Microbial production of fatty diols

Abstract

This invention provides fatty diols and recombinant microorganisms for producing them. [Solution] A recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, wherein the recombinant microorganism is engineered to express a nucleic acid sequence encoding a polypeptide that includes (a) thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and (b) carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 026,573 filed July 18, 2014, the full disclosure of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. This ASCII copy was created on 17 July 2015, is named LS00052PCT_SL.txt, and has a file size of 50,064 bytes.

[0003] field This disclosure relates to fatty diols and methods for producing them. In this specification, this disclosure relates to recombinant microorganisms engineered to produce fatty diols by fermentation. Further encompassing are processes using microorganisms to produce fatty diols from simple carbon sources. [Background technology]

[0004] Fatty alcohols have numerous commercial uses as components of industrial substances and processes, particularly as such components in the manufacture of detergents and surfactants. In cosmetics and food products, fatty alcohols are used as emulsifiers, softeners, and thickeners, as well as industrial solvents and plasticizers. Fatty alcohols can be produced from petrochemical or oleochemical raw materials. Petrochemicals are chemical products derived from petroleum. Oleochemicals are refined oils derived from natural sources such as vegetable and animal fats.

[0005] The chemical synthesis pathways for fatty alcohols are energy-intensive, environmentally harmful, and require the use of hazardous reagents. For example, ethylene can be oligomerized using triethylaluminum followed by air oxidation. This process produces even-chain fatty alcohols and is known as the Ziegler process. Alternatively, ethylene can be oligomerized to form an alkene mixture, which is then subjected to hydroformylation to form odd-chain aldehydes. These aldehydes are then hydrogenated to produce fatty alcohols. Another chemical process converts olefin products into fatty aldehydes, which are then converted into fatty alcohols. Olefin products are produced by the Shell Higher Olefin Process, which was commercialized by Royal Dutch Shell in 1977 (for example, producing over one million tons of olefins annually).

[0006] While the natural synthesis pathway for fatty alcohols is considered an environmentally friendly process, it remains more expensive than chemical pathways. Traditionally, fatty alcohols were derived from fatty esters or wax esters, which were initially extracted from whale oil and later from animal fats (e.g., bovine or sheep fat). Jojoba is an alternative plant source for wax esters. Currently, fatty alcohols can also be produced from oleochemical-derived raw materials (e.g., refined vegetable oils), such as rapeseed oil, mustard oil, coconut oil, or palm kernel oil. These vegetable oils are primarily composed of triacylglycerols (TAGs), which contain glycerol esterified with three fatty acids (FAs). Vegetable oils are used for different purposes depending on the FA composition of their TAGs. For example, soap production requires a high proportion of lauric acid (12:0), while oils rich in oleic acid (18:1) are recommended for cooking. TAG can be subjected to an esterification reaction to form an ester, and then this ester is hydrogenated to produce a fatty alcohol. Animal fat is mostly C 16 -C 18However, the carbon chain lengths derived from plant sources are more variable than that (e.g., C6-C 24 ). Long-chain alcohols (e.g., C 20 -C 22 ) can be obtained from rapeseed or mustard seeds, while medium-chain fatty alcohols (e.g., C 12 -C 14 ) can be obtained from coconut or palm kernel oil. Coconut and palm kernel oil are rich in lauric acid (C 12 ) and myristic acid (C 14 ). In 2000, due to the epidemic of bovine spongiform encephalopathy (i.e., mad cow disease) in Europe, animal fats have generally been replaced by vegetable oleic fatty acids derived from palm oil and soybean oil.

[0007] Fatty diols or aliphatic diols are examples of fatty alcohols and can be produced by chemical methods. For example, 1,3-diols can be synthesized from ethylene and carboxylic acid chlorides (see, for example, the translation from Kirchanov et al. (1981), Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya 4:909-911 (Non-Patent Document 1)). 1,3-diols can also be made by the hydration reaction of α,β-unsaturated ketones and aldehydes, and the ketoalcohol formed in this reaction is hydrogenated. Another chemical synthesis of 1,3-diols involves the hydroformylation of epoxides followed by the hydrogenation of aldehydes (e.g., when making 1,3-propanediol from ethylene oxide). A more specialized route for 1,3-diols involves the reaction of alkenes with formaldehyde and the use of β-hydroxyketones. 1,3-diols have been associated with their usefulness as food additives (see, for example, U.S. Patent No. 3,806,615 (Patent Document 1)). The 1,3-dihydroxy configuration is related to the non-toxic properties of these chemical components.

[0008] 1,3-diols are bifunctional and can be used as linking molecules between other molecules, for example, in polymer production. For example, 1,3-propanediol is used as a monomer in polymer production. 1,3-fatty diols can also be used as precursors for surfactants, such as "gemini" surfactants. In "gemini" surfactants, both alcohol moieties are chemically modified (e.g., ethoxylation, glycosylation, sulfation, etc.). The 3-hydroxy moiety of 1,3-fatty diols is also chiral, which makes 1,3-fatty diols useful as synths for the production of monomers, pharmaceuticals, nutritional supplements, insecticides, herbicides, flavorings, fragrances, solvents, and other compounds where chirality is important.

[0009] Since fatty diols are important components of industrial substances and processes, it is desirable to produce high-quality fatty diols that adequately meet industrial demand while keeping environmental impacts low. This disclosure addresses this need. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 3,806,615 [Non-patent literature]

[0011] [Non-Patent Document 1] Kirchanov et al. (1981), Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya 4:909-911 [Overview of the project] [Means for solving the problem]

[0012] One aspect of the present disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism comprising a nucleic acid sequence encoding a polypeptide having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity. In one aspect, the 1,3-lipid diols are produced in vivo. In another aspect, the 1,3-lipid diols include C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 Examples include, but are not limited to, 1,3-fatty diols. In yet another embodiment, the simple carbon source is derived from renewable raw materials. In one embodiment, the disclosure provides a recombinant microorganism comprising a nucleic acid sequence encoding a polypeptide having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and the microorganism produces 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source. In another embodiment, the nucleic acid sequence is exogenous. In another embodiment, the nucleic acid sequence comprises one or more nucleic acid sequences.

[0013] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism comprising a pathway engineered to express a nucleic acid sequence encoding a polypeptide having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity. In one aspect, the 1,3-lipid diols are produced in vivo. In another aspect, as 1,3-lipid diols, C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 Examples include, but are not limited to, 1,3-fatty diols. In yet another embodiment, the simple carbon source is derived from renewable raw materials. In one embodiment, the disclosure provides a recombinant microorganism having a pathway engineered to express a nucleic acid sequence encoding a polypeptide having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, which produces 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source. In another embodiment, the nucleic acid sequence is exogenous. In another embodiment, the nucleic acid sequence comprises one or more nucleic acid sequences.

[0014] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism being engineered to express one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and optionally alcohol dehydrogenase (EC1.1.1.) activity. In one aspect, the 1,3-lipid diols are produced in vivo. In another aspect, the 1,3-lipid diols include C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 Examples include, but are not limited to, 1,3-fatty diols. In yet another embodiment, the simple carbon source is derived from renewable raw materials. In one embodiment, the disclosure provides a recombinant microorganism engineered to express one or more nucleic acid sequences encoding a polypeptide having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and alcohol dehydrogenase (EC1.1.1.) activity, which produces 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source. In another embodiment, the one or more nucleic acid sequences are exogenous.

[0015] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism comprising a pathway engineered to express one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and optionally alcohol dehydrogenase (EC1.1.1.) activity. In one aspect, the 1,3-lipid diols are produced in vivo. In another aspect, as 1,3-lipid diols, C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 Examples include, but are not limited to, 1,3-lipid diols. In yet another embodiment, the simple carbon source is derived from renewable raw materials. In one embodiment, the disclosure provides a recombinant microorganism having a pathway engineered to express one or more nucleic acid sequences encoding a polypeptide having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and alcohol dehydrogenase (EC1.1.1.) activity, which produces 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source. In another embodiment, the one or more nucleic acid sequences are exogenous.

[0016] Another aspect of the present disclosure provides recombinant microorganisms for producing 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source, the simple carbon source being derived from renewable raw materials.

[0017] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source, the microorganism expressing one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity. In one embodiment, the thioesterases include, but are not limited to, fatB1, TE_EEI82564, TE_CAD63310, phaG, and tesA. In another embodiment, the carboxylic acid reductase is carB. In yet another embodiment, the one or more nucleic acid sequences are exogenous.

[0018] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source, the microorganism comprising a pathway engineered to express one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity. In one embodiment, the thioesterase is fatB1, TE_EEI82564, TE_CAD63310, phaG, and tesA, and is not limited thereto. In another embodiment, the carboxylic acid reductase is carB. In yet another embodiment, the one or more nucleic acid sequences are exogenous.

[0019] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism expressing one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and alcohol dehydrogenase (EC1.1.1.) activity. In one embodiment, the thioesterase is fatB1, TE_EEI82564, TE_CAD63310, phaG, and tesA, and is not limited thereto. In another embodiment, the carboxylic acid reductase is carB. In yet another embodiment, the alcohol dehydrogenase is alrA. In yet another embodiment, the one or more nucleic acid sequences are exogenous.

[0020] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source, the microorganism comprising a pathway engineered to express one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and alcohol dehydrogenase (EC1.1.1.) activity. In one embodiment, the thioesterase is fatB1, TE_EEI82564, TE_CAD63310, phaG, and tesA, and is not limited thereto. In another embodiment, the carboxylic acid reductase is carB. In yet another embodiment, the alcohol dehydrogenase is alrA. In yet another embodiment, the one or more nucleic acid sequences are exogenous.

[0021] The disclosure further encompasses cell cultures comprising recombinant microorganisms for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source. In one embodiment, the microorganisms are engineered to express nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3, or EC1.2.1.42) activity. In another embodiment, the microorganisms are engineered to express nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3, or EC1.2.1.42) activity and alcohol dehydrogenase (EC1.1.1.-) activity. In yet another embodiment, the cell cultures produce 1,3-lipid diols. In another aspect, the cell culture contains C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diol, C 19 It produces 1,3-lipidiols, including 1,3-lipidiols. In one embodiment, the nucleic acid sequence is exogenous. In another embodiment, the nucleic acid sequence comprises one or more types of nucleic acid sequences.

[0022] This disclosure further includes a method for producing 1,3-lipid diols, comprising the microorganisms described above (see above).

[0023] Another aspect of the present disclosure provides a method for producing 1,3-fatty diols, the method comprising the steps of: preparing recombinant microorganisms in a fermentation broth, wherein the microorganisms express one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity, carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity, and optionally alcohol dehydrogenase (EC1.1.1.-) activity; and isolating 1,3-fatty diols from the fermentation broth. In one embodiment, the method further comprises the step of adding a simple carbon source to the fermentation broth. In yet another embodiment, the simple carbon source is derived from renewable raw materials. In another embodiment, the present disclosure provides a method for producing 1,3-fatty diols, the method comprising the steps of: preparing recombinant microorganisms in a fermentation broth, wherein the microorganisms are operated to express one or more nucleic acid sequences encoding polypeptides having thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity and carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity; and isolating 1,3-fatty diols from the fermentation broth. In one embodiment, 1,3-fatty diols include C51,3-fatty diol, C61,3-fatty diol, C71,3-fatty diol, C81,3-fatty diol, C91,3-fatty diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 Examples include, but are not limited to, 1,3-fatty diols. In one embodiment, the method further comprises the step of adding a simple carbon source to the fermentation broth. In yet another embodiment, the simple carbon source is derived from renewable raw materials.

[0024] Another aspect of the present disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism expressing a nucleic acid sequence encoding a polypeptide having acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity. In one aspect, the 1,3-lipid diols are produced in vivo. In another aspect, the lipid diols include C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 Examples include, but are not limited to, 1,3-lipid diols. In one embodiment, the nucleic acid sequence is exogenous.

[0025] A further aspect of this disclosure provides a recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, the microorganism expressing one or more nucleic acid sequences encoding polypeptides having acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity and alcohol dehydrogenase (EC1.1.1.-) activity. In one aspect, the 1,3-lipid diols are produced in vivo. In another aspect, the lipid diols include C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C14 1,3 fatty diol, C 15 1,3 fatty diol, C 16 1,3 fatty diol, C 17 1,3 fatty diol, C 18 1,3 fatty diol, and C 19 Examples include, but are not limited to, 1,3 fatty diols. In one embodiment, the one or more nucleic acid sequences are exogenous.

[0026] The present disclosure further contemplates a cell culture comprising a recombinant microorganism for producing 1,3 fatty diol when grown in a fermentation broth containing a simple carbon source, the microorganism being engineered to express one or more nucleic acid sequences encoding a polypeptide having acyl-ACP reductase (EC 1.2.1.80 or EC 1.2.1.42) activity and optionally alcohol dehydrogenase (EC 1.1.1.-) activity. In one aspect, the cell culture produces 1,3 fatty diol. In another aspect, as the fatty diol, C5 1,3 fatty diol, C6 1,3 fatty diol, C7 1,3 fatty diol, C8 1,3 fatty diol, C9 1,3 fatty diol, C 10 1,3 fatty diol, C 11 1,3 fatty diol, C 12 1,3 fatty diol, C 13 1,3 fatty diol, C 14 1,3 fatty diol, C 15 1,3 fatty diol, C 16 1,3 fatty diol, C 17 1,3 fatty diol, C 18 1,3 fatty diol, and C 19 Examples include, but are not limited to, 1,3 fatty diols. In one embodiment, the one or more nucleic acid sequences are exogenous.

