WO2018172331A1

WO2018172331A1 - Diterminal oxidation of alkanes

Info

Publication number: WO2018172331A1
Application number: PCT/EP2018/056973
Authority: WO
Inventors: Youri Michel VAN NULAND; Ruud Alexander Weusthuis; Gerrit Eggink
Original assignee: Wageningen Universiteit
Current assignee: Wageningen Universiteit
Priority date: 2017-03-23
Filing date: 2018-03-20
Publication date: 2018-09-27
Anticipated expiration: 2019-09-23

Abstract

A method for the preparation of amono- and/or di-esterified aliphatic α,ω-diol, a mono- and/or di-esterified aliphatic α,ω-dicarboxylic acidora mono-and/ordi-esterified aliphatic ω-hydroxycarboxylic acid by conversion of a substrate selected from an alkane, 1-alkanol, alkanal, alkanoateand/or an alkyl alkanoate, e.g. an ethyl alkanoate or a propylalkanoate, is provided.

Description

Title: Diterminal oxidation of alkanes Field of the invention

The present invention is in the field of the production of diterminally oxidized alkyl chains. Such compounds are useful in polymer chemistry, petrochemistry, and the like.

Background of the invention

Substituted alkanes, for example alcohols, aldehydes, ketones, and carboxylic acids, represent a class of compounds popular in industry. Alkyls having two or more substitutions are essential for many uses, for example as cross-linkers in the production of industrial polymers; however, these compounds are even more difficult to achieve, particularly using biotechnological approaches. It is known in the art how an alkane can be converted into the corresponding 1 -alkanol, but production of the corresponding α,ω-diol (i.e., an alkyl chain with the two terminal carbon atoms each carrying one hydroxyl group) is more difficult to achieve.

The lack of selective and mild (oxidation) processes for (activation of) terminal methyl groups restricts conversion of abundant and inexpensive alkanes and alkanols into structural chemicals. This has long been considered to be a 'hot problem' that has been researched for about half a century. The solution could lead to a paradigm shift to a more sustainable petrochemistry. Several chemical means exist to achieve methyl oxidation of alkanes, via organometallic C- H activation or by heterogeneous catalysis. Due to the relative inertness of terminal C-H bonds compared to subterminal C-H bonds, alkane oxidation often results in subterminal oxidation. The selectivity of diterminal oxidation is low because the C-H bonds near the first functionalized carbon atoms are prone to further oxidation. Synthesis of alkanols or alkanediols is even more challenging because the hydroxy groups are easily further oxidized to aldehydes and carboxylic acids. The application of molecular sieves has greatly improved terminal selectivity, but conversions with molecular sieves are far less selective than monooxygenases. Monooxygenases such as CYP52 or AlkB have been applied for terminal oxidation of n- alkanes with 100% selectivity. CYP52 has been used for the conversion of long-chain alkanes to dicarboxylic acids. This has resulted in production of high titers of α,ω- bifunctional monomers via whole-cell biocatalysis. AlkB was applied for terminal oxidation of medium-chain alkanes. However, AlkB has a tendency to overoxidize the substrate to aldehydes and carboxylic acids. Moreover, monooxygenases function poorly regarding diterminal oxidation of medium-chain compounds because the first functionalized group interferes with further oxidation.

Therefore, two critical breakthroughs have to be realized to enable the selective oxidation of medium chain alkanes: enabling diterminal oxidation and preventing overoxidation. There is a need in the art for new methods of producing diterminally oxidized alkyl chains, preferably of medium-chain length, particularly by means of biocatalysis.

Summary of the invention

The present inventors have shown that in vivo esterification is a promising tool to achieve diterminal oxidation of n-alkanes. The use of a whole-cell biocatalyst allowed for reaching this goal under mild conditions, in a one-pot fashion. The application of monooxygenase AlkB ensured 100 % terminal selectivity. The modular approach of the pathways allowed for steering of the product type. From medium-chain n-alkanes different classes of products were produced, including mono-alkyl dicarboxylic acids, di-alkyl dicarboxylic acids, alkyl acetates, and diacetoxy alkanes. Esterified α,ω-dicarboxylic acids were produced directly from n-alkanes and in vivo esterification in presence of ethanol by Eeb1 improved the diterminal oxidation C6-C10 alkanes. AtfA facilitated diterminal oxidation of n-hexane in absence of exogenously added alcohols. From longer n-alkanes, it mostly formed alkyl alkanoates. The C6 to C10 n-alkanes were generally accepted as substrate, which means that this technology can be applied for a wide variety of molecules. This technology could also be exploited for selective hydroxylation for the production of chiral molecules.

Co-expressing AlkBGTL and Atf1 was the most efficient approach to di-terminal oxidation of n-alkanes. The formed diacetoxyalkanes can be hydrolysed to give α,ω-diols, compounds that are challenging to produce. The α,ω-diols can also be further oxidized to α,ω- dicarboxylic acids. Whole-cell production of α,ω-diols from n-alkanes has been reported before, but only low titers (0.26 g/L maximum) were achieved with high biomass

concentrations. The present inventors managed to produce up to 175.83 mM of

diacetoxyalkane in the organic phase, with 1 .0 g_CCiw/L of resting cells and a phase ratio of 0.1 :1 organic:aqueous. By varying the amount of added n-alkane, the reaction could be steered towards diacetoxyalkanes. ln a first aspect, the present disclosure provides a method for the preparation of mono- esters and/or di-esters of an aliphatic α,ω-diol, aliphatic α,ω-dicarboxylic acid, and/or aliphatic ω-hydroxycarboxylic acid, comprising the steps of:

- providing a substrate selected from an alkane, 1 -alkanol, alkanal, alkanoate and/or an alkyl alkanoate, e.g. an ethyl alkanoate or a propyl alkanoate;

- contacting said alkane, 1 -alkanol, alkanal, alkanoate, and/or an alkyl alkanoate, e.g. an ethyl alkanoate or a propyl alkanoate with:

- a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module.

