Methods for producing isobutene from a carbon source: (a) A step of culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; (b) A step of thermochemically converting the 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) to isobutene at a temperature of 180°C to 400°C; (c) A step of recovering the produced isobutene and Features including, A method for producing isobutene from a carbon source.

2. Step (b) is the incubation step, (a) a container without a gas supply; or (b) A container with gas supplied at <0.1 vvm (container volume per minute) using an inlet gas. The method according to claim 1, which is performed by [the specified method].

3. The method according to claim 1 or 2, wherein the 3-methylcrotonic acid is isolated or purified from the liquid culture medium before step (b) of claim 1.

4. The inlet gas is air, an inert gas, or a mixture of air and an inert gas, and the inert gas is nitrogen, helium, argon, neon, CO2 ２ The method according to claim 2, wherein a mixture of these gases is selected.

5. The method according to any one of claims 1 to 4, wherein the carbon source is metabolized to acetyl-CoA before its enzymatic conversion to 3-methylcrotonic acid.

6. The method according to any one of claims 1 to 5, wherein the carbon source is selected from the group consisting of glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid, and mixtures thereof.

7. The method according to any one of claims 1 to 6, wherein the microorganism used in claim 1(b)(i) is pre-cultured in a suitable liquid culture medium under suitable conditions prior to the conversion step (b)(i) of claim 1.

8. The method according to any one of claims 1 to 7, further comprising the step of purifying / concentrating the recovered isobutene.

9. The method according to any one of claims 1 to 8, wherein the microorganism is a bacterium, yeast, fungus, or algae.

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