[0027] In yet another embodiment, the disclosure provides a method for producing 1,3-fatty diols, the method comprising the steps of: preparing a recombinant microorganism in a fermentation broth, wherein the microorganism is operated to express a nucleic acid sequence encoding a polypeptide having acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity; and isolating 1,3-fatty diols from the fermentation broth. In one embodiment, the microorganism further expresses a nucleic acid sequence encoding a polypeptide having alcohol dehydrogenase (EC1.1.1.-) activity. In another embodiment, the method further comprises the step of adding a simple carbon source to the fermentation broth. In yet another embodiment, the simple carbon source is derived from renewable raw materials. The method produces C51,3-fatty diols, C61,3-fatty diols, C71,3-fatty diols, C81,3-fatty diols, C91,3-fatty diols, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 It produces fatty diols including, but not limited to, 1,3-lipid diols.

[0028] In yet another embodiment, the disclosure further comprises the secretion and recovery of 1,3-fatty diols from any of the recombinant microorganisms described above (see above). In one embodiment, the 1,3-fatty diols are secreted into a fermentation broth. In another embodiment, the 1,3-fatty diols are recovered via oil-water separation, for example, by gravity sedimentation, centrifugation, decantation, etc.

[0029] This disclosure further encompasses adiol composition. In one embodiment, the composition comprises one or more diols, including a 1,3-diol.

[0030] Another aspect of this disclosure provides the use of fatty diols in the manufacture of surfactants, including ethoxylates.

[0031] This disclosure further encompasses chiral 1,3-fatty diols, their enantiomers, and chiral mixtures. Further intended are compositions of 1,3-fatty diols, their enantiomers, and chiral mixtures. [Invention 1001] Recombinant microorganisms that produce 1,3-fatty diols when grown in a fermentation broth containing a simple carbon source, (a) Thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity; and (b) Carboxylate reductase (EC6.2.1.3 or EC1.2.1.42) activity It is being manipulated to express a nucleic acid sequence encoding a polypeptide containing, The recombinant microorganisms mentioned above. [Invention 1002] A recombinant microorganism according to the present invention 1001, which produces the aforementioned 1,3-lipid diol in vivo. [Invention 1003] The aforementioned 1,3-lipid diols are C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 A recombinant microorganism according to the present invention 1001, selected from the group consisting of 1,3-lipid diols. [Invention 1004] A recombinant microorganism according to the present invention 1001, further expressing a nucleic acid sequence encoding a polypeptide containing alcohol dehydrogenase (EC1.1.1.-) activity. [Invention 1005] The recombinant microorganism of the present invention 1001, wherein the simple carbon source is derived from renewable raw materials. [Invention 1006] The recombinant microorganism of the present invention 1001, wherein the thioesterase is selected from the group consisting of fatB1, TE_EEI82564, TE_CAD63310, and phaG. [Invention 1007] The recombinant microorganism of the present invention 1001, wherein the carboxylic acid reductase is carB. [Invention 1008] The recombinant microorganism of the present invention 1004, wherein the alcohol dehydrogenase is alrA. [Invention 1009] A cell culture containing any of the microorganisms described in invention 1001 to 1008. [Invention 1010] A cell culture according to the present invention 1009 that produces 1,3-lipid diols. [Invention 1011] The aforementioned 1,3-lipid diols are C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 A cell culture according to the present invention 1010, selected from the group consisting of 1,3-lipid diols. [Invention 1012] A method for producing 1,3-lipid diols, comprising the microorganism of the present invention 1001. [Invention 1013] (a) A step of preparing recombinant microorganisms in a fermentation broth, wherein the microorganisms express a nucleic acid sequence encoding a polypeptide comprising thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity; carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity; and optionally alcohol dehydrogenase (EC1.1.1.) activity; and (b) A step of isolating 1,3 fatty diols from the fermentation broth, wherein the fermentation broth contains a simple carbon source. A method for producing 1,3-lipid diols, including [the specified substance]. [Invention 1014] The aforementioned 1,3-lipid diols are C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 A method of the present invention 1013, selected from the group consisting of 1,3-lipid diols. [Invention 1015] A recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, wherein the recombinant microorganism is engineered to express a nucleic acid sequence encoding a polypeptide containing acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity. [Invention 1016] A recombinant microorganism according to the present invention 1015, further expressing a nucleic acid sequence encoding a polypeptide containing alcohol dehydrogenase (EC1.1.1.-) activity. [Invention 1017] A recombinant microorganism according to the present invention 1015, which produces the aforementioned 1,3-lipid diol in vivo. [Invention 1018] The aforementioned 1,3-lipid diols are C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 A recombinant microorganism according to the present invention 1017, selected from the group consisting of 1,3-lipid diols. [Invention 1019] The aforementioned 1,3-lipid diol, C 12 A recombinant microorganism of the present invention 1017, which is a 1,3-fatty diol. [Invention 1020] The recombinant microorganism of the present invention 1015, wherein the simple carbon source is derived from renewable raw materials. [Invention 1021] A cell culture containing any microorganism according to invention 1015 to 1020. [Invention 1022] A cell culture according to the present invention 1021 that produces 1,3-lipid diols. [Invention 1023] The aforementioned 1,3-lipid diols are C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 171,3-lipid diol, C 18 1,3-lipid diols, and C 19 A cell culture according to the present invention 1022, selected from the group consisting of 1,3-lipid diols. [Invention 1024] A method for producing 1,3-lipid diols, comprising the microorganism of the present invention 1015. [Invention 1025] (a) a step of preparing recombinant microorganisms in a fermentation broth, wherein the microorganisms are operated to express a nucleic acid sequence encoding a polypeptide having acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity; and (b) A step of isolating 1,3 fatty diols from the fermentation broth, wherein the fermentation broth contains a simple carbon source. A method for producing 1,3-lipid diols, including [the specified substance]. [Invention 1026] The method of the present invention 1025, further expressing a nucleic acid sequence encoding a polypeptide containing alcohol dehydrogenase (EC1.1.1.-) activity. [Invention 1027] The aforementioned 1,3-lipid diols are C51,3-lipid diol, C61,3-lipid diol, C71,3-lipid diol, C81,3-lipid diol, C91,3-lipid diol, C 10 1,3-lipid diol, C 11 1,3-lipid diol, C 12 1,3-lipid diol, C 13 1,3-lipid diol, C 14 1,3-lipid diol, C 15 1,3-lipid diol, C 16 1,3-lipid diol, C 17 1,3-lipid diol, C 18 1,3-lipid diols, and C 19 A method of the present invention 1025, selected from the group consisting of 1,3-lipid diols.

[0032] This disclosure is best understood in conjunction with the accompanying drawings, which illustrate some preferred embodiments. However, it should be understood that this disclosure is not limited to the specific embodiments disclosed in the drawings. [Brief explanation of the drawing]

[0033] [Figure 1] An example of a 1,3-diol synthesis pathway, including enzymatic function, is shown. [Figure 2] An example of a 1,3-diol production pathway is shown, and an example of enzyme function is provided for illustrative purposes. [Figure 3] This paper describes alternative synthesis routes for 1,3-diols, including their enzymatic functions. [Figure 4] GC / MS chromatography of extracts from recombinant E. coli strains expressing TE_EEI82564 and CarB is shown. All samples were derivatized with BSTFA + 1% TMCS. Peak (1) is derivatized 1,3-octanol, and peak (2) is derivatized 1,3-decanol. [Figure 5] Figure 4 shows the mass spectra of derivatization peaks 1 and 2, which originate from recombinant E. coli strains expressing TE_EEI82564 and CarB. The derivatizing agent was BSTFA + 1% TMCS. [Figure 6] This shows the ion fragmentation pattern of 1,3-decanediol derivatized with BSTFA + 1% TMCS. [Figure 7] The composition of 1,3-diols (diols) and fatty alcohols (FALCs) produced by recombinant Escherichia coli strains expressing TE_CAD63310 and CarB is shown.

[0034] The following abbreviations will be used in Figures 8-11. FAS: Fatty acid biosynthesis / fatty acid synthase TE: Thioesterase ACS: Acyl-CoA Synthase TL:3-Ketoacyl-CoA thiolase (reversible) (S)3HACS: (S)-3-hydroxyacyl-CoA dehydrogenase (reversible) (S)2ECOH:(S)-2-enoyl-CoA hydratase / (S)-3-hydroxylacyl-CoA dehydratase CAR: Carboxylic acid reductase FAR: Fatty acyl-CoA / ACP reductase and fatty alcohol-forming fatty acyl-CoA / ACP reductase ACR: Acyl-CoA reductase AAR: Acyl ACP / CoA Reductase [Figure 8] This diagram shows the biochemical pathways from acyl-ACP to 1,3-lipid diols. Pathway 1 uses the enzymatic functions of TE, CAR, and ADH to produce 1,3-diols. Pathway 2 uses TE, ACS, ACR, and ADH to produce 1,3-diols. Pathway 3 uses AAR and ADH to produce 1,3-diols. Pathway 4 uses FAR and ADH to produce 1,3-diols. Pathway 5 uses FAR to produce 1,3-diols. [Figure 9] This diagram shows the biochemical pathways from acyl-CoA to 1,3-lipid diols. Pathway 1 uses the enzymatic functions of TE, CAR, and ADH to produce 1,3-diols. Pathway 2 uses ACR and ADH to produce 1,3-diols. Pathway 3 uses AAR and ADH to produce 1,3-diols. Pathway 4 uses FAR and ADH to produce 1,3-diols. Pathway 5 uses FAR to produce 1,3-diols. [Figure 10] This pathway exhibits (R)-1,3-lipid diol production. Pathway 1 uses the enzyme functions of TE, CAR, and ADH to produce right-handed chiral 1,3-diols. Pathway 2 uses TE, ACR, and ADH to produce right-handed chiral 1,3-diols. Pathway 3 uses AAR and ADH to produce right-handed chiral 1,3-diols. Pathway 4 uses FAR and ADH to produce right-handed chiral 1,3-diols. Pathway 5 uses FAR to produce right-handed chiral 1,3-diols. [Figure 11] This pathway exhibits (S)-1,3-lipid diol production. Pathway 1 uses the enzyme functions of TE, CAR, and ADH to produce left-handed chiral 1,3-diols. Pathway 2 uses ACR and ADH to produce left-handed chiral 1,3-diols. Pathway 3 uses AAR and ADH to produce left-handed chiral 1,3-diols. Pathway 4 uses FAR and ADH to produce left-handed chiral 1,3-diols. Pathway 5 uses FAR to produce left-handed chiral 1,3-diols. Pathway 6 uses TE, ACS, FadE, and (S)2ECOH to produce left-handed chiral 1,3-diols. Pathway 7 uses fatty acids and ACS to produce left-handed chiral 1,3-diols. Pathway 8 uses TE, ACS, TL, and (S)3HACS to produce left-handed chiral 1,3-diols. [Modes for carrying out the invention]

[0035] Detailed explanation Summary The development of novel and environmentally friendly methods for producing fatty diols will bring improvements to industry. This method enables the production of fatty diols from simple carbon sources derived from renewable raw materials, including, but not limited to, carbohydrates such as corn, sugarcane, natural gas, or lignocellulosic biomass; waste products such as municipal solid waste, glycerol, flue gas, synthesis gas, and carbon dioxide; or carbon flux resulting from the modification of organic materials such as biomass, natural gas, or other carbonaceous materials. Furthermore, this method enables the production of fatty diols from CO2 and light using photosynthetic organisms such as cyanobacteria and algae. This method is better for the environment because it does not produce toxic by-products that occur in petrochemical processes.

[0036] More specifically, this disclosure provides recombinant microorganisms engineered to convert simple carbon sources derived from renewable raw materials into fatty diols. 1,3-diols are an example of fatty diols, which are colorless, odorless, and stable chemical components. Microbially produced 1,3-diols are expected to have numerous industrial applications, such as detergents, surfactants, emulsifiers, softeners, solvents, plastics, flavorings, fragrances, and components of bioactive compounds. Microbially produced 1,3-diols are also expected to find applications in the food industry as substitutes (or additives) for natural foods, because they are readily metabolized, non-toxic, non-volatile, high in energy, and have a long shelf life.

[0037] The recombinant microorganisms of this disclosure are used in fermentation processes for the production of fatty diols. In this specification, this disclosure encompasses microbial fatty acid metabolism and the conversion of its intermediates to 1,3-diols. One advantage of this disclosure is the adoption of a cleaner production method, namely a simpler fermentation process. The use of renewable raw materials protects the environment because such raw materials rely on renewable and sustainable raw materials that do not deplete natural resources. The use of industrial waste products (e.g., glycerol) as raw materials supports better waste management and recycling. Another advantage is that it provides an option for producing products for new industrial purposes, namely fatty diol compositions as mixtures of selective chain length, chirality, and specific ratios, or as mixtures with derivatives.

[0038] definition As used herein, the terms “1,3-fatty diol,” “1,3-diol,” “1,3-dialcohol,” “3-OH fatty alcohol,” “3-hydroxy fatty alcohol,” “1,3-dihydroxy alcohol,” or “1,3-aliphatic diol” are used synonymously herein and refer to chemical components having a chain length of at least 5 carbon atoms, arising from microbial fatty acid metabolism via fatty acylthioester intermediates, and having at least two OH groups, i.e., one OH group at position 1 and one OH group at position 3 of the carbon chain.

[0039] "1,3-diol" is produced by recombinant microorganisms or recombinant microbial host cells when used herein.

[0040] A "1,3-diol composition" typically contains at least a 1,3-diol together with another component.

[0041] The term "Enzyme Classification (EC) number" refers to a number that designates a specific polypeptide sequence or enzyme. EC numbers classify enzymes by the reactions they catalyze. EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), and explanations are available on the IUBMB Enzyme Nomenclature website on the World Wide Web.

[0042] The term "thioesterase" refers to enzyme activity characterized by EC number 3.1.2.14, EC number 3.1.1.5, or EC number 3.1.2.

[0043] The term "carboxylic acid reductase (CAR)" refers to the enzyme activity characterized by EC number 6.2.1.3, EC number 1.2.1.42, or EC number 1.2.99.6.

[0044] The terms "aldehyde reductase" and "alcohol dehydrogenase" are used synonymously herein and refer to the enzyme activity characterized by EC number 1.1.-.-.