Such mono-esters and/or di-esters of an aliphatic α,ω-diol, aliphatic α,ω-dicarboxylic acid, and/or aliphatic ω-hydroxycarboxylic acid may be used as intermediates in the production of the corresponding aliphatic α,ω-diol, aliphatic α,ω-dicarboxylic acid, and/or aliphatic ω- hydroxycarboxylic acid.

In an embodiment, the alkane, 1 -alkanol, alkanal, alkanoate, and/or alkyl alkanoate may be converted to the corresponding mono- and/or di-esterified α,ω-diol, and/or ω-hydroxyacid In such embodiment, said alkane, 1 -alkanol, alkanal, alkanoate, and/or alkyl alkanoate may be contacted with a monooxygenase module and an esterification module.

The esterification module may use a carboxylic acid abundantly available in a host cell, such as acetyl-CoA. The esterification module may comprise, or consist of, Atf1 . The monooxygenase module may comprise AlkBGT. The monooxygenase module may further comprise AlkL.

The substrate may be selected from a C3-16 alkane, 1 -C3-16 alkanol, C3-C16 alkanal, C3- 16 alkanoate, and/or a C2-16 alkyl alkanoate.

In another embodiment, the alkane, 1 -alkanol, alkanal, alkanoate, and/or alkyl alkanoate may be converted to the mono- and/or di-ester of the corresponding aliphatic α,ω

dicarboxylic acid or aliphatic ω-hydroxycarboxylic acid. In such embodiment, said alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate may be contacted with a monooxygenase module, a dehydrogenase module, and an

esterification module. The esterification module may then use an alcohol, which may be added exogenously or produced endogenously, preferably wherein said alcohol is ethanol or propanol.

The esterification module may comprise, or consist of, an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and an AtfA alcohohacyltransferase, such as from

Acinetobacter baylyi; or an alcohol :acetyltransferase such as Atf1 , preferably from

Saccharomyces cerevisiae; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from Saccharomyces cerevisiae.

The monooxygenase module may comprise AlkBGT, and may optionally further comprise AlkL

The dehydrogenase module may comprise, or consist of, an alcohol dehydrogenase, such as AlkJ, preferably from Pseudomonas putida GPo1 , and an aldehyde dehydrogenase, such as AlkH, preferably from Pseudomonas putida GPo1 .

The substrate may be selected from a C3-16 alkane, 1 -C3-16 alkanol, and/or an alkanoate C3-16 alkylester.

In an embodiment, at least one of the monooxygenase module, esterification module, and/or dehydrogenase module is provided in the form of a host cell expressing said modules.

In an embodiment, all of the monooxygenase module, esterification module, and, optionally, dehydrogenase module are provided in the form of a host cell expressing said modules. The host cell may be a prokaryotic cell or a eukaryotic cell.

In an aspect, the present disclosure further provides a genetically modified microorganism capable of selective terminal and/or diterminal oxidation of an aliphatic compound, comprising:

- a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module. Said microorganism may comprise at least one exogenous nucleic acid encoding:

- a monooxygenase module;

- optionally, a dehydrogenase module; or

- an esterification module.

In an embodiment, said microorganism comprises at least one exogenous nucleic acid encoding AlkBGT, preferably derived from Pseudomonas putida GPo1 .

Said microorganism may comprise at least one exogenous nucleic acid encoding an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and/or an AtfA

alcohohacyltransferase, such as from Acinetobacter baylyi; or an alcohohacetyltransferase with a wide substrate spectrum such as Atf1 , preferably from Saccharomyces cerevisiae; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from Saccharomyces cerevisiae.

Said microorganism may comprise at least one exogenous nucleic acid encoding AlkJ, preferably from Pseudomonas putida GPo1 , and/or an aldehyde dehydrogenase, such as AlkH, preferably from Pseudomonas putida GPo1 . Detailed description of the invention

As used herein, the terms "comprising" and "to comprise", and their conjugations, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb "to consist of".

In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references referred to herein are incorporated by reference in their entirety. Method for the production of α,ω-oxidized alkyl chains

The present disclosure provides for a method for the preparation of mono- and/or di-esters of an aliphatic α,ω-diol, aliphatic α,ω-dicarboxylic acid, aliphatic ω-hydroxycarboxylic acid, comprising the steps of:

- providing a substrate selected from an alkane, 1 -alkanol, alkanal, alkanoate and/or an alkyl alkanoate, preferably an ethyl alkanoate or a propyl alkanoate;

- contacting said alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate, such as an ethyl alkanoate or a propyl alkanoate, with:

- a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module.

The term "alkane", as used herein, refers to any compound represented by the formula C_nH_2n+2, wherein n is or is more than 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably a C3-C16 alkane, more preferably a C3-C12 alkane, more preferably a C4-C10 alkane. For example, the alkane may be propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, or the like. The term "1 -alkanol", as used herein, refers to any compound represented by the formula C_nH_2n+20, wherein n is or is more than 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably a C3-C16 alkanol, more preferably a C3-C12 alkanol, more preferably a C4-C10 alkanol, even more preferably a C4- C7 alkanol. The term includes any cycloalkanol. For example, the 1 -alkanol may be 1 - propanol, 1 -butanol, 1 -pentanol, 1 -hexanol, 1 -heptanol, 1 -octanol, 1 -nonanol, 1 -decanol, 1 - undecanol, 1 -dodecanol, or the like.

The term "alkyl alkanoate", as used herein, refers to any alkyl ester of an alkanoate.

Preferably, the alkyl chain on either side of the ester bond comprises, independently, between 1 and 30 carbon atoms, preferably between 2 and 16, more preferably between 2 and 12, even more preferably between 2 and 10 carbon atoms. For example, the alkyl alkanoate may be an ethyl alkanoate, a propyl alkanoate, or any other alkyl ethanoate.