[0045] The term "acyl-ACP reductase (AAR)" refers to the enzyme activity characterized by EC number 1.2.1.80 or EC number 1.2.1.42.

[0046] The term "acetyl-CoA carboxylase" refers to the enzyme activity characterized by EC number 6.4.1.2.

[0047] The terms “accession number,” “NCBI accession number,” and “GenBank accession number” are used synonymously herein and refer to numbers that designate a specific nucleic acid sequence. The sequence accession numbers described herein are obtained from databases provided by the NCBI (National Center for Biotechnology Information), maintained by the National Institutes of Health in the United States, and from the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databases provided by the Swiss Institute for Bioinformatics (also referred to as UniProtKB accession numbers).

[0048] As used herein, the term “nucleotide” refers to the monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate groups. Natural bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are typical purine or pyrimidine derivatives, but it should be understood that natural and unnatural base analogs are also included. Natural sugars are pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), but it should be understood that natural and unnatural sugar analogs are also included. Nucleic acids typically form nucleic acids or polynucleotides by linking via phosphate bonds, but many other linkages are known in the field (e.g., phosphorothioates, boranophosphates, etc.).

[0049] As used herein, the term “polynucleotide” refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which may be single-stranded or double-stranded and may contain unnatural or modified nucleotides. The terms “polynucleotide,” “nucleic acid sequence,” and “nucleotide sequence” are used synonymously herein and refer to polymeric nucleotides of any length, which are either RNA or DNA. These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. The terms include, but are not limited to, analogues of either RNA or DNA made from nucleotide analogs, and modified polynucleotides, such modifications including methylated and / or capped polynucleotides. Polynucleotides may be in any form, such as plasmids, viral, chromosomal, ESTs, cDNA, mRNA, and rRNA, but are not limited to these forms.

[0050] The terms “endogenous polynucleotide,” “endogenous DNA,” and “endogenous nucleic acid sequence” are used synonymously herein and refer to DNA that originates within a host cell.

[0051] The terms “exogenous polynucleotide,” “exogenous DNA,” and “exogenous nucleic acid sequence” are used synonymously herein and refer to DNA that originates outside the host cell. For example, it is possible to insert a gene from host cell A into host cell B. However, a gene originating from host cell A can be manipulated or modified (inside or outside host cell A) and then reinserted into the same host cell A.

[0052] The terms “modified polynucleotide,” “modified DNA,” and “modified nucleic acid sequence” are used synonymously herein and refer to DNA that has been modified in any way from its original or natural state. This modification may affect the stability, expression, activity, or function of the DNA, or the gene product (e.g., polypeptide or protein) that the DNA encodes. In one embodiment, the expression of the encoded polypeptide is increased. In another embodiment, the expression of the encoded polypeptide is decreased. In yet another embodiment, the encoded polypeptide is not expressed.

[0053] As used herein, the terms “polypeptide” and “protein” and “polypeptide sequence” and “protein sequence” are synonymous and refer to polymers of amino acid residues. The term “recombinant polypeptide” refers to a polypeptide produced by genetic recombination techniques, which generally involve inserting DNA or RNA encoding the protein to be expressed into a suitable expression vector, and then using this vector to transform a host cell to produce the polypeptide.

[0054] As used herein, the terms “homologous” and “homologous” refer to a polynucleotide or polypeptide comprising a sequence that is at least about 50% identical to a corresponding polynucleotide or polypeptide sequence. Preferably, a homologous polynucleotide or polypeptide has a polynucleotide or amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% homology to a corresponding amino acid or polynucleotide sequence. As used herein, the terms “sequence homology” and “sequence identity” are used synonymously. Those skilled in the art are well aware of methods for determining homology between two or more sequences. In short, the calculation of "homology" between two sequences can be performed as follows: The sequences are aligned to the best extent for comparison purposes (for example, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the first sequence aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, even more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the length of the second sequence. Then, amino acid residues or nucleotides are compared at the corresponding amino acid or nucleotide positions of the first and second sequences. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then these molecules are identical at that position. The homology percentage between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that needed to be introduced for optimal alignment of the two sequences and the length of each gap.Sequence comparison and determination of homology percentage between two sequences can be achieved using mathematical algorithms such as BLAST (Altschul et al. (1990) J. Mol. Biol. 215(3): 403-410). Homology percentage between two amino acid sequences can also be determined using the Needleman and Wunsch algorithm, which is integrated into the GAP program of the GCG software package and uses either a Blossum62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, (1970) J. Mol. Biol. 48:444-453). Homology percentage between two nucleotide sequences can also be determined using the GAP program of the GCG software package, using NWSgapdna. A CMP matrix and 40, 50, 60, 70, or 80 gap weights and 1, 2, 3, 4, 5, or 6 length weights. Those skilled in the art can perform an initial homology calculation and adjust the algorithm parameters accordingly. A preferred combination of parameters (and to be used when it is unclear to the calculator which parameters to use to determine whether the molecule is within the required homology limits) is a Blossum62 score matrix combined with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Further methods of sequence alignment are known in the biotechnology field (see, e.g., Rosenberg (2005) BMC Bioinformatics 6:278); Altschul et al. (2005) FEBS J. 272(20): 5101-5109).

[0055] "Endogenous" polypeptides refer to polypeptides encoded by the genome of the host cell (e.g., parent microbial cell) from which recombinant cells are manipulated or from which recombinant cells originate.

[0056] An "exogenous" polypeptide refers to a polypeptide that is not originally encoded by the genome of the parental or host microbial cell (e.g., the host cell). Mutant polypeptides are an example of exogenous polypeptides. Another example of exogenous polypeptides is a protein that occurs in the cell naturally but is the result of modified expression, such as the expression of an exogenous polynucleotide (e.g., a vector or plasmid containing a gene that is identical to the original gene but has been engineered to be overexpressed in the host cell; such a gene may optionally be inserted into the host DNA).

[0057] The term "heterogeneous" generally means originating from a different species, a different organism, or a different source. As used herein, this term refers to a nucleotide sequence or polypeptide sequence that does not exist naturally in a particular organism. Heterogeneous expression means that a protein or polypeptide is expressed in a cell that does not normally express that protein. Thus, heterogeneous means that the introduced protein originally originated from a different cell type, a different species, or a different source than the recipient. For example, a polynucleotide sequence that is endogenous to plant cells can be introduced into a bacterial host cell by a genetic engineering method, so that the plant polynucleotide is a heterogeneous polynucleotide in the recombinant bacterial host cell. Another example of a heterogeneous polypeptide is a protein that occurs naturally in the cell but is the result of modified expression, e.g., heterogeneous polynucleotide expression (e.g., a vector or plasmid containing a gene identical to the natural gene but engineered to be overexpressed in the host cell; such a gene may optionally be inserted into host DNA).

[0058] As used herein, the term "fragment" of a polypeptide refers to a shorter portion of a full-length polypeptide or protein, ranging in size from four amino acid residues to the total amino acid sequence minus one amino acid residue. In certain embodiments of this disclosure, a fragment refers to the total amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate-binding domain or a catalytic domain).

[0059] As used herein, the term “mutatogenesis” refers to the process by which the genetic information of an organism is altered in a stable manner. Mutaogenesis of proteins that encode nucleic acid sequences produces mutant proteins. Mutaogenesis also refers to alterations in the untranslated region of the nucleic acid sequence that result in changes in protein activity.

[0060] As used herein, the term “gene” means a nucleic acid sequence that codes for either an RNA product or a protein product, as well as an operablely linked nucleic acid sequence that affects the expression of the RNA or protein (e.g., promoter sequences or enhancer sequences) or an operablely linked nucleic acid sequence that codes for a sequence that affects the expression of the RNA or protein (e.g., ribosome binding sites or translational regulatory sequences).

[0061] Regulatory sequences are known in this field and include, for example, promoters, enhancers, polyadenylation signals, transcription termination factors, and intra-sequence ribosome entry sites (IRESs), which lead to the expression of polynucleotide sequences in host cells. Regulatory sequences interact specifically with cellular proteins involved in transcription (Maniatis et al. (1987) Science 236:1237-1245). Examples of regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0062] The term "multiple" refers to a number of at least two (for example, multiple polynucleotide sequences mean at least two polynucleotide sequences).

[0063] In the methods of this disclosure, the regulatory sequence is operably ligated to the polynucleotide sequence. The term "operably ligated" means that the polynucleotide sequence and the regulatory sequence are linked in such a manner that gene expression becomes possible when an appropriate molecule (e.g., a transcription-activating protein) is bound to the regulatory sequence. The operably ligated promoter is located upstream of the selected polynucleotide sequence with respect to the direction of transcription and translation. The operably ligated enhancer may be located upstream, within, or downstream of the selected polynucleotide.

[0064] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it is ligated. One useful type of vector is an episome (i.e., a nucleic acid capable of extrachromosomal replication). Useful vectors are those that enable the autonomous replication and / or expression of the nucleic acids to which they are ligated. A vector capable of operatively ligating the expression of a gene is referred to herein as an “expression vector.” Generally, useful expression vectors in recombinant DNA techniques are often in the form of a “plasmid,” which generally refers to a circular double-stranded DNA loop that is not bound to a chromosome in its vector form. Since plasmids are the most commonly used form of vectors, the terms “plasmid” and “vector” are used synonymously herein. However, other forms of expression vectors that perform equivalent functions, and other forms of expression vectors that may become known in the art in the future, are also included. In some embodiments, the recombinant vector further includes a polynucleotide sequence and an operatively ligated promoter. In some embodiments, the promoter is a developmentally regulated promoter, an organelle-specific promoter, a tissue-specific promoter, an inducible promoter, a constitutive promoter, or a cell-specific promoter. A recombinant vector typically comprises at least one sequence including (a) an expression control sequence operably linked to a polynucleotide sequence; (b) a selection marker operably linked to a polynucleotide sequence; (c) a marker sequence operably linked to a polynucleotide sequence; (d) a purified portion operably linked to a polynucleotide sequence; (e) a secretion sequence operably linked to a polynucleotide sequence; and (f) a targeting sequence operably linked to a polynucleotide sequence. In certain embodiments, the nucleotide sequence is stably integrated into the genomic DNA of a host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region. The expression vectors described herein contain the polynucleotide sequences described herein in a form suitable for expression of the polynucleotide sequence in a host cell.Those skilled in the art will understand that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired polypeptide expression level. The expression vectors described herein, upon introduction into host cells, can produce polypeptides containing fusion polypeptides encoded by the polynucleotide sequences described herein. Expression of polypeptide-encoding genes in prokaryotes, such as Escherichia coli, is most often carried out using vectors containing constitutive or inducible promoters that lead to the expression of either fusion or non-fusion polypeptides. Fusion vectors add several amino acids to the polypeptide encoded therein, usually to the amino or carboxyl terminus of the recombinant polypeptide. Such fusion vectors typically serve one or more of the following three purposes: (1) increasing the expression of the recombinant polypeptide; (2) increasing the solubility of the recombinant polypeptide; and (3) assisting in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, fusion expression vectors introduce proteolytic cleavage sites at the junction between the fusion region and the recombinant polypeptide. This allows for the separation of the recombinant polypeptide from the fusion region after purification of the fusion polypeptide. In certain embodiments, the polynucleotide sequences of this disclosure are operably linked to a bacteriophage T5-derived promoter. In certain embodiments, the host cell is a yeast cell, and the expression vector is a yeast expression vector. Examples of expression vectors in the yeast S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et al. (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), pYES2 (Invitrogen Corp., San Diego, CA), and picZ (Invitrogen Corp., San Diego, CA).In other embodiments, the host cell is an insect cell, and the expression vector is a baculovirus expression vector. Examples of baculovirus vectors available for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al. (1989) Virology 170:31-39). In yet another embodiment, the polynucleotide sequences described herein can also be expressed in mammalian cells using mammalian expression vectors. Other suitable expression systems are well known in the art for both prokaryotic and eukaryotic cells; see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," second edition, Cold Spring Harbor Laboratory, (1989).

[0065] As used herein, “acyl-CoA” refers to an acylthioester formed between the carbonyl carbon of an alkyl chain and the sulfhydryl group of the 4'-phosphopanthethionyl moiety of coenzyme A(CoA), the ester having the formula RC(O)S-CoA, where R is any alkyl group having at least four carbon atoms.

[0066] As used herein, “acyl-ACP” refers to an acyl thioester formed between the carbonyl carbon of an alkyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of an acyl carrier protein (ACP). The phosphopantetheinyl moiety is post-translationally ligated to a conserved serine residue on the ACP by the action of a holo-acyl carrier protein synthase (ACPS), a type of phosphopantetheinyl transferase that uses coenzyme A as a substrate, and a phosphopantetheinyl donor. In some embodiments, acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACP. In other embodiments, acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACP. In some embodiments, the carbon chain has approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of these acyl-ACPs is a substrate for an enzyme that converts them to a fatty acid derivative. As will be apparent to those skilled in the art, the 4'-phosphopanthethionyl moiety of holoACP is derived from coenzyme A. That is, enzymes that utilize acyl-ACP as a substrate often have some activity toward acyl-CoA, and enzymes that utilize acyl-CoA as a substrate often have some activity toward acyl-ACP.

[0067] As used herein, the term “fatty acid biosynthetic pathway” means a biosynthetic pathway that produces fatty acids, fatty acid thioesters, and / or derivatives thereof. The fatty acid biosynthetic pathway may include additional enzymes or polypeptides having enzymatic activity other than those considered herein in order to produce fatty acid derivatives having desired properties.

[0068] As used herein, the term “clone” typically refers to a cell or group of cells that are descendants of a single common ancestor and are essentially genetically identical to that ancestor, such as a bacterium in a cloned bacterial colony that arose from a single bacterial cell.

[0069] As used herein, the term “culture” typically refers to a liquid medium containing living cells. In one embodiment, a culture comprises cells growing in a given medium under controlled conditions, for example, a culture of recombinant host cells grown in a liquid medium containing a selected carbon source and nitrogen. “Culture” or “culturing” refers to growing a population of recombinant host cells in a liquid or solid medium under appropriate conditions. In certain embodiments, culturing refers to a fermentative biotransformation from substrate to end product. Culture media are well known, and the individual components of such media are available from distributors, for example, under the trademarks of Difco® and BBL®. In one non-limiting example, an aqueous nutrient medium is a “nutrient-rich medium” containing a complex source of nitrogen, salt, and carbon, for example, YP medium as such a medium containing 10 g / L of peptone and 10 g / L of yeast extract per medium. The host cells of the culture can be further genetically engineered to efficiently absorb carbon and utilize cellulosic materials as a carbon source, according to the methods described in U.S. Patent Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030; and WO2010127318. In some embodiments, the host cells are also genetically engineered to express invertase so that sucrose can be used as a carbon source.

[0070] As used herein, the term "conditions effective for expressing a genetically modified polynucleotide sequence" means any conditions that enable a host cell to express the relevant enzymatic function for the purpose of producing a desired fatty acid derivative, such as a fatty diol. Suitable conditions include, for example, fermentation conditions.

[0071] The term “recombinant microorganism” refers to a genetically modified or engineered host cell in which certain enzyme activity within the host cell is altered, added, and / or deleted compared to the parental or native host cell. Genetically modified or engineered host cells are examples of recombinant microorganisms. Therefore, “altered or altered levels of protein activity,” such as enzymes, in recombinant host cells refers to differences in one or more properties of activity, determined by comparison with the parental or native host cell in which the same alteration does not exist. Typically, differences in activity are determined between a recombinant host cell with altered activity and a corresponding wild-type host cell without altered activity (e.g., comparison of a culture of recombinant host cells with the corresponding wild-type host cells). The modified activity may result from, for example, an altered amount of protein expressed by a recombinant host cell (e.g., as a result of an increased or decreased copy number of the protein-coding DNA sequence, an increased or decreased number of the protein-coding mRNA transcript, and / or an increased or decreased amount of protein translation from mRNA to protein); a change in protein structure (e.g., a change in primary structure, e.g., a change in substrate specificity, a change in the protein-coding sequence resulting in observed changes in dynamic parameters, etc.); and a change in protein stability (e.g., an increase or decrease in proteolysis). In some embodiments, the polypeptide is a mutant or variant of any of the polypeptides described herein. In certain cases, the coding sequence of the polypeptides described herein is codon-optimized for expression in a particular host cell. For example, one or more codons may be optimized for expression in E. coli (e.g., as described in Grosjean et al. (1982) Gene 18:199-209). In one embodiment, the recombinant microorganism produces a desired product such as a fatty acid derivative (e.g., a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty diol). In one particular embodiment, the recombinant microorganism produces a 1,3-diol.

[0072] As used herein, the term “regulatory sequence” typically refers to a sequence of DNA bases that is operably linked to a protein-coding DNA sequence and ultimately controls the expression of that protein. Examples of regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, transcription modulators (such as enhancer elements), nucleotide sequences that affect RNA stability, and translational regulatory sequences (e.g., ribosome binding sites (e.g., the Shine-Dalgarno sequence in prokaryotes, or the Kozak sequence in eukaryotes), start codons, stop codons, etc.).

[0073] The terms “modified level of expression” and “altered level of expression” are used synonymously and refer to the presence of a polynucleotide, polypeptide, metabolite, or product (e.g., a fatty acid derivative) in a genetically modified host cell at a different concentration compared to its concentration in the corresponding wild-type cell under the same conditions. Examples of fatty acid derivatives include fatty acids, 3-hydroxy fatty acids, fatty aldehydes, 3-hydroxy fatty aldehydes, fatty alcohols, and 1,3-fatty diols.

[0074] As used herein, the term “potency” refers to the amount of fatty acid derivatives, such as fatty diols (e.g., 1,3-diols), produced per unit volume of host cell culture, and is generally expressed in mass / volume units, e.g., 10 g / L. In one embodiment, potency may refer to a specific 1,3-diol produced by a given recombinant host cell culture, or to a combination of 1,3-diols. In another embodiment, potency may also refer to a fatty diol composition (e.g., a 1,3-diol composition) produced by a given recombinant host cell culture.

[0075] Where used herein, "yield of adiol (e.g., 1,3-diol) produced by host cells" refers to the efficiency with which an input carbon source is converted into a product (e.g., adiol) in the host cell, while "mass yield" is expressed as a percentage in units of mass(product) / mass(carbon source). For example, a 30% mass yield indicates that 30 g of product is produced from 100 g of carbon source; a 20% mass yield indicates that 20 g of product is produced from 100 g of carbon source; and a 10% mass yield indicates that 10 g of product is produced from 100 g of carbon source. The yield may refer to a specific 1,3-diol produced by a given recombinant host cell culture, or it may refer to a combination of 1,3-diols.

[0076] As used herein, the term “productivity” refers to the amount of adiol (e.g., 1,3-diol) or derivative produced per unit time per unit volume of host cell culture (e.g., expressed in g / L / hour). Productivity may refer to a specific 1,3-diol produced by a given recombinant host cell culture, or to a combination of 1,3-diols.

[0077] As used herein, the term "glucose utilization rate" refers to the amount of glucose used by the culture per unit time, expressed in grams per liter per hour (g / L / hour).

[0078] "Raw materials" are raw materials used in the manufacture of products or for industrial processes. "Renewable raw materials" are raw materials derived from renewable materials such as biomaterials, e.g., plants, that are replaceable through natural means (e.g., corn, sugarcane, lignocellulosic biomass) or waste, e.g., municipal solid waste, glycerol, free fatty acids, flue gas, or synthesis gas; carbon dioxide. In contrast, "non-renewable raw materials" are raw materials that are depleted by use (e.g., crude oil, coal, nuclear fuel) and cannot be recycled.

[0079] As used herein, the term “simple carbon source” refers to a substrate or compound suitable for use as a food source for the growth of prokaryotic or simple eukaryotic cells. Suitable sources as simple carbon sources can be in a variety of forms, including, but are not limited to, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO2). Examples of simple carbon sources include, but are not limited to, monosaccharides such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides such as fructooligosaccharides and galactooligosaccharides; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides such as sucrose, maltose, cellobiose, and turanose; cellulosinous materials and derivatives such as hemicellulose, methylcellulose, and sodium carboxymethylcellulose; saturated or unsaturated fatty acids, succinic acid compounds, lactic acid compounds, and acetic acid compounds; alcohols such as ethanol, methanol, and propanol; glycerol, etc., or mixtures thereof. In one embodiment, the simple carbon source is derived from corn, sugarcane, sorghum, beets, switchgrass, storage hay, straw, wood, pulp, sewage, food waste, cellulosinous municipal waste products, flue gas, synthesis gas, or carbon dioxide. Simple carbon sources can also be photosynthetic products such as glucose. In one embodiment, the simple carbon source is derived from renewable raw materials. In one particular embodiment, the simple carbon source is derived from renewable raw materials, such as carbohydrates such as corn, sugarcane, or lignocellulosic biomass; or waste products, such as glycerol, fatty acids, flue gas, or synthesis gas; or organic materials, such as biomass; or carbon dioxide fixed by photosynthesis.In another embodiment, the simple carbon source is selected from glucose, fructose, mannose, galactose, xylose, arabinose, fructooligosaccharides, galactooligosaccharides, starch, cellulose, pectin, xylan, sucrose, maltose, cellobiose, turanose, hemicellulose, methylcellulose, sodium carboxymethylcellulose, succinic acid compounds, lactic acid compounds, acetic acid compounds, ethanol, methanol, glycerol, and mixtures thereof. In a particular embodiment, the simple carbon source is derived from biomass. Examples of biomass sources include plants or vegetation, such as corn, sugarcane, or switchgrass. Another example of a biomass source is metabolic waste products, such as animal matter (e.g., cow manure fertilizer). Further examples of biomass sources include algae and other marine plants. Biomass includes, but is not limited to, waste products from industry, agriculture, forestry, and households, such as fermentation waste, stored hay, straw, wood, sewage, food waste, cellulosic municipal waste, and leftover food. The term "biomass" also refers to carbon sources such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).

[0080] As used herein, the term “isolated” with respect to a product (such as a 1,3-diol or derivative) refers to a product isolated from cellular components, cell culture media, or a chemical or synthetic precursor. Fatty acids (e.g., 1,3-diols) and related compositions produced by the methods described herein may be relatively immiscible in fermentation broth and in the cytoplasm. Therefore, fatty diol compositions can be collected in the organic phase either intracellularly or extracellularly. In one embodiment, the 1,3-diol composition is collected extracellularly.

[0081] As used herein, the terms “purify,” “refined,” or “purified” mean removing or isolating a molecule from its environment, for example, by isolation or separation. A “substantially purified” molecule is free of at least about 60% of other components associated with it (e.g., at least about 70%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%). As used herein, these terms also indicate the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of fatty diols in a sample. For example, if 1,3-diols are produced in recombinant host cells, 1,3-diols can be purified by the removal of host cell proteins. The percentage of 1,3-diols in the purified sample will increase. The terms “purify,” “refined,” and “purified” are relative terms that do not require complete purity. Therefore, for example, when 1,3-diols are produced in recombinant host cells, the purified 1,3-diol is a 1,3-diol substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).

[0082] The term "produce adiols (e.g., 1,3-diols) in vivo," as used for the purposes of the specification and claims, means producing adiols from a simple carbon source in living and / or recombinant and / or genetically modified host cells, wherein the simple carbon source is added to the fermentation broth so that the host cells can take up and metabolize the simple carbon source during fermentation. In one embodiment, the simple carbon source is derived from renewable raw materials.

[0083] Manipulation of host cell lines for screening Fatty acid biosynthesis is one of the most conserved systems in bacterial biosynthesis. The fatty acid synthase (FAS) multienzyme complex is present in all bacteria and eukaryotes. Most FAS-related genes are required for cell growth and survival. Eukaryotic and bacterial FAS drive essentially the same type of biochemical transformation. In eukaryotes, FAS is called FASI, and most of its catalytic domain is encoded by a single polypeptide chain (inseparable). In prokaryotes such as bacteria, FAS is called FASII, and its individual enzymes and carrier proteins are encoded by separate genes that encode separate (separable) proteins.

[0084] Acyl carrier proteins (ACPs), in conjunction with enzymes in the FAS pathway, regulate the length, saturation, and branching of fatty acids produced in natural organisms. Steps in this pathway are catalyzed by fatty acid biosynthesis (FAB) and enzymes of the acetyl-CoA carboxylase (ACC) gene family. For example, enzymes that may be included in the manipulated FAS pathway include acetyl-CoA carboxylase (e.g., AccABCD), malonyl-CoA:ACP transacylase (e.g., FabD), 3-ketoacyl-ACP synthase III (e.g., FabH), 3-ketoacyl-ACP reductase (e.g., FabG), 3-hydroxyacyl-ACP dehydratase / isomerase (e.g., FabA), 3-hydroxyacyl-ACP dehydratase (e.g., FabZ), trans-2-enoyl-ACP reductase (e.g., FabI or fabL or fabK), trans-2-enoyl-ACP isomerase (e.g., FabM), 3-ketoacyl-ACP synthase I (e.g., FabB), and 3-ketoacyl-ACP synthase II (e.g., FabF). Depending on the desired product, one or more of these genes can be attenuated or overexpressed. Therefore, host cells are engineered to increase the production of fatty acid derivatives (e.g., fatty diols, fatty alcohols) and fatty acid derivative intermediates (e.g., fatty aldehydes) by supplying a simple carbon source, which may be derived from renewable raw materials. In this specification, the primary objective is to enhance the activity of key regulatory enzymes that regulate the production of fatty acid derivatives such as fatty diols in order to transform bacterial strains into microbial factories for fatty diol production. In one embodiment, the bacterial strain produces fatty diols such as 1,3-diols. In another embodiment, the bacterial strain produces fatty diols such as 1,3-diols together with fatty alcohols. In yet another embodiment, the bacterial strain is further modified to increase specific ketoacyl-ACP reductase activity and / or decrease 3-hydroxyacyl-ACP dehydratase activity, thereby resulting in increased 1,3-fatty diol production.

[0085] Host cells are pre-programmed to increase the production of other fatty acid derivatives, including fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), and fatty alcohols (FALC) (see, for example, U.S. Patent No. 8,283,143, which is incorporated herein by reference). As will be understood by those skilled in the art, fatty acid synthesis can also occur through the elongation of acyl-CoA using acyl-ACP-independent fatty acid biosynthesis. FAS enzymes responsible for the relevant fatty acid biosynthesis reactions, such as condensation, reduction, dehydration, etc., can also be used to synthesize acyl thioesters that can be used as substrates for the production of fatty acid derivatives, including, but not limited to, fatty acids, fatty aldehydes, fatty alcohols, and 1,3-fatty diols and their 3-hydroxy derivatives. As is known to those skilled in the art, the biochemical reactions responsible for the oxidation of fatty acids (β-oxidation cycles) can function in reverse to support the synthesis of fatty acid thioesters. These acylthioesters can be used as substrates for the production of fatty acid derivatives, including, but not limited to, fatty acids, fatty aldehydes, fatty alcohols, and 1,3-fatty diols and their 3-hydroxy derivatives. Furthermore, in some organisms, fatty acid biosynthesis can occur without ACP, for example, through the synthesis of acyl-CoA (see, e.g., U.S. Patent Application Publication 2014 / 0051136(A1); U.S. Patent Application Publication 2014 / 0273114(A1); and Dellomonaco et al. (2011) Nature 476(7360):355-9). In one embodiment, these diverse components of the FAS system can be co-expressed within the same cell and work cooperatively to produce fatty acylthioesters and derivatives, including, but not limited to, fatty acids, fatty aldehydes, fatty alcohols, and 1,3-fatty diols and their 3-hydroxy derivatives.