The substrate may be saturated or unsaturated, may be branched or unbranched, and the non-terminal carbon atoms in the substrate may be substituted. ln an embodiment, the monooxygenase module comprises, or consists of, one or more enzymes classified under EC1 .14.14.- or EC1 .14.15.-. Said monooxygenase module may, for example, comprise AlkBGT (Pseudomonas putida GPo1 ), AlkB-related monooxygenases {Acinetobacter, Alcanivorax, Burkholderia, Mycobacterium, Pseudomonas, Rhodococcus) a cytochrome P450 alkane hydroxylase from the CYP153 family, Acinetobacter sp. OC4, Acinetobacter sp. EB104, Polaromonas sp. JS666 monooxygenase, Mycobacterium sp. HXN-1500 monooxygenase, Mycobacterium austroafricanum monooxygenase, a cytochrome P450 alkane hydroxylase from the CYP52 family (Candida maltosa, Candida tropicalis, Yarrowia lipolytica), Pseudomonas butanovora butane monooxygenase, propane monooxygenases (Rhodococcus sp. strain BCP1 , Mycobacterium vaccae JOB5, Gordonia sp. TY-5, Nocardioides CF8 long-chain alkane monooxygenases (Geobacillus

thermooleovorans, Pseudomonas fluorescens CHAO, Dietzia sp.), or the like.

The optional dehydrogenase module may comprise, or consist of, one or more

dehydrogenases, such as an alcohol dehydrogenase, e.g. from the class EC-1 .1 .1 .-, EC 1 .1 .5. -e.g. AlkJ from Pseudomonas putida GPo1 , FA01 from Candida tropicalis, chnD, yjgB from Acinetobacter sp. SE19, ccdC from Rhodococcus ruber, ADHA10 from Candida tropicalis, and/or an (acetylating) aldehyde dehydrogenase, e.g. from the class EC 1 .2.1 .-, such as AlkH from Pseudomonas putida GPo1 , puuC, gabD, feaB, patD, astD from

Escherichia coli, chnE from Acinetobacter sp. SE19, ALDH3A2 from Homo sapiens.

In an embodiment, the esterification module comprises, or consists of, a combination of an acyl-CoA ligase, e.g. from the class EC6.2.1 .-, such as AlkK from Pseudomonas putida GPo1 , and an alcohohacyltransferase, e.g. from the class EC 2.3.1 .- such as AtfA from Acinetobacter baylyr, or an alcohol :acetyltransferase with a wide substrate spectrum such as Atf1 , preferably from Saccharomyces cerevisiae; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from Saccharomyces cerevisiae; or an ethanol acetyltransferase, such as Eat1 from Wickerhamomyces anomalus,

Kluyveromyces marxianus, Saccharomyces cerevisiae; or a chloramphenicol acyltransferase

The reactions of the pathways described herein can be performed in one or more host cells naturally expressing one or more of the relevant modules, or genetically engineered to express one or more of the relevant modules, or both naturally expressing one or more of the relevant modules and genetically engineered to express one or more relevant modules. Alternatively, relevant enzymes comprised within modules can be extracted from one or more host cells and used in a purified or semi-purified form. Extracted enzymes may be immobilized to the floors and/or walls of appropriate reaction vessels. Moreover, host cell lysates may be used as sources of relevant enzymes. In the methods taught herein all the steps can be performed in cells (such as host cells), all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.

At least one of the monooxygenase module, esterification module, and/or dehydrogenase module may be provided in the form of a host cell expressing said modules. The term "host cell" as used herein, is understood as meaning an intact, viable, an metabolically active host cell which provides the desired enzyme activity of the monooxygenase module, esterification module, and/or dehydrogenase module.

In an embodiment, all of the monooxygenase module, esterification module, and, optionally, dehydrogenase module are provided in the form of a single host cell expressing said modules.

The host cell is usually held in an aqueous solution. The skilled person is familiar with numerous aqueous solutions, usually referred to as media, that may be used to grow and sustain host cells, for example LB medium in the case of E. coli. The aqueous solution is kept under aerobic conditions. For growing cells to be used as host cells, it is advantageous that they are cultured in a complex medium to allow for an increase in growth rate compared to a minimal medium.

For carrying out any of the methods taught herein, it is advantageous to use a simple buffer or a minimal medium (i.e., a medium comprising only the minimal set of salts and nutrients indispensable for keeping the host cell in a metabolically active and/or viable state). For example, M9 medium may be used as a minimal medium.

If the substrate to be oxidized has limited solubility in water, an organic solvent or detergent such as Tween or Triton may be added to the aqueous solution, or a hydrophobic solvent may be used to solubilise the substrate to be oxidized. The person skilled in the art is familiar with the preparation of various aqueous and organic solutions.

The conversions may be carried out in a batch mode or in a continuous mode. The person skilled in the art is familiar with suitable fermenters and/or reaction vessels. Fatty acids could be used directly as substrate, by ethylating them first in vivo, followed by ω-oxidation by AlkBGTHJ. The formation of diethyl esters showed that the esterification module was also able to esterify the monoethyl dicarboxylic acids. To efficiently produce esterified α,ω-dicarboxylic acids from alkanes a similar approach was adopted (Fig 1 ). Escherichia coli was equipped with a monooxygenase module, a dehydrogenase module and an esterification module. The monooxygenase module consisted of the AlkBGTL enzymes. The dehydrogenase module consisted of AlkH and AlkJ. The esterification module comprised the AlkK acyl-CoA ligase from Pseudomonas putida GPo1 and the AtfA alcohohacyltransferase from Acinetobacter baylyi. These modules were able to convert n-alkanes with ethanol as co-substrate first into ethylated fatty acids, and in a second round into diethyl esters.

A similar system was adopted for esterified α,ω-alkanediol production, in which the formed alcohols were converted into esters with acetyl-CoA. Atf1 from Saccharomyces cerevisiae is known as a wide substrate spectrum alcohohacetyltransferase with excellent activity with acetyl-CoA and was therefore selected for the esterification module. It was tested in combination with a monooxygenase module consisting of AlkBGTL. Acetoxyalkanes and their ω-oxygenates were the products resulting from this combination.

It is clear from Figure 1 that any intermediate produced from alkanes en route to diterminally oxidated alkanes may be used as a substrate for producing diterminally oxidized alkanes. This includes any aliphatic substrate containing at least one oxidizable terminal methyl group. The aliphatic substrate may comprise one oxidizable terminal methyl group and an, optionally esterified, primary alcohol group or terminal carboxylic acid group.

In an aspect, the method taught herein may comprise the steps of:

Providing an aliphatic substrate containing an oxidizable terminal methyl group and a primary alcohol group or terminal carboxylic acid group, or an ester thereof;

- If not yet esterified (i.e., optionally), converting the primary alcohol group or terminal carboxylic acid group in said substrate into an esterified substrate using an esterification module;

Converting the oxidizable terminal methyl group of the esterified substrate to an ω- hydroxy a-ester using a monooxygenase module; and

- Optionally, converting the ω-hydroxy a-ester to an α,ω-diester using the esterification module. The aliphatic substrate containing an oxidizable terminal methyl group and a primary alcohol group or terminal carboxylic acid group may be prepared by contacting an aliphatic substrate containing two terminal methyl groups with a monooxygenase module. The ω-hydroxy a-ester or α,ω-diester may subsequently be converted to an α,ω-alkane diol using methods well-known to the skilled person.

Methods for producing an esterified α,ω-diol

In an aspect, the present disclosure provides a method for producing mono- and/or di-esters of an α,ω-diol and/or ω-hydroxyacid. Such mono- and/or di-esters of an α,ω-diol and/or ω- hydroxycarboxylic acid can easily be converted into the corresponding α,ω-diol or ω- hydroxycarboxylic acid by conventional hydrolysis. Thus, such mono- or di-esters may be intermediates in the production of the corresponding α,ω-diol and/or ω-hydroxyacid. In this case, the dehydrogenase module is preferably absent, and the alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate is preferably contacted with a monooxygenase module and an esterification module only.

Production of α,ω-diols is known to be highly challenging. Due to overoxidation of the terminal alcohol and poor ω-oxidation capacity of AlkB, carboxylic acids are often the main product. The present inventors managed to prevent overoxidation by converting the produced alcohol directly to an ester, which could subsequently be ω-oxidized.

For production of the ester, a carboxylic acid is necessary, preferably one that is abundantly available in the host, preferably as a CoA-ester. Acetyl-CoA meets both criteria, and therefore is one preferred carboxylic acid for use in the esterification. In a preferred embodiment, said esterification module comprises, or consists of, the alcohol acetyl transferase Atf1 in case production of esterified α,ω-diols is desirable. For production of esterified α,ω-diols, the monooxygenase module may comprise or consist of any enzyme capable of (di)terminal oxidation of an alkane, alkanol or akyl alkanoate. The monooxygenase may, for example, comprise AlkBGT, as shown herein. The skilled person is capable of selecting a suitable monooxygenase module.

An AlkL may further be employed to facilitate uptake of apolar molecules into a host cell. The substrate may be any substrate as taught herein, and may preferably be selected from a C3-16 alkane, 1 -C3-16 alkanol, and/or an C3-16 alkyl alkanoate as taught above with respect to the general method. For example, precursors of the important commodity chemicals 1 ,4-butanediol and 1 ,5-pentanediol could be produced from 1 -butanol or 1 - pentanol, respectively. Depending on the desired compound to be produced by the method taught herein, different substrates may be selected. The skilled person is capable of selecting a suitable starting material to steer production towards the desired compound.

When an α,ω-dialkoxyalkane, ω-alkoxyacid, and/or a ω-alkoxyacid ester are produced, these compounds can easily be converted into the corresponding α,ω-diol or ω-hydroxy fatty acid using routine methods available to the skilled person.

Methods for producing an esterified α,ω dicarboxylic acid

In an aspect, the present disclosure provides a method for producing mono-esters and/or di- esters of an α,ω dicarboxylic acid, and/or an ω-hydroxycarboxylic acid. Such mono- and/or di-esters of an α,ω-dicarboxylic acid and/or ω-hydroxycarboxylic acid can easily be converted into the corresponding α,ω-diol or ω-hydroxyacid by conventional hydrolysis. These mono-esters or di-esters may be used as intermediates in the production of the corresponding α,ω dicarboxylic acid, and/or an ω-hydroxycarboxylic acid,

In this case, since "over"-oxidation of the terminal groups is desired, the substrate is contacted with a monooxygenase module, a dehydrogenase module, and an esterification module. The dehydrogenase module ensures that the terminally oxidized groups become carboxylic acid groups that are subsequently available for esterification by the esterification module.

The esterification module may use an alcohol available in a host cell, or a suitable alcohol may be added for esterification purposes. In a preferred embodiment, the esterification module uses ethanol or propanol. The esterification module comprises, or consists of, an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and an AtfA

In a suitable embodiment, the monooxygenase module comprises, or consists of, AlkBGT. An AlkL may further be employed to facilitate uptake of apolar molecules into a host cell.

In an embodiment, the dehydrogenase module comprises, or consists of, an alcohol dehydrogenase, such as AlkJ, preferably from Pseudomonas putida GPo1 , and an aldehyde dehydrogenase, such as AlkH, preferably from Pseudomonas oleovorans.

The substrate may be any substrate as taught herein, and may preferably be selected from a C3-16 alkane, 1 -C3-16 alkanol, and/or an C3-16 alkyl alkanoate as taught above with respect to the general method.

Genetically modified microorganism capable of producing α,ω-oxidized aliphatic molecules In an aspect, the present disclosure provides a genetically modified microorganism (herein also referred to as "host cell") capable of selective terminal and/or diterminal oxidation of an aliphatic compound, comprising:

- a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module.

The genetically modified microorganism may include endogenous pathways that can be manipulated such that it is capable of selective terminal and/or diterminal oxidation of an aliphatic compound, particularly an alkane, 1 -alkanol, and/or an alkyl alkanoate. In an endogenous pathway the host cell naturally expresses all of the enzymes catalysing the reactions within the pathway. A host cell containing an engineered pathway does not naturally express all of the enzymes catalysing the reactions with the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the host cell. In this case, exogenous enzymes have been introduced.

The term "endogenous" as used herein with reference to a nucleic acid or protein and a host refers to a nucleic acid or protein that does occur in (and can be obtained from) that particular host as it is found in nature.