[0086] Chiral molecules A molecule is said to be chiral if it can exist as stereoisomers (i.e., enantiomers) that are mirror images of each other but cannot be superimposed. This is relevant because a biological response to a particular molecule often depends on how that molecule matches a specific site on the organism's receptor molecule. Chiral molecules, including chiral alcohols and diols, are the basic units for the synthesis of certain compounds, such as pharmaceuticals, nutritional supplements, and other active compounds. In pharmaceutical and nutritional supplement applications, it is necessary to know which enantiomer is active and which matches the intended receptor.

[0087] One way to obtain compounds as pure active isomers is to produce the chemicals by employing organisms such as microorganisms. This is because biomolecular production in organisms is stereospecific (i.e., this yields specific stereoisomers). For example, amino acids, vitamins, and hormones are naturally produced by yeast during the fermentation of sugars and can be harvested from yeast. The properties of enzymes as chiral catalysts are recognized by those skilled in the art, and the increasing demand for drugs of high optical purity has increased interest in enzymes for the purpose of precise chemical synthesis. In contrast to producing chiral molecules in organisms, when chiral molecules are produced by chemical procedures, enantiomer mixtures (i.e., racemic mixtures) are obtained.

[0088] Current methods for enantiomer analysis include non-chromatographic techniques such as optical rotation, nuclear magnetic resonance, isotope dilution, calorimetry, and enzymatic techniques. These techniques require pure samples and do not involve the separation of enantiomers. The quantification (which does not require pure samples) and separation of enantiomers can be performed simultaneously by chiral chromatography using a chiral column, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) (see Stereochemistry of Organic Compounds, Ernest L. Elil / Sanuel H. Wilen, 1994, John Wiley & Sons, Inc.). It is possible to synthesize chiral compounds using biocatalysts, and the chiral purity of the product can be determined using chiral chromatography methods, such as chiral HPLC or LC / MS (see U.S. Patent Publications 2008 / 0248539(A1) and 2013 / 0052699(A1)).

[0089] Chirality of 3-hydroxy fatty acid derivatives A unique characteristic of 3-hydroxy fatty acid derivatives (e.g., 3-hydroxy fatty acids, 3-hydroxy fatty esters, 3-hydroxy fatty aldehydes, 3-hydroxy fatty alcohols, etc.) is that each molecule is chiral. The 3-hydroxy functional group is a stereocenter, providing a chiral center to each compound. Chirality can be a useful molecular property in defining the uses of a molecule, and such properties include, but are not limited to, polymeric properties, bioactivity, and pharmaceutical efficacy. The stereoisomers of 3-hydroxy fatty acid derivatives depend on the selectivity of the fatty acid biosynthesis (FAS) that produces the isomers. By manipulating which FAS enzyme is responsible for synthesizing the 3-hydroxy fatty acid derivative, the chirality of the resulting 3-hydroxy fatty acid derivative can be controlled. For example, if natural E. coli FAS is used for 1,3-lipiddiol biosynthesis, (R)-1,3-lipiddiol is likely to be produced. Its chiral center is created by the activity of (R)-3-hydroxylacylACP-forming 3-ketoacyl-ACP reductase, which is catalyzed by E. coli FabG (or its homolog in other microorganisms). (R)-3-hydroxylacylACP is a substrate for alcohol biosynthesis polypeptides, including those shown in pathways 1-5 in Figure 10 (but not limited to these), and pathways 1-5 convert it to (R)-1,3-lipiddiol. Furthermore, (S)-3-hydroxyacyl-CoA is an intermediate in fatty acid degradation via the β-oxidation pathway.Free fatty acids are converted to acyl-CoA by acyl-CoA synthase, which is catalyzed by FadD in E. coli and homologs in other microorganisms; acyl-CoA is oxidized to trans-2-enoyl-CoA by fatty acyl-CoA dehydrogenase, which is catalyzed by FadE in E. coli and homologs in other microorganisms; then trans-2-enoyl-CoA is hydrated by 2-trans-enoyl-CoA hydratase / (S)-3-hydroxyacyl-CoA dehydratase to (S)-3-hydroxy- It is converted to acyl-CoA, which is catalyzed by FadB in E. coli and homologs in other microorganisms; then (S)-3-hydroxyacyl-CoA is further oxidized by 3-ketoacyl-CoA dehydrogenase to 3-ketoacyl-CoA, which is also catalyzed by FadB in E. coli and homologs in other microorganisms; finally, 3-ketoacyl-CoA is thiolated by 3-ketoacyl-CoA thiolase to acyl-CoA and acetyl-CoA, which is catalyzed by FadA in E. coli and homologs in other microorganisms. Strains in which the β-oxidation (S)-3-hydroxyacyl-CoA dehydrogenase activity is selectively disrupted, for example, by a mutation in histidine 450 in E. coli FadB (or functional homologs in or from different microorganisms), will accumulate (S)-3-hydroxyacyl-CoA when provided with free fatty acids (pathways 6 and 7 in Figure 11). Histidine 450 is the catalytic residue of L-3-hydroxyacyl coenzyme A dehydrogenase associated with the large α-subunit of the E. coli-derived fatty acid oxidation polyenzyme complex (see He et al. (1996) Biochemistry 35(29):9625-9630). (S)-3-hydroxyacyl-CoA can then be converted to (S)-1,3-lipidiols through the action of fatty alcohol-forming polypeptides, such as those described in pathways 1-5 in Figure 11. Free fatty acids can be supplied to cells from an external source (pathway 7 in Figure 11), or they can be generated within cells, for example, by hydrolysis of acyl ACP by thioesterase (pathway 6 in Figure 11).In one embodiment, the acyl-CoA intermediate of the above reaction is extended by 3-ketoacyl-CoA thiolase to 3-ketoacyl-CoA (pathway 8 in Figure 11), which is catalyzed by Escherichia coli FadA and homologs of other microorganisms; the 3-ketoacyl-CoA is then reduced by a mutant of FadB in which hydratase / dehydratase activity is selectively disrupted (e.g., by mutations in Glu119 (or its homolog of a related enzyme) of Escherichia coli FadB). This results in the accumulation of (S)-3-hydroxylacyl-CoA, which can then be converted to (S)-1,3-lipidiol by a lipid diol-forming polypeptide, such as those described in pathways 1-5 in Figure 11. The large alpha-subunit glutamate-119 is the catalytic site in the hydration of 2-trans-enoyl-coenzyme A catalyzed by a multi-enzyme complex of fatty acid oxidation derived from E. coli (see He et al. (1997) Biochemistry 36(36):11044-11049). The large alpha-subunit glutamate-139 is the catalytic site in the dehydration of both D- and L-3-hydroxyacyl-coenzyme A, but is not the catalytic site in the isomerization of delta-3 and delta-2-enoyl-coenzyme A catalyzed by a multi-enzyme complex of fatty acid oxidation derived from E. coli (see Yang et al. (1995) Biochemistry 34(19):6441-6447). In another embodiment, the acyl-CoA intermediate of the above reaction is extended by 3-ketoacyl-CoA thiolase to 3-ketoacyl-CoA, which is catalyzed by FadA from E. coli and homologs from other microorganisms. Subsequently, 3-ketoacyl-CoA is reduced by (S)-3-hydroxyacyl-CoA dehydrogenase (e.g., from EC1.1.1.35) (Pathway 8, Figure 11). This leads to the accumulation of (S)-3-hydroxyacyl-CoA, which can then be converted to (S)-1,3-lipidiol by lipid diol-forming polypeptides such as those described in Pathways 1-5, Figure 11.

[0090] Genetic modifications can be performed on host cells for the purpose of manipulating them to express a specific enzyme function (see Table 1 below). In some embodiments, a polynucleotide (or gene) sequence is provided to host cells in the form of a recombinant vector. The recombinant vector includes a promoter operably ligated to the polynucleotide sequence. In some embodiments, the promoter is a developmentally regulated promoter, an organelle-specific promoter, a tissue-specific promoter, an inducible promoter, a constitutive promoter, or a cell-specific promoter. In some embodiments, the recombinant vector includes at least one sequence selected from: an expression control sequence operably ligated to the polynucleotide sequence; a selection marker operably ligated to the polynucleotide sequence; a marker sequence operably ligated to the polynucleotide sequence; a purified portion operably ligated to the polynucleotide sequence; a secretion sequence operably ligated to the polynucleotide sequence; and a targeting sequence operably ligated to the polynucleotide sequence. Expression vectors described herein contain the polynucleotide sequence in a form suitable for expression of the polynucleotide sequence in host cells. It will be understood by those skilled in the art that the design of the expression vector may depend on factors such as the selection of host cells to be transformed and the desired polypeptide expression level. The expression vectors described herein, upon introduction into host cells, can produce polypeptides containing fusion polypeptides encoded by the polynucleotide sequence described above (see above). In prokaryotes, such as E. coli, the expression of polypeptide-encoding genes is most commonly carried out using vectors containing constitutive or inducible promoters that lead to the expression of either fusion or non-fusion polypeptides. Fusion vectors typically involve adding several amino acids to the polypeptide encoded therein, usually at the amino or carboxyl terminus of the recombinant polypeptide.Such fusion vectors typically serve one or more of the following three purposes: to increase the expression of recombinant polypeptides; to enhance the solubility of recombinant polypeptides; and to assist in the purification of recombinant polypeptides by acting as ligands in affinity purification. Often, fusion expression vectors have a proteolytic cleavage site introduced at the junction between the fusion site and the recombinant polypeptide. This allows for the separation of the recombinant polypeptide from the fusion site after the purification of the fusion polypeptide. Examples of such enzymes and their homologous recognition sequences include factor Xa, thrombin, and enterokinase. Examples of fusion expression vectors include the pGEX vector (Pharmacia Biotech, Inc., Piscataway, NJ; Smith et al. (1988) Gene 67:31-40), the pMAL vector (New England Biolabs, Beverly, MA), and the pRITS vector (Pharmacia Biotech, Inc., Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the target recombinant polypeptide.

[0091] Examples of inducible non-fusion E. coli expression vectors include the pTrc vector (Amann et al. (1988) Gene 69:301-315) and the pET 11d vector (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector depends on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector depends on transcription from a T7 gn10-lac fusion promoter mediated by co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by resident λ prophages possessing the T7 gn1 gene under transcriptional regulation by the lacUV5 promoter in host strains such as BL21(DE3) or HMS174(DE3). Expression systems suitable for both prokaryotic and eukaryotic cells are well known in the field (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory). Examples of inducible non-fusion E. coli expression vectors include the pTrc vector (Amann et al. (1988) Gene 69:301-315) and the pET 11d vector (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). In certain embodiments, the polynucleotide sequence of this disclosure is operably linked to a promoter derived from bacteriophage T5. In one embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector.Vectors can be introduced into prokaryotic or eukaryotic cells via various techniques recognized in the field for introducing exogenous nucleic acids (e.g., DNA) into host cells. Suitable methods for transforming or transmuting host cells can be found, for example, in Sambrook et al. (see above). Regarding the stable transformation of bacterial cells, it is known that only a small percentage of cells take up the expression vector and replicate (depending on the expression vector and transformation technique used). For the purpose of identifying and selecting these transformants, genes encoding selection markers (e.g., antibiotic resistance) can be introduced into host cells along with the gene of interest. Examples of selection markers include, but are not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline, which confer resistance to such drugs. The nucleic acid encoding the selection marker can be introduced into host cells on the same vector as the polypeptide encoding the polypeptides described herein, or on a separate vector. Cells stably transformed by the introduced nucleic acid can be identified by proliferation in the presence of an appropriate selective drug. The manipulated or recombinant host cells described herein are cells used to produce fatty acid derivative compositions, such as lipodiol compositions. In any aspect of the disclosure described herein, the host cells can be selected from eukaryotic plants, bacteria, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophilic bacteria, yeasts, fungi, manipulated organisms thereof, or synthetic organisms. In some embodiments, the host cells are light-dependent or carbon-fixing. In some embodiments, the host cells are autotrophic. Lipodiols can be produced using a variety of host cells as described herein.

[0092] The host cells or microorganisms of this disclosure include host strains or host cells that can be genetically engineered or modified to have modifications for the purpose of testing the efficiency of specific enzyme activity. A variety of optional genetic engineering and modifications can be used interchangeably per host cell, depending on which native enzyme pathways are present in the original host cell. Host strains may include several genetic modifications for the purpose of testing specific variable parameters, such as culture conditions including fermentation components, carbon sources (e.g., raw materials), temperature, pressure, conditions for reducing culture contamination, and oxygen levels.

[0093] In one embodiment, the host strain optionally includes attenuation or deletion of one or more enzymes involved in fatty acid β-oxidation and / or phage binding sites. These gene modifications are designed to reduce intracellular fatty acid degradation or to enhance resistance to bacteriophages. In one embodiment, the host strain is Escherichia coli, and the gene modification is attenuation or deletion of fadE and / or fhuA. Acyl-CoA dehydrogenase (FadE in E. coli) is a key enzyme in fatty acid metabolism. This enzyme catalyzes the second step of fatty acid degradation (β-oxidation), which is the process of metabolizing fatty acid thioesters (acyl-CoA) into acetyl-CoA molecules and NAD(P)H. More specifically, the second step of the β-oxidation cycle of fatty acid degradation in bacteria is the oxidation of acyl-CoA to 2-enoyl-CoA, and this oxidation is catalyzed by FadE. In E. coli or other bacteria, when FadE or fatty acyl-CoA dehydrogenase is deleted or attenuated, these bacteria are unable to grow on fatty acids as a carbon source with little or no ability. The lack of availability of fatty acids of any chain length is consistent with the reported fadE strain phenotype, i.e., fadE mutants in which FadE function is disrupted. The fadE gene can be optionally knocked out or attenuated, thereby ensuring the accumulation of acyl-CoA in the cell, which can potentially become intermediates in the fatty acid derivative pathway, so that all acyl-CoA can be efficiently converted to fatty acid derivatives. However, fadE attenuation may be optional when using sugars as a carbon source under unrestricted conditions, because under such conditions, FadE expression may be suppressed, and therefore FadE may be present only in small amounts and unable to efficiently compete with ester synthases or other enzymes for acyl-CoA substrates. Under these circumstances, FadE can be considered suppressed by catabolic metabolite repression.E. coli and many other microorganisms preferentially consume sugars over fatty acids; therefore, when both carbon sources are available, sugars are expected to be consumed first as a result of the suppression of the fad regulon (see D. Clark, J Bacteriol. (1981) 148(2):521-6). Furthermore, the presence of fatty acids in the absence of sugars induces FadE expression. Acyl-CoA intermediates may be lost in the β-oxidation pathway because proteins expressed by the fad regulon (including FadE) become upregulated and efficiently compete for acyl-CoA. Therefore, having a knocked-out or attenuated fadE gene may be beneficial. The carbon source can be carbohydrates, thus selectively attenuating FadE.