The term "exogenous" as used herein with reference to a nucleic acid or protein and a host refers to a nucleic acid or protein that does not occur in (and can be obtained from) that particular host as it is found in nature. Thus, a non-naturally occurring nucleic acid is considered to be exogenous to a host once in the host. Importantly, non-naturally occurring nucleic acids may contain nucleic acid sub-sequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid containing a genomic DNA sequence within an expression vector is a non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, as that nucleic acid as a whole does not exist in nature. Genetically modified microorganism may naturally express some of the enzymes of the pathways taught herein. Thus, a pathway within a genetically modified microorganism may include all exogenous enzymes, all endogenous enzymes, or both endogenous and exogenous enzymes. Endogenous genes of the host cell may be disrupted to prevent formation of undesirable metabolites or prevent the loss of intermediates in the pathway through action of other enzymes.

In an embodiment, the host cell comprises at least one exogenous nucleic acid encoding:

- a monooxygenase module;

- optionally, a dehydrogenase module; or

- an esterification module.

For example, the host cell may comprise at least one exogenous nucleic acid encoding AlkBGT, preferably derived from Pseudomonas putida GPo1 .

The host cell may further comprise at least one exogenous nucleic acid encoding AlkL, preferably derived from Pseudomonas putida GPo1 or Psuedomonas oleovorans. Alternatively or additionally, the host cell may comprise at least one exogenous nucleic acid encoding an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and/or an AtfA alcohohacyltransferase, such as from Acinetobacter baylyi; or an

alcohohacetyltransferase with a wide substrate spectrum such as Atf1 , preferably from Saccharomyces cerevisiae; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from Saccharomyces cerevisiae.

Alternatively or additionally, the host cell may comprise at least one exogenous nucleic acid encoding AlkJ, preferably from Pseudomonas putida GPo1 , and/or an aldehyde

dehydrogenase, such as AlkH, preferably from Pseudomonas oleovorans.

In an embodiment, the host cell is a prokaryote. For example, the host cell can be a bacterium from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus

Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans, from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis or from the genus Rhodococcus such as Rhodococcus equi. Such prokaryotes can also be a source of genes to construct host cells as taught herein that are capable of terminal oxidation of alkanes.

In another embodiment, the recombinant host is a eukaryote, e.g., a eukaryote from the genus Candida such as Candida tropicalis, from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica, from the genus Issatchenkia such as Issatchenkia orientalis, from the genus Debaryomyces such as Debaryomyces hansenii, from the genus Arxula such as Arxula adenoinivorans, or from the genus Kluyveromyces such as Kluyveromyces lactis. Such eukaryotes can also be a source of genes to construct host cells as taught herein that are capable of terminal oxidation of alkanes.

Suitable embodiments:

1 . Method for the preparation of mono- and/or di-esters of an aliphatic α,ω-diol, aliphatic α,ω-dicarboxylic acid, aliphatic ω-hydroxycarboxylic acid, comprising the steps of:

- providing a substrate selected from an alkane, 1 -alkanol, alkanal, alkanoate and/or an alkyl alkanoate, e.g. an ethyl alkanoate or an propyl alkanoate;

- contacting said alkane, 1 -alkanol alkanal, alkanoate and/or an alkyl alkanoate, e.g. an ethyl alkanoate or an propyl alkanoate with:

- a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module.

2. Method according to embodiment 1 , wherein the alkane, 1 -alkanol, alkanal, alkanoate, and/or alkyl alkanoate is converted to mono- and/or di-esters of the

corresponding α,ω-diol, or ω-hydroxyacid. 3. Method according to embodiment 2, wherein said alkane, 1 -alkanol, alkanal, alkanoate and/or alkanoate alkylester is contacted with a monooxygenase module and an esterification module. 4. Method according to embodiment 3, wherein the esterification module uses a carboxylic acid abundantly available in a host cell.

5. Method according to embodiment 4, wherein said carboxylic acid is acetyl-CoA. 6. Method according to embodiment 4 or 5, wherein said esterification module comprises, or consists of, Atf1 .

7. Method according to any of embodiments 2-6, wherein the monooxygenase module comprises AlkBGT.

8. Method according to embodiment 7, wherein said monooxygenase module further comprises AlkL.

9. Method according to any of embodiments 2-8,wherein the substrate is selected from a C3-16 alkane, 1 -C3-16 alkanol, and/or an alkanoate C3-16 alkylester.

10. Method according to embodiment 1 , wherein the alkane, 1 -alkanol, alkanal, alkanoate, and/or alkyl alkanoate is converted to mono- and/or di-esters of the

corresponding aliphatic α,ω dicarboxylic acid or aliphatic ω-hydroxycarboxylic acid.

1 1 . Method according to embodiment 10, wherein said alkane, 1 -alkanol, alkanal, alkanoate, and/or alkyl alkanoate is contacted with a monooxygenase module, a dehydrogenase module, and an esterification module. 12. Method according to embodiment 1 1 , wherein the esterification module uses an alcohol, which may be added exogenously or produced endogenously, preferably wherein said alcohol is ethanol or propanol.

13. Method according to any one of the preceding embodiments, wherein the esterification module comprises, or consists of, an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and an AtfA alcohohacyltransferase, such as from

Acinetobacter baylyi; or an alcohohacetyltransferase such as Atf1 , preferably from Saccharomyces cerevisiae; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from Saccharomyces cerevisiae.

14. Method according to any one of embodiments 10-13, wherein the esterification module comprises, or consists of, an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and an AtfA alcohohacyltransferase, such as from Acinetobacter baylyr, or an alcohohacetyltransferase with a wide substrate spectrum such as Atf1 ; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from

Saccharomyces cerevisiae.

15. Method according to any one of embodiments 10-14, wherein the monooxygenase module comprises AlkBGT.

16. Method according to any one of embodiments 10-15, wherein said monooxygenase module further comprises AlkL.

17. Method according to any one of embodiments 10-16, wherein the dehydrogenase module comprises, or consists of, an alcohol dehydrogenase, such as AlkJ, preferably from Pseudomonas putida GPo1 , and an aldehyde dehydrogenase, such as AlkH, preferably from Pseudomonas putida GPo1 .

18. Method according to any one of embodiments 2-8,wherein the substrate is selected from a C3-16 alkane, 1 -C3-16 alkanol, and/or an alkanoate C3-16 alkylester. 19. Method according to any one of the preceding embodiments, wherein at least one of the monooxygenase module, esterification module, and/or dehydrogenase module is provided in the form of a host cell expressing said modules.

20. Method according to any one of the preceding embodiments, wherein all of the monooxygenase module, esterification module, and, optionally, dehydrogenase module are provided in the form of a host cell expressing said modules.

21 . Method according to any one of embodiments 19 or 20, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

22. A genetically modified microorganism capable of selective terminal and/or diterminal oxidation of an aliphatic compound, comprising: - a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module. 23. A genetically modified microorganism according to embodiment 22 comprising at least one exogenous nucleic acid encoding:

- a monooxygenase module;

- optionally, a dehydrogenase module; or

- an esterification module.

24. A genetically modified microorganism according to embodiment 22 or 23 comprising at least one exogenous nucleic acid encoding AlkBGT, preferably derived from

Pseudomonas putida GPo1 . 25. A genetically modified microorganism according to any one of embodiments 22-24 comprising at least one exogenous nucleic acid encoding an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and/or an AtfA alcohohacyltransferase, such as from Acinetobacter baylyr, or an alcohol :acetyltransferase with a wide substrate spectrum such as Atf1 , preferably from Saccharomyces cerevisiae; or a medium-chain fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from Saccharomyces cerevisiae.

26. A genetically modified microorganism according to any one of embodiments 22-25 comprising at least one exogenous nucleic acid encoding AlkJ, preferably from

Pseudomonas putida GPo1 , and/or an aldehyde dehydrogenase, such as AlkH, preferably from Pseudomonas putida GPo1 .

The invention is further illustrated by the following figures and non-limiting examples from which further features, embodiments, aspects and advantages of the present disclosure may be taken.

Brief description of the Figures

Figure 1 shows the proposed pathway for the production of esterified α,ω-bifunctional monomers from n-alkanes or other substrates. The monooxygenase module is responsible for terminal hydroxylation, AlkBGT was selected for this module. The dehydrogenase module converts the formed terminal alcohols to carboxylic acids. Alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH were selected for this module. The esterification module generates esters. The esters enter the cycle again, for ω-functionalization. (a) Dicarboxylic acid pathway. In this pathway, the acyltransferase module consists of two enzymes. AlkK is a medium-chain acyl-CoA ligase, which converts carboxylic acids to acyl- CoA's. AtfA is a broad-specificity wax synthase, which takes the acyl-CoA product from AlkK together with in vivo produced terminal alcohols to produce an alkyl ester. Eeb1 is a more specific alcohol acyltransferase, with a preference for ethanol as alcohol donor. Alkyl esters go through the three modules again, which gives access to esterified dicarboxylic acids and ω-hydroxy acids, (b) Diol pathway. The acyltransferase module in this pathway is Atf1 , which utilizes acetyl-CoA from central carbon metabolism and in vivo produced terminal alcohols. This yields alkyl acetates. Alkyl acetates go through the two modules again, resulting in diacetoxyalkane production.

Figure 2 shows conversion of n-alkanes by induced, resting cells of E. coli pBGTL (negative control) or E. coli pBGTL-atfl (diol pathway). Alkanes were added to 1 % v/v. Applied biomass concentrations were 1 .0 g_Cdw/L.

Figure 3 shows conversion of n-alkanes by induced £ coli pBGTL-aff/ (diol pathway) resting cells. Alkane concentrations in BEHP were varied. The ratio of organic phase to aqueous phase was 1 :2. Applied biomass concentration was 1 .0 g_Cdw/L.

Figure 4 shows conversion of n-alkanes by E. coli pBGTL-atfl (diol pathway) resting cells. The ratio of organic phase to aqueous phase was varied, as well as the alkane

concentration in BEHP. Applied biomass concentration was 1 .0 g_Cd L.

Figure 5 shows conversion of n-alkanes by resting cells of various E. coli strains, as indicated. Alkanes were added to 5 mM in reactions containing ethanol (2.5 %). Applied biomass concentrations were 1 .0 g_cciw/l-.

Figure 6 shows conversion of n-alkanes by resting cells of various E. coli strains, as indicated. Alkanes were added to 1 % v/v. Applied biomass concentrations were 1 .0 g_Cd L. Examples

Materials and methods

Strains. £ coli TOP10 was used for cloning purposes. £. coli NEBT7 (a BL21 derivative) was used for conversion studies. For strains containing a single plasmid 50 μg/mL kanamycin was added to the medium. For strains with two plasmids 25 μς/ΓτιΙ. kanamycin and 50 μς/ΓτιΙ. ampicillin was added to the medium.

Plasmids. The desired genes were cloned in the pCOMI O plasmid by standard digestion 5 and ligation techniques. If no straightforward digestion and ligation was possible, the Golden Gate method was applied. atfA and atf 1 were codon optimized for E. coli and synthesized by GenScript.

Cultivation and gene expression. Strains were inoculated from glycerol stocks stored at -80 10 °C in LB containing the appropriate antibiotic(s) and incubated overnight at 30 °C in a rotary shaker set to 250 rpm. The overnight culture was diluted 100 times in M9^* mineral medium, M9 medium containing 0.5 % glucose and 1 mL/L trace elements US^Fe. This culture was again incubated overnight. The next day, this second preculture was used to inoculate M9^* mineral medium to an OD600nm of 0.167. This culture was directly induced with 0.025 % v/v 15 dicyclopropylketone to induce recombinant gene expression. After 4 h induction at 30 °C, 250 rpm, the cells were harvested by centrifugation for 5 min. at 4255g.