[0094] For example, in E. coli, either the fadE gene (encoding acyl-CoA dehydrogenase) or the fadD gene (encoding acyl-CoA synthetase) can be deleted. Such strains are unable to break down fatty acids, or do so very little, and therefore increase the amount of fatty acids available within the cell. This allows such fatty acids to be more readily available for conversion into products such as fatty acid derivatives. Fatty acids can also be made available by deleting other fatty acid-degrading enzymes, such as fadA or fadB. Deletion of either of these genes is optional and can be performed when free fatty acids are supplied from an external source or are intermediates in the pathway to products. Table 1 (below) presents a comprehensive list of enzyme activity in metabolic pathways, and this table includes various fatty acid-degrading enzymes that can be attenuated to increase the availability of fatty acids in host strains.

[0095] In E. coli, the gene fhuA encodes the TonA protein, an energy-coupled transporter and receptor on the E. coli outer membrane (V. Braun (2009) J Bacteriol. 191(11):3431-3436). Deletion of fhuA is optional. By deleting fhuA, cells can become more resistant to phage attack. Phage attack can be detrimental in commercial fermentation. Therefore, deleting fhuA in host cells that are likely to be subject to potential contamination during fermentation may be desirable. Similarly, homologous proteins from other organisms, as well as phage binding sites, may be candidates for deletion to improve phage resistance.

[0096] In another embodiment, the host strain (see above) also optionally includes the overexpression of one or more genes, including fadR, fabA, fabD, fabG, fabH, fabV, and / or fabF. Examples of such genes include fadR from Escherichia coli, fabA (NP_460041), fabD (NP_460164), fabG (NP_460165), fabH (NP_460163) from Salmonella typhimurium, fabV (YP_001217283) from Vibrio cholera, and fabF (NP_350156) from Clostridium acetobutylicum. Overexpression of one or more of these genes encoding enzymes and regulators of fatty acid biosynthesis may help increase the titer of fatty acid derivative intermediates, including fatty aldehydes, and end products such as fatty diols, under various culture conditions.

[0097] In one embodiment, an Escherichia coli strain is used as a host cell for fatty diol production. These host cells may optionally contain the overexpression of one or more biosynthetic genes (i.e., genes encoding enzymes and regulators of fatty acid biosynthesis) that can further increase or enhance the titer of fatty acid derivative compounds such as fatty acid derivative intermediates (e.g., fatty aldehydes) and end products (e.g., fatty diols, fatty alcohols) under various culture conditions. Such genes include, but are not limited to, fadR, fabA, fabD, fabG, fabH, fabV, and / or fabF. Examples of genetically modified genes include fadR from Escherichia coli, fabA (NP_460041) from Salmonella tiphimuria, fabD (NP_460164) from Salmonella tiphimuria, fabG (NP_460165) from Salmonella tiphimuria, fabH (NP_460163) from Salmonella tiphimuria, fabV (YP_001217283) from Vibrio cholerae, and fabF (NP_350156) from Clostridium acetobutyricum. In some embodiments, synthetic operons containing these biosynthetic genes can be manipulated and expressed in cells for the purpose of testing the overexpression of fatty acid derivative intermediates under various culture conditions and / or further enhancing fatty diol production. Such synthetic operons contain one or more biosynthetic genes. For example, the ifab138 operon is an engineered operon containing an optional fatty acid biosynthesis gene, including Vibrio cholerae fabV, Salmonella tiphimuria fabH, S. tiphimuria fabD, S. tiphimuria fabG, S. tiphimuria fabA, and / or Clostridium acetobutyricum fabF, which can be used to promote the overexpression of fatty acid derivatives and intermediates for the purpose of testing specific culture conditions. One advantage of such synthetic operons is that they can further increase or improve the rate of production of fatty acid derivatives (e.g., fatty acids, fatty aldehydes, fatty alcohols, fatty diols, etc.) in cells containing them.

[0098] In some embodiments, the host cells or microorganisms used to produce acyl thioesters (such as acyl-CoA or acyl-ACP) and biosynthetic enzymes (e.g., TE, CAR, AR, ADH, ACC, AAR, FAR, ACR; see Figures 1-3 and 8-11) further express genes encompassing certain enzymatic activity that can increase the production of one or more specific fatty acid derivatives, such as fatty acids, 3-hydroxy fatty acids, fatty alcohols, 1,3-fatty diols, fatty aldehydes, and 3-hydroxy fatty aldehydes. In one embodiment, the host cells have thioesterase (TE) activity (EC3.1.2.- or EC3.1.2.14 or EC3.1.1.5) for the production of fatty acids and 3-hydroxy fatty acids, and this production can be increased by gene overexpression. In another embodiment, the host cell has thioesterase (TE) activity (EC3.1.2.- or EC3.1.2.14 or EC3.1.1.5) and carboxylic acid reductase (CAR) activity (EC6.2.1.3 or EC1.2.1.42 or EC1.2.99.6) for the production of fatty alcohols and / or fatty diols. In another embodiment, the host cell has thioesterase (TE) activity (EC3.1.2.- or EC3.1.2.14 or EC3.1.1.5) and carboxylic acid reductase (CAR) activity (EC6.2.1.3 or EC1.2.1.42 or EC1.2.99.6) and alcohol dehydrogenase (ADH) / aldehyde reductase (AR) activity (EC1.1.1.-) for the production of fatty alcohols and / or fatty diols. In another embodiment, the host cell has acyl-ACP reductase (AAR) activity (EC1.2.1.80 or EC1.2.1.42) for the production of fatty aldehydes and / or 3-hydroxy-fatty aldehydes. In another embodiment, the host cell has acyl-ACP reductase (AAR) activity (EC1.2.1.80 or EC1.2.1.42) and alcohol dehydrogenase (ADH) / aldehyde reductase (AR) activity (EC1.1.1.-) for the production of fatty alcohols and / or fatty diols.The combination of genes can be overexpressed or underexpressed by appropriately manipulating the microorganism. In one embodiment, one or more overexpressed genes are endogenous. In another embodiment, one or more overexpressed genes are exogenous.

[0099] In an alternative embodiment, the host cell has acyl-ACP reductase (AAR) activity (EC1.2.1.80 or EC1.2.1.42) and / or acyl-ACP / acyl-CoA reductase (AAR / ACR) activity (EC1.2.1.80 or EC1.2.1.42 or EC1.2.1.50) and / or alcohol dehydrogenase activity (EC1.1.-.-) and / or fatty alcohol-forming acyl-CoA / acyl-ACP reductase (FAR) activity (EC1.1.1.-) and / or carboxylic acid reductase (CAR) activity (EC6.2.1.3 or EC1.2.1.42 or EC1.2.99.6) and / or thioesterase (TE) activity (EC3.1.2.- or EC3.1.2.14 or EC3.1.1.5) for the production of fatty alcohols. In other alternative embodiments, host cells possess acyl-CoA reductase activity (EC1.2.1.50), acyl-CoA synthase (FadD) activity (EC2.3.1.86), and thioesterase (TE) activity (EC3.1.2.- or EC3.1.2.14 or EC3.1.1.5) for the production of fatty alcohols. The expression of these alternative enzyme activities in microorganisms and microbial cells is taught in U.S. Patents 8,097,439; 8,110,093; 8,110,670; 8,183,028; 8,268,599; 8,283,143; 8,232,924; 8,372,610; and 8,530,221, which are incorporated herein by reference. In other embodiments, the host cells or microorganisms used to produce acyl-ACP and / or acyl-CoA and other biosynthetic enzymes contain certain innate enzyme activity that is upregulated or overexpressed for the purpose of producing one or more specific fatty acid derivatives, such as fatty aldehydes and / or fatty alcohols and / or fatty diols. In one embodiment, the host cells have innate thioesterase (TE) activity for fatty acid production, which can be increased by overexpressing the thioesterase gene.

[0100] This disclosure includes host strains or microorganisms that express genes encoding biosynthetic enzymes (see above). Recombinant host cells produce fatty acid derivative intermediates such as fatty aldehydes and fatty acid derivative final products such as fatty alcohols and / or fatty diols, as well as compositions and blends thereof. The fatty acid derivative final products are typically recovered from a culture medium and / or isolated from host cells. In one embodiment, fatty diols and / or fatty alcohols are recovered from a culture medium (extracellular). In another embodiment, fatty diols and / or fatty alcohols are isolated from host cells (intracellular). In yet another embodiment, fatty diols and / or fatty alcohols are recovered from a culture medium and isolated from host cells. In yet another embodiment, fatty diols and / or fatty alcohols are extracellular, associated with host cells, and isolated from host cells. Fatty diol compositions produced by host cells can be analyzed using methods known in the art, such as GC-FID, for the purpose of determining the distribution of specific fatty diols, as well as the chain length and saturation of the components of the fatty diol composition.

[0101] Examples of host cells that function as microorganisms (e.g., microbial cells) include the genera Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, and Myceliophtra. Examples of host cells include, but are not limited to, those of the genera *Liophtora*, *Penicillium*, *Phanerochaete*, *Pleurotus*, *Trametes*, *Synechococcus*, *Synechocystis*, *Lactococcus*, *Chrysosporium*, *Saccharomyces*, *Stenotrophamonas*, *Schizosaccharomyces*, *Yarrowia*, or *Streptomyces*. In some embodiments, the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell. In some embodiments, the host cell is an *Escherichia coli* cell. In some embodiments, the host cell is an E. coli B cell, an E. coli C cell, an E. coli K cell, or an E. coli W cell.In other embodiments, the host cells include Bacillus lentus cells, Bacillus brevis cells, Bacillus stearothermophilus cells, Bacillus lichenoformis cells, Bacillus alkalophilus cells, Bacillus coagulans cells, Bacillus circulans cells, Bacillus pumilis cells, Bacillus thuringiensis cells, Bacillus clausii cells, Bacillus megaterium cells, and Bacillus subtilis cells. These are subtilis cells, or Bacillus amyloliquefaciens cells.In further embodiments, the host cells include Trichoderma koningii cells, Trichoderma viride cells, Trichoderma reesei cells, Trichoderma longibrachiatum cells, Aspergillus awamori cells, Aspergillus fumigates cells, Aspergillus foetidus cells, Aspergillus nidulans cells, Aspergillus niger cells, Aspergillus oryzae cells, and Humicola insolence cells. The host cells are *Insolens* cells, *Humicola lanuginose* cells, *Rhodococcus opacus* cells, *Rhizomucor miehei* cells, or *Mucor michei* cells. In yet another embodiment, the host cells are *Streptomyces lividans* cells or *Streptomyces murinus* cells. In yet another embodiment, the host cells are actinomycete cells. In some embodiments, the host cells are *Saccharomyces cerevisiae* cells. In other embodiments, the host cell is derived from eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophilic bacteria, yeasts, fungi, organisms manipulated therefrom, or synthetic organisms. In some embodiments, the host cell is light-dependent or carbon-fixing. In some embodiments, the host cell is autotrophic. In some embodiments, the host cell is photoautotrophic, such as in the presence of light.In some embodiments, the host cells are heterotrophic or mixed trophic in the absence of light. In a particular embodiment, the host cells include Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcus braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus species PCC7002, Synechococcus species PCC7942, Synechocystis species PCC6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum, and Chlorojlexus aurantiacus. auranticus), Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, Pseudomonas ptida These are cells derived from *Putida* or *Zymomonas mobilis*.In one particular embodiment, the microbial cells are derived from cyanobacteria, including, but not limited to, the genera Prochlorococcus, Synechococcus, Synechocystis, Cyanothece, and Nostoc punctiforme. In another embodiment, the microbial cells are derived from specific cyanobacterial species, including, but not limited to, Synechococcus elongatus PCC7942, Synechocystis species PCC6803, and Synechococcus species PCC7001.