Conversions. The cell pellet was resuspended in resting cell buffer, which contained 1 % glucose, 2 mM MgS04, and 50 mM KPi pH 7.4. Of this resting cell suspension, 0.5 mL was

20 transferred to 13 mL pyrex tubes for conversions. Reactions were started by adding pure n- alkane, n-alkane dissolved in BEHP (bis (2-ethylhexyl)phthalate) or n-alkane dissolved in ethanol (final concentration ethanol 2.5%). Conversions with BEHP were done with an organic:aqueous phase ratio of 1 :2, unless stated otherwise. Tubes were tightly capped. Conversions were done in a rotary shaker, set to 30 °C, 250 rpm. Reactions were stopped

25 by adding phosphoric acid to 1 % v/v and immediately transferring the reaction to ice.

GC sample preparation. Reactions were extracted by adding 2 volumes of CHCI3 containing 0.2 mM tetradecane or 0.2 mM dodecane as internal standard. The extraction was done for -5 min. with a rotator. The organic phase was derivatized with 10% v/v of a 0.2 M TMSH solution in MeOH. Qualitative analysis was done with GC-MS, quantitative analysis with GC-

30 FID.

GC-MS. GC-MS analysis was done with a Trace GC Ultra coupled to a DSQII mass spectrometer. 1 μί sample was injected in splitless mode, with the inlet set at 350 °C. The temperature program was as follows: 50 °C hold 3 min. ,7.5 °C/min ramp to 350 °C, hold for 35 10 min. GC-FID. GC-FID analysis was done with a 7890A (Agilent). 1 μΙ_ sample was injected in splitless mode, with the following temperature program: 50 °C hold 1 min., 15 °C/min to 180 °C, 7 °C/min to 230 °C, 30 °C/min to 350 °C hold 3 min. Quantification was done by using available standards. If standards were not commercially available, quantification was done on basis of structurally related compounds.

Example 1 : Production of esterified α,ω-diols

Results

Production of α,ω-diols is known to be highly challenging. Due to overoxidation of the terminal alcohol and poor ω-oxidation capacity of AlkB, carboxylic acids are the main product. This was confirmed by tests with only the monooxygenase module (£. coli pBGTL) in presence of 1 % n-alkanes: The major products in these tests were overoxidized products: fatty acids and ω-hydroxy fatty acids. Minor amounts of 1 -alcohols and were detected, but for all chain lengths, at least 95% of the products was overoxidized. We attempted to stop overoxidation by converting the produced alcohol directly to an ester. Potentially this ester can subsequently be ω-oxidized. For production of the ester, a carboxylic acid is necessary that is abundantly available in the host, preferably as a CoA-ester. Acetyl-CoA meets both criteria. Atf1 was selected for the esterification module, without an acyl-CoA ligase. Atf1 is capable of producing alkyl acetates, from a large variety of alcohols and acetyl-CoA. E. coli was tested expressing AlkBGTL and Atf1 with n-alkanes as substrate (Fig 1 ). For n-heptane to n-decane, the major products were alkyl acetates. These alkyl acetates were product of transesterification of the 1 -alcohols generated by AlkB and acetyl-CoA. All alkyl acetates produced from the different alkane chain lengths were ω-oxidized, as in most samples ω- hydroxy alkyl acetates were detected. Although it is known that AlkB accepts a wide range of aliphatic substrates, no reports have been made that it can ω-hydroxylate alkyl acetates. Surprisingly, ω-hydroxy alkyl acetates were again transesterified with acetyl-CoA to yield α,ω-diacetoxyalkanes. These diacetoxyalkanes were detected for all tested chain lengths. In n-hexane tests, this was the major product after 19 h incubation, with a concentration of 9.20 mM. Both ω-hydroxy alkyl acetates and α,ω-diacetoxyalkanes can serve as precursor for α,ω-diols. Overoxidation only occurred to a limited extent; 1 -alcohols were overoxidized to fatty acids, but also ω-hydroxy alkyl acetates were overoxidized to ω-acetoxy acids. The highest percentage of overoxidized products was 26% for n-decane tests, in n-hexane tests only 3%. Thus, converting the products into acetate esters efficiently protected the molecule from overoxidation. Since 1 ,4-butanediol and 1 ,5-pentanediol are important commodity chemicals, we checked if 1 ,4-diacetoxybutane and 1 ,5-diacetoxypentane could be produced by adding 0.26% v/v 1 - butanol or 1 -pentanol. Butyl acetate was produced successfully (24.49 mM), and served as substrate for diacetoxybutane production (1 .98 mM). 1 -pentanol was very efficiently converted into 1 ,5-diacetoxypentane, which accumulated to 24.18 mM (also ω-hydroxy pentyl acetate accumulated). Thus, with this platform also precursors of short-chain α,ω- diols can be produced.

We then aimed to improve the product titers of diacetoxyalkanes. Adding n-alkanes in BEHP allowed us to improve the product titers (Fig. 3).

We also examined the effect of the ratio organic phase:aqueous phase, with varying concentrations of n-hexane in BEHP (Fig. 4). Lowering this ratio increased the 1 ,6- diacetoxyhexane product share, up to 48% of total product where the ratio organic:aqueous was 0.1 :1 . These findings suggest that the K_m of AlkB is lower for n-hexane than for hexyl acetate, and hexyl acetate ω-oxidation is hampered by the presence of bulk n-alkane.

Example 2: Production of esterified α,ω-dicarboxylic acids and ω-hydroxy acids Induced, resting £ coli pBGTHJL cells were incubated with medium-chain n-alkanes.

Alkanes ranging from n-hexane to n-decane were added to 5 mM from a concentrated stock in ethanol (final ethanol concentration 2.5 % v/v).

£ coli pBGTHJL produced fatty acids from all n-alkanes (Fig. 5). Diterminal oxidation also occurred for all tested alkanes except n-hexane, since ω-hydroxy fatty acids and to a limited extent dicarboxylic acids accumulated. The oxidation of ω-hydroxy fatty acids to dicarboxylic acids was not efficient, as only 0.04 mM accumulated after 19 h incubation. In n-hexane and n-heptane conversions, there was no production of dicarboxylic acid. The tests were repeated with £ coli strains that, besides the monooxygenase and dehydrogenase modules, also expressed an esterification module (Fig. 5). £. coli pBGTHJKL-aif4 contains the alk operon as it is found in P. putida GPo1 , and from there expresses acyl-CoA ligase AlkK. Unspecific acyltransferase atfA is situated on the same plasmid, with an extra P_aikB promoter. £ coli pBGTHJKL-aif4 produced mono-ethyl dicarboxylic acids, showing that esterification was successful. No ethyl esters accumulated after 2 or 19 h, indicating that ethyl esters were efficiently converted to mono-ethyl dicarboxylic acids. This strain produced up to 0.39 mM mono-ethyl dicarboxylic acid, whereas £ coli pBGTHJL produced at most 0.04 mM dicarboxylic acid. Hence, complete oxidation to the ω-acid was more efficient in presence of an acyltransferase. AtfA also coupled 1 -alcohols, that accumulated from n-alkane hydroxylation, to acyl-CoA. This resulted in trace alkyl alkanoate production.

£ coli pBGTHJL + pE-ll has alkK and atfA on a separate plasmid, where both alkK and atfA have their own P_a|_kB promoter. This resulted in higher expression levels of AlkK. This strain produced mostly esters, but was less efficient regarding terminal oxidation. This resulted in lower product titers, and a high concentration of alkyl alkanoates.

£ coli pBGTHJL-eefc>7 did not produce alkyl alkanoates, since Eeb1 is specific for short alcohols. Ethyl ester synthesis was more efficient in this strain, and as a consequence more mono-ethyl dicarboxylic acid accumulated, up to 0.87 mM from n-octane. Regarding products with two carboxylic functionalities, product titers increased ~22-fold. This strain also enabled £. coli to produce 0.36 mM mono-ethyl adipate from n-hexane.

Diterminal oxidation preferentially occurred after esterification, since n-hexane was only diterminally oxidized by E. coli pBGTHJL + pE-ll and E. coli pBGTHJKL-eebl . E. coli pBGTHJL only produced hexanoic acid, implying that hexanoic acid is hardly or not ω- oxidized by AlkB. Furthermore, E. coli pBGTHJKL-eebl produced more diterminally oxidized product than E. coli pBGTHJL.

In order to produce more alkyl alkanoates, and potentially their terminal oxygenates, ethanol was omitted from the reaction (Fig. 6) and higher alkane concentrations were applied (1 % v/v). This boosted alkyl alkanoate titers, up to 1 .61 mM with n-heptane as substrate. Fatty acid concentrations far exceeded alkyl alkanoate concentrations in £ coli pBGTHJKL-aif4 tests. On the contrary, £ coli pBGTHJL + pE-ll produced mainly alkyl alkanoates. This strain also terminally oxidized hexyl hexanoate and subsequently esterified this molecule to yield the di-esters di-hexyl adipate and hexyl 6-(hexanoyloxy)hexanoate. AlkB can oxidize either end of hexyl hexanoate, resulting in a mixture of products, which was quantified as the sum, since gas chromatography did not allow separation of these highly similar compounds.

Claims

- contacting said alkane, 1 -alkanol, alkanal, alkanoate and/or an alkyl alkanoate, e.g. an ethyl alkanoate or a propyl alkanoate with:

- a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module.

2. Method according to claim 1 , wherein the alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate is converted to mono- and/or di-esters of the corresponding α,ω-diol, or ω-hydroxyacid.

3. Method according to claim 2, wherein said alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate is contacted with a monooxygenase module and an esterification module.

4. Method according to claim 2 or 3, wherein said esterification module comprises, or consists of, Atf1 .

5. Method according to any one of claims 2-4, wherein the monooxygenase module comprises AlkBGT.

6. Method according to claim 1 , wherein the alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate is converted to mono- and/or di-esters of the corresponding aliphatic α,ω dicarboxylic acid or aliphatic ω-hydroxycarboxylic acid.

7. Method according to claim 6, wherein said alkane, 1 -alkanol, alkanal, alkanoate and/or alkyl alkanoate is contacted with a monooxygenase module, a dehydrogenase module, and an esterification module.

8. Method according to claim 7, wherein the esterification module uses an alcohol, which may be added exogenously or produced endogenously, preferably wherein said alcohol is ethanol.

9. Method according to any one of claims 6-8, wherein the esterification module comprises, or consists of, an AlkK acyl-CoA ligase, such as from Pseudomonas putida GPo1 , and an AtfA alcohohacyltransferase, such as from Acinetobacter baylyi; or an alcohohacetyltransferase with a wide substrate spectrum such as Atf1 ; or a medium-chain

5 fatty acid ethyl ester synthase/esterase 1 , such as Eeb1 or Eht1 , preferably from

Saccharomyces cerevisiae.

10. Method according to any one of claims 6-9, wherein the monooxygenase module comprises AlkBGT.

10

1 1 . Method according to any one claims 6-10, wherein the dehydrogenase module comprises, or consists of, an alcohol dehydrogenase, such as AlkJ, preferably from

Pseudomonas putida GPo1 , and an aldehyde dehydrogenase, such as AlkH, preferably from Pseudomonas putida GPo1 .

15

12. Method according to any one of the preceding claims, wherein at least one of the monooxygenase module, esterification module, and/or dehydrogenase module is provided in the form of a host cell expressing said modules.

20 13. Method according to any one of the preceding claims, wherein all of the

monooxygenase module, esterification module, and, optionally, dehydrogenase module are provided in the form of a host cell expressing said modules.

14. Method according to claim 1 , wherein the alkanoate is converted to mono- and/or di- 25 esters of the corresponding dicarboxylic acid or ω-hydroxycarboxylic acid using ethanol or propanol for the esterification.

15. A genetically modified microorganism capable of selective terminal and/or diterminal oxidation of an aliphatic compound, comprising:

30 - a monooxygenase module;

- optionally, a dehydrogenase module; and

- an esterification module.

16. A genetically modified microorganism according to claim 15 comprising at least one 35 exogenous nucleic acid encoding:

- a monooxygenase module;

- optionally, a dehydrogenase module; or - an esterification module.