[0102] Recombinant host cells engineered to produce 1,3-diols This disclosure identifies polynucleotides encoding enzymatically functional polypeptides for the purpose of altering enzymatic pathways for the production of desired compounds, such as fatty diols (e.g., 1,3-diols). These polypeptides are identified herein by their enzyme accession number (EC number, see Table 1 below) and are useful for manipulating fatty acid pathways that lead to fatty diol production. More specifically, Figures 1-3 and 8-11 show pathways manipulated to produce 1,3-diols. As shown in the figures, 3'-hydroxyacyl carrier proteins (ACPs) (acyl-ACP or 3-hydroxyacyl-ACP) possessing acyl intermediates can be converted to 1,3-diols by employing 3'-hydroxy fatty acids (3'OHFAs) and 3'-hydroxy fatty aldehydes (3'OH fatty aldehydes) as intermediates. In one embodiment, the manipulated pathway is shown in Figures 1-3 and 8-11 and produces 1,3-diols. In this specification, a simple carbon source such as glucose is first converted to 3'-hydroxyacyl-ACP by microorganisms (e.g., Escherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus, Synechocystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Fumycola, Rhizomucor, Kluiveromyces, Pichia, Mucor, Miseliophtra, Penicillium, Fanelocheate, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophomonas, Schizosaccharomyces, Yarowia, or Streptomyces). In some embodiments, universally and highly conserved acyl-ACP or 3'-hydroxyacyl-ACP is produced by a natural microbial pathway. In one embodiment, a 3'-hydroxyacyl-ACP can be used to initiate the manipulated pathway. For example, 3'-hydroxyacyl-ACP can be converted to an intermediate such as 3'OH FA by an enzyme having thioesterase (TE) activity (see Table 2 below). The intermediate 3'OH FA can then be converted to another intermediate such as 3'OH aldehyde by an enzyme having carboxylic acid reductase (CAR) activity (see Table 3 below).Next, enzymes possessing alcohol dehydrogenase (ADH) or aldehyde reductase (AR) activity (see Table 4 below) can convert 3'OH aldehydes to 1,3-diols. For the purpose of further illustrating such pathways, Figure 2 presents examples of specific enzymes possessing thioesterase activity (e.g., fatB1, tesA, phaG); CAR activity (e.g., carB); and ADH / AR activity (e.g., alrA). Further examples of thioesterase (TE) enzymes capable of carrying out the reaction to convert 3'OH acyl-ACP to 3'OH FA are shown in Table 2. In one embodiment, the genes encoding these thioesterases are tesA, tesB, fatB, fatB1, fatB2, fatB3, TE_EEI82564, TE_CAD63310, and phaG. In another embodiment, the genes encoding these thioesterases are TE_EEI82564 and / or TE_CAD63310, which were previously considered unrelated to the ability to convert 3'OH acyl-ACP to 3'OH FA (see, e.g., Jing et al. (2011) BMC Biochemistry 12(44):1471-2091). Further examples of CAR enzymes capable of converting 3'OH FA to 3'OH aldehyde are shown in Table 3. In one embodiment, the gene encoding the CAR enzyme is carB. Further examples of ADH / AR enzymes capable of converting 3'OH aldehyde to 1,3-diol are shown in Table 4. In one embodiment, the genes encoding these ADH / AR enzymes are alrA and / or yqhD.

[0103] In another embodiment, an engineered pathway that also produces a 1,3-diol is shown in Figure 3. Similarly, a simple carbon source such as glucose is first converted to 3'-hydroxyacyl-ACP by microorganisms (e.g., Escherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus, Synechocystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Fumycola, Rhizomucor, Kluiveromyces, Pichia, Mucor, Miseliophtra, Penicillium, Fanelocheate, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophomonas, Schizosaccharomyces, Yarowia, or Streptomyces). In some embodiments, the universally and highly conserved 3'-hydroxyacyl-ACP is produced by the natural pathways of microorganisms. As described above, 3'-hydroxyacyl-ACP can be used to initiate engineered pathways. For example, 3'-hydroxyacyl-ACP is converted to intermediates such as 3'OH fatty aldehydes by enzymes having acyl-ACP reductase (AAR) activity (see Table 1). The production of fatty alcohols and / or fatty aldehydes by AARs may be enhanced through heterologous expression of a gene called accABCD that encodes acetyl-CoA carboxylase. Examples of AAR enzymes capable of converting 3'OH acyl-ACP to 3'OH aldehydes include, but are not limited to, those of Synechococcus elongatus, Cyanoseis species, Synechocystis species, and Prochlorococcus marinus. Subsequently, enzymes having alcohol dehydrogenase (ADH) or aldehyde reductase (AR) activity (see Table 4) can convert 3'OH aldehydes to fatty diols such as 1,3-diols. Therefore, this disclosure provides recombinant microorganisms that can efficiently and selectively produce fatty diols, including 1,3-diols, in vivo. Most cells naturally produce enzymes that can reduce aldehydes, as these can be cytotoxic.Therefore, heterologous expression of AR and ADH may not be necessary for the production of fatty alcohols and diols, but it may improve the efficiency of fatty alcohol and diol production.

[0104] Furthermore, polynucleotides encoding polypeptides with fatty acid-degrading enzyme activity can be selectively attenuated in host cells. Non-limiting examples of such polypeptides include acyl-CoA synthetases (e.g., E. coli FadD) and acyl-CoA dehydrogenases (e.g., E. coli FadE). Table 1 provides a comprehensive list of enzyme activities in illustrative metabolic pathways, including various fatty acid-degrading enzymes that can be selectively attenuated according to methods known in the art (see, e.g., U.S. Patent No. 8,283,143, above). For example, FadR (see Table 1) is an important regulator involved in fatty acid degradation and fatty acid biosynthesis pathways in E. coli (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)). The E. coli enzyme FadD (see Table 1) and the fatty acid transport protein FadL are components of the fatty acid uptake system. FadL and its homologs mediate the transport of fatty acids into bacterial cells, while FadD and its homologs mediate the formation of acyl-CoA esters. An alternative heterologous uptake system for fatty acids and fatty acid derivatives is AlkL, an outer membrane protein of the Pseudomonas genus (Julsing et al. (2012) Appl. Environ. Microbiol. 78:5724-5733). When other carbon sources are unavailable, exogenous fatty acids are taken up by bacteria and converted to acyl-CoA esters. These esters can bind to the transcription factor FadR to repress the expression of the fad gene, which encodes proteins responsible for fatty acid transport (FadL), activation (FadD), and β-oxidation (FadA, FadB, and FadE). When alternative carbon sources are available, bacteria synthesize fatty acids as acyl-ACPs, which are used for phospholipid synthesis but are not substrates for β-oxidation. Therefore, both acyl-CoA and acyl-ACP are independent fatty acid sources that can yield different end products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169(2004)).FadR and / or FabB, as well as their functional homologs, can enhance the production of fatty acid derivatives in host cells (e.g., E. coli), but their overexpression is optional. This specification intends to suggest that FabB overexpression may improve the elongation rate (fatty acid chain synthesis), and that FadR overexpression may increase the expression of FabA and FabB. The latter is possible because FadR is considered to be a positive regulator of FabA and FabB.

[0105] (Table 1) Enzyme activity TIFF2026069552000002.tif46170TIFF2026069552000003.tif215170TIFF20260695520 00004.tif221170TIFF2026069552000005.tif221170TIFF2026069552000006.tif98170

[0106] (Table 2) Thioesterase activity TIFF2026069552000007.tif78157

[0107] (Table 3) Carboxylic acid reductase (CAR) activity TIFF2026069552000008.tif32164

[0108] (Table 4) Alcohol dehydrogenase (ADH) or aldehyde reductase (AR) activity TIFF2026069552000009.tif51157

[0109] This disclosure identifies polynucleotides encoding polypeptides with useful enzymatic activity in recombinant host cells and production methods. These enzymatic polypeptides contribute to the production of compositions containing fatty diol compounds. It is generally recognized that complete sequence identity for such polynucleotides is not required. For example, modifications can be made to specific polynucleotide sequences (e.g., polynucleotides encoding enzymatic polypeptides), and the encoded polypeptides can be screened for activity. Such modifications typically include conservative mutations and silent mutations (e.g., codon optimization). Genetically engineered or modified polynucleotides and encoded mutant polypeptides can be screened for desired functions using methods known in the art, including, but not limited to, improved catalytic activity, improved stability, or reduced inhibition (e.g., reduced feedback inhibition).

[0110] Furthermore, this disclosure identifies the enzyme activity involved in various steps (i.e., reactions) of the manipulated pathway involved in the production of fatty diols (see above) as described herein by enzyme classification (EC) numbers, and presents examples of polypeptides (e.g., enzymes) classified by such EC numbers and examples of polynucleotides encoding such polypeptides. Such exemplary polypeptides and polynucleotides, identified herein by accession numbers and / or sequence identification numbers (SEQ ID NOs), are useful for manipulating the fatty acid pathway that leads to the production of fatty diols, including 1,3-fatty diols, in parental host cells to obtain recombinant or genetically modified host cells as described herein. The polypeptides and polynucleotides described herein are illustrative and not limiting. The sequences of homologous polypeptides described herein are available to those skilled in the art through various databases (for example, the Entrez database provided by the National Center for Biotechnology Information (NCBI), the ExPasy database provided by the Swiss Institute for Bioinformatics, the BRENDA database provided by Braunschweig University of Technology, and the KEGG database provided by the Bioinformatics Center of the Institute for Chemical Research, Kyoto University and the University of Tokyo, all of which are available on the World Wide Web).

[0111] Fermentation and fatty diol production As used herein, fermentation, in a broad sense, refers to the conversion of organic material into a target substance by recombinant host cells. For example, fermentation may include the conversion of a carbon source to fatty acid derivatives such as fatty diols by recombinant host cells by growing a culture of recombinant host cells in a medium containing a carbon source. Conditions that allow the production of target substances such as fatty diols and / or fatty alcohols are any conditions that enable the host cells to produce the desired product, such as a fatty diol composition. Similarly, these conditions may include any conditions that enable the host cells to synthesize a target polypeptide, such as a polynucleotide sequence of a vector expressed in the host cells. Typical fermentation conditions are, for example, suitable conditions. Fermentation conditions can include many parameters, such as temperature range, pH level, aeration level, feed rate, and medium composition, but are not limited to these. Each of these conditions, individually or in combination, enables the growth of host cells. Fermentation can be aerobic, anaerobic, or modified thereof (such as microaerobic). Examples of culture media include broth (liquid) or gel (solid). Generally, culture media contain a carbon source that can be directly metabolized by host cells (e.g., a simple carbon source derived from renewable raw materials). Enzymes can also be used in the culture medium to facilitate fluidization (e.g., depolymerization of starch or cellulose into fermentable sugars) and subsequent metabolism of the carbon source.

[0112] For small-scale production, engineered host cells can be grown; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide encoding a polypeptide with specific enzyme activity (e.g., TE, CAR, ADH, FAR, ACR, ACC, and / or AAR enzyme activity). For large-scale production, engineered host cells can be grown; fermented; and induced to express any desired polynucleotide sequence, such as a polynucleotide encoding a polypeptide with specific enzyme activity (e.g., TE, CAR, ADH, FAR, ACR, ACC, and / or AAR enzyme activity). The fatty acid diol compositions described herein can be found in the extracellular environment of recombinant host cell cultures and can be readily isolated from the culture medium. Fatty acid derivatives such as fatty acid diols and / or fatty alcohols may be secreted by recombinant host cells and transported into the extracellular environment of recombinant host cell cultures, or passively transported into the extracellular environment of recombinant host cell cultures. The fatty acid diol compositions may be isolated from recombinant host cell cultures using established methods known in the art.

[0113] Several modifications have been made to the producing host cells for the purpose of producing fatty diols (see above). That is, this disclosure provides recombinant host cells that have been engineered to provide a biosynthetic pathway compared to unmanipulated, i.e., natural host cells (e.g., wild-type host cells that function as control cells), which is achieved, for example, by improving a particular strain. Microorganisms, such as bacteria, cyanobacteria, yeasts, algae, or filamentous fungi, can be used as producing hosts. Non-limiting examples of microorganisms that can be used as producing hosts include Escherichia coli and S. cerevisiae. The microbial strains efficiently convert glucose or other renewable raw materials into fatty acid derivatives, including fatty alcohols and fatty diols. To achieve this, the strains have been carefully engineered to express key enzymes with specific functions. High-density fermentation protocols and procedures for producing various compounds have been established (see, for example, U.S. Patents 8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028; 8,110,670; 8,110,093; and 8,097,439, which are incorporated herein by reference).

[0114] Notably, to date, there is no method for directly and efficiently producing fatty diols, including 1,3-diols, from glucose or other renewable raw materials. Nevertheless, these fatty diols have applications as components in detergents, surfactants, emulsifiers, softeners, solvents, plastics, and food additives. The fermentation-based methods for producing fatty diols and their compositions presented herein provide an environmentally friendly alternative to the chemical methods employed in the art. In some embodiments, host cells are cultured in a culture medium (e.g., a fermentation medium) containing a carbon source (e.g., a simple carbon source) at an initial concentration of about 20 g / L to about 900 g / L. In other embodiments, the culture medium contains the carbon source at initial concentrations of about 2 g / L to about 10 g / L; about 10 g / L to about 20 g / L; about 20 g / L to about 30 g / L; about 30 g / L to about 40 g / L; or about 40 g / L to about 50 g / L. In some embodiments, the available carbon source level in the culture medium can be observed during the progress of fermentation. In some embodiments, the method further includes the step of adding a supplemental carbon source to the culture medium when the initial carbon source level in the culture medium falls below about 0.5 g / L. In some embodiments, the supplemental carbon source is added to the culture medium when the carbon source level in the culture medium falls below about 0.4 g / L, about 0.3 g / L, about 0.2 g / L, or about 0.1 g / L. In some embodiments, the supplemental carbon source is added to maintain the carbon source level between about 1 g / L and about 25 g / L. In some embodiments, the supplemental carbon source is added to maintain the carbon source level above about 2 g / L (e.g., above about 2 g / L, above about 3 g / L, above about 4 g / L). In a particular embodiment, the supplemental carbon source is added to maintain the carbon source level below about 5 g / L (e.g., below about 5 g / L, below about 4 g / L, below about 3 g / L). In some embodiments, supplemental carbon sources are added to maintain the carbon source level at approximately 2 g / L to 5 g / L, 5 g / L to 10 g / L, or 10 g / L to 25 g / L.

[0115] In one embodiment, the carbon source for fermentation is derived from renewable raw materials. In some embodiments, the carbon source is glucose. In other embodiments, the carbon source is glycerol. Other possible carbon sources include, but are not limited to, fructose, mannose, galactose, xylose, arabinose, starch, cellulose, pectin, xylan, sucrose, maltose, cellobiose, and turanose; cellulosic materials and derivatives, e.g., hemicellulose, methylcellulose, and sodium carboxymethylcellulose; saturated or unsaturated fatty acids, succinic acid compounds, lactic acid compounds, and acetic acid compounds; alcohols, e.g., ethanol, methanol, and glycerol, or mixtures thereof. In one embodiment, the carbon source is derived from corn, sugarcane, sorghum, beets, switchgrass, storage hay, straw, wood, pulp, sewage, food waste, cellulosic municipal waste, flue gas, synthesis gas, or carbon dioxide. Simple carbon sources can also be photosynthetic products, e.g., glucose or sucrose. In one embodiment, the carbon source is derived from waste products, such as glycerol, flue gas, or synthesis gas; or from organic materials, such as the modification of biomass; or from natural gas or methane, or the modification of these materials into synthesis gas; or from carbon dioxide fixed by photosynthesis, for example, 1,3-diol may be produced by recombinant cyanobacteria that grow by photosynthesis and use CO2 as a carbon source. In a particular embodiment, the carbon source is derived from biomass. Examples of biomass sources are plants or vegetation, such as corn, sugarcane, or switchgrass. Another example of a biomass source is metabolic waste products, such as animal matter (e.g., cow manure fertilizer). Further examples of biomass sources include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry, and households, and such waste products include, but are not limited to, fermentation waste, stored hay, straw, wood, sewage, food waste, cellulosic municipal waste, municipal solid waste, and leftover food.

[0116] In some embodiments, fatty diols (e.g., 1,3-diols) are produced at concentrations of about 0.5 g / L to about 40 g / L. In some embodiments, fatty diols are produced at concentrations of about 1 g / L or more (e.g., about 1 g / L or more, about 10 g / L or more, about 20 g / L or more, about 50 g / L or more, about 100 g / L or more). In some embodiments, fatty diols are produced at concentrations of about 1 g / L to about 170 g / L, about 1 g / L to about 10 g / L, about 40 g / L to about 170 g / L, about 100 g / L to about 170 g / L, about 10 g / L to about 100 g / L, about 1 g / L to about 40 g / L, about 40 g / L to about 100 g / L, or about 1 g / L to about 100 g / L.

[0117] In some embodiments, the fatty diols are approximately 25 mg / L, approximately 50 mg / L, approximately 75 mg / L, approximately 100 mg / L, approximately 125 mg / L, approximately 150 mg / L, approximately 175 mg / L, approximately 200 mg / L, approximately 225 mg / L, approximately 250 mg / L, approximately 275 mg / L, approximately 300 mg / L, approximately 325 mg / L, approximately 350 mg / L, approximately 375 mg / L, approximately 400 mg / L, approximately 425 mg / L, approximately 450 mg / L, approximately 475 mg / L, approximately 500 mg / L, approximately 525 mg / L, approximately 550 mg / L, and approximately 57 mg / L. 5mg / L, approximately 600mg / L, approximately 625mg / L, approximately 650mg / L, approximately 675mg / L, approximately 700mg / L, approximately 725mg / L, approximately 750mg / L, approximately 775mg / L, approximately 800mg / L, approximately 825mg / L, approximately 850mg / L, approximately 875m g / L, approximately 900mg / L, approximately 925mg / L, approximately 950mg / L, approximately 975mg / L, approximately 1000mg / L, approximately 1050mg / L, approximately 1075mg / L, approximately 1100mg / L, approximately 1125mg / L, approximately 1150mg / L, approximately 1175mg / L, approximately 1200mg / L, about 1225mg / L, about 1250mg / L, about 1275mg / L, about 1300mg / L, about 1325mg / L, about 1350mg / L, about 1375mg / L, about 1400mg / L, about 1425mg / L, about 1450mg / L, about 1475mg / L, about 1500mg / L, about 1525mg / L, about 1550mg / L, about 1575mg / L, about 1600mg / L, about 1625mg / L, about 1650mg / L, about 1675mg / L, about 1700mg / L, about 1725mg / L, about 1 It is produced with a potency in the range of 750 mg / L, approximately 1775 mg / L, approximately 1800 mg / L, approximately 1825 mg / L, approximately 1850 mg / L, approximately 1875 mg / L, approximately 1900 mg / L, approximately 1925 mg / L, approximately 1950 mg / L, approximately 1975 mg / L, approximately 2000 mg / L (2 g / L), 3 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, or any two of these values.In other embodiments, the fatty diol (e.g., 1,3-diol) is produced at titers greater than 100 g / L, greater than 200 g / L, greater than 300 g / L, or higher, such as 500 g / L, 700 g / L, 1000 g / L, 1200 g / L, 1500 g / L, or 2000 g / L. Preferred titers of fatty diols such as 1,3-diol produced by recombinant host cells by the method of the present disclosure are 5 g / L to 200 g / L, 10 g / L to 150 g / L, 20 g / L to 120 g / L, and 30 g / L to 100 g / L, 100 g / L to 150 g / L, and 120 g / L to 180 g / L. In one embodiment, the titer of adipodiols such as 1,3-diols produced by recombinant host cells by the method of the present disclosure is approximately 1 g / L to approximately 250 g / L, more specifically, 90 g / L to approximately 120 g / L. The titer may refer to a specific 1,3-diol produced by a given recombinant host cell culture, or to a combination of 1,3-diols of various chain lengths or various functions.

[0118] In other embodiments, host cells manipulated to produce adiols such as 1,3-diols according to the method of the present disclosure have a yield in the range defined by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30%, or at least 40%, or any two of these values. In other embodiments, fatty diols such as 1,3-diols are produced in yields of over 30%, over 40%, over 50%, over 60%, over 70%, over 80%, over 90%, or higher. Alternatively, or in addition, the yield may be about 30% or less, about 27% or less, about 25% or less, or about 22% or less. Thus, the yield can be defined by any two of the above limit values. For example, the yield of fatty diols such as 1,3-diols produced by recombinant host cells according to the method of this disclosure can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. In a particular embodiment, the yield of fatty diols such as 1,3-diols produced by recombinant host cells is about 10% to about 40%. In another specific embodiment, the yield of lipid diols such as 1,3-diols produced by recombinant host cells is approximately 25% to 30%. The yield may refer to a specific 1,3-diol produced by a given recombinant host cell culture, or to a combination of 1,3-diols with various chain lengths or different functions. The yield is also likely to depend on the raw materials used.

[0119] In some embodiments, the production capacity of lipid diols such as 1,3-diols produced by recombinant host cells is at least 100 mg / L / hour, at least 200 mg / L / hour, at least 300 mg / L / hour, at least 400 mg / L / hour, at least 500 mg / L / hour, at least 600 mg / L / hour, at least 700 mg / L / hour, at least 800 mg / L / hour, at least 900 mg / L / hour, at least 1000 mg / L / hour, at least 1100 mg / L / hour, and at least 12 The production rate is 00 mg / L / hour, at least 1300 mg / L / hour, at least 1400 mg / L / hour, at least 1500 mg / L / hour, at least 1600 mg / L / hour, at least 1700 mg / L / hour, at least 1800 mg / L / hour, at least 1900 mg / L / hour, at least 2000 mg / L / hour, at least 2100 mg / L / hour, at least 2200 mg / L / hour, at least 2300 mg / L / hour, at least 2400 mg / L / hour, or at least 2500 mg / L / hour. For example, the production capacity of fatty diols such as 1,3-diols produced by recombinant host cells according to the method of this disclosure may be 500 mg / L / hour to 2500 mg / L / hour, or 700 mg / L / hour to 2000 mg / L / hour. In one particular embodiment, the production capacity is approximately 0.7 mg / L / hour to approximately 3 g / L / hour. The production capacity may also refer to specific fatty diols, such as 1,3-diols, produced by a given recombinant host cell culture.

[0120] In some embodiments, the host cells used in the fermentation procedures described herein (see above) are mammalian cells, plant cells, insect cells, yeast cells, fungal cells, filamentous fungal cells, algal cells, cyanobacteria cells, and bacterial cells. In certain embodiments, the host cells are selected from the genera Escherichia, Bacillus, Pseudomonas, Lactobacillus, Rhodococcus, Synechococcus, Synechocystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Fumicola, Rhizomucor, Kluiveromyces, Pichia, Mucor, Myserioftra, Penicillium, Fanelocheate, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophomonas, Schizosaccharomyces, Yarowia, or Streptomyces. In other embodiments, the host cells are Bacillus lentus cells, Bacillus brevis cells, Bacillus stearothermophilus cells, Bacillus licheniformis cells, Bacillus alkarophilus cells, Bacillus coagulans cells, Bacillus circulans cells, Bacillus pumilus cells, Bacillus thuringiensis cells, Bacillus clausii cells, Bacillus megatherium cells, Bacillus subtilis cells, or Bacillus amyloriquefaciens cells. In other embodiments, the host cells are Pseudomonas ptida cells. In a particular embodiment, the host cells are Synechococcus species PCC7002, Synechococcus elongatas PCC7942, Synechocystis species PCC6803, Synechococcus elongatas PCC6301, Prochlorococcus marinus CCMP1986 (MED4), Anabaena variabilis ATCC29413, Nostoc punctateforme ATCC29133 (PCC73102), and Gloebacter violaceus. These include *Violaceus* ATCC29082 (PCC7421), *Nostoc* species ATCC27893 (PCC7120), *Cyanoseis* species PCC7425 (29141), *Cyanoseis* species ATCC51442, or *Synechococcus* species ATCC27264 (PCC7002).In other embodiments, the host cells are Trichoderma coningii cells, Trichoderma viride cells, Trichoderma liesei cells, Trichoderma longibrachiatum cells, Aspergillus awamori cells, Aspergillus fumigatus cells, Aspergillus foetidus cells, Aspergillus nidurans cells, Aspergillus niger cells, Aspergillus oryzae cells, Humicola insolence cells, Humicola lanuginosa cells, Rhodococcus opacus cells, Rhizomucor myehei cells, or Mucor myehei cells. In other embodiments, the host cells are actinomycete cells. In yet another embodiment, the host cells are Streptomyces lividans cells or Streptomyces murinus cells. In yet another embodiment, the host cells are Saccharomyces cerevisiae cells.

[0121] In yet another embodiment, the host cell is a cell derived from a eukaryotic plant, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophilic bacteria, yeast, fungi, organisms that have been manipulated therefrom, or synthetic organisms. In certain embodiments, the host cells are derived from Arabidopsis taliana, Panicum virgatum, Miscanthus giganteus, Zea maize, Botryococcus brownii, Chlamydomonas reinhardi, Dunaliella salina, Thermosynechococcus elongatus, Synechococcus elongatus, Synechococcus species, Synechocystis species, Chlorobium tepidam, Chloroflexus aurantiacus, Chromatium binosum, Rhodospiryllum labrum, Rhodobacter capsulatus, Rhodopseudomonas parstris, Clostridium lyngdarii, Clostridium thermocerum, or Penicillium chrysogenum. In certain other embodiments, the host cells are derived from Pichia pastris, Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Pseudomonas fluorescens, Pseudomonas putida, or Zymomonas mobilis. In further embodiments, the host cells are derived from Synechococcus species PCC7002, Synechococcus species PCC7942, or Synechocystis species PCC6803. In some embodiments, the host cells are CHO cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells. In certain embodiments, the host cells are Escherichia coli cells. In some embodiments, the Escherichia coli cells are strains B, C, K, or W.

[0122] Compositions and formulations of fatty diols Bioproducts containing biologically produced organic compounds (e.g., fatty diol compositions produced in accordance with this disclosure), and in particular fatty diol compositions produced using the fatty acid biosynthesis pathway disclosed herein, are produced from renewable sources (e.g., simple carbon sources derived from renewable raw materials) and are therefore novel compositions. These novel bioproducts are obtained by dinucleate carbon isotope fingerprinting or 14 Based on 14C dating, it can be distinguished from organic compounds derived from petrochemical carbon. Furthermore, the specific source of biogenic carbon (e.g., comparison of glucose and glycerol) can also be determined by dinucleate carbon isotope fingerprinting (see, for example, U.S. Patent No. 7,169,588). The ability to distinguish bioproducts such as fatty diols of this disclosure from petroleum-derived organic compounds is beneficial for tracking these materials in distribution. For example, organic compounds or chemicals containing both biogenic and petroleum-derived carbon isotope compositions can be distinguished from organic compounds and chemicals made solely from petroleum-derived materials. Therefore, bioproducts produced herein can be tracked or traced in distribution based on their unique carbon isotope composition. Bioproducts are determined by the stable carbon isotope ratio in each sample ( 13 C / 12 By comparing C), it can be distinguished from petroleum-derived organic compounds. 13 C / 12 The C ratio is the ratio of carbon dioxide in the atmosphere at the time carbon dioxide is fixed. 13 C / 12 This is the result of the C ratio. This ratio also accurately reflects the metabolic pathway. Regional variations also occur. Petroleum, C3 plants (broadleaf plants), C4 plants (grasses), and marine carbonates are all, 13 C / 12 C and corresponding δ 13 They show a clear difference in C value. Both C4 and C3 plants exhibit various characteristics. 13 C / 12This shows the 14C isotope ratio, with typical values ​​being approximately -7 to -13 per mille for C4 plants and approximately -19 to -27 per mille for C3 plants (see, for example, Stuiver et al., Radiocarbon 19:355 (1977)). Non-renewable energy sources such as coal and oil generally fall within the latter range.

[0123] δ 1 3C(‰)=[( 13 C / 12 C) Sample-( 13 C / 12 C) Standard material] / ( 13 C / 12 C) Standard material x 1000 The IAEA, USGS, NIST, and other selected international isotope institutes are collaborating to develop a series of alternative RMs. The per mille deviation notation from the PDB is δ 13 The measurement is performed on CO2 by high-precision stable isotope ratio mass spectrometry (IRMS) for molecular ions of mass 44, 45, and 46. The compositions described herein include compositions and products of fatty diols produced by any of the methods described herein. Specifically, the fatty diol compositions or products have a δ of about -28 or greater, about -27 or greater, -20 or greater, -18 or greater, -15 or greater, -13 or greater, -10 or greater, or -8 or greater. 13 C may be present. For example, a fatty diol composition or product may have δ at about -30 to about -15, about -27 to about -19, about -25 to about -21, about -15 to about -5, about -13 to about -7, or about -13 to about -10. 13 It may have C. In other cases, the fatty diol composition or product may have δ about -10, -11, -12, or -12.3. 13 It may have C. The fatty diol compositions and products produced in accordance with this disclosure in this specification are each of the compounds 14 By comparing the amount of carbon, it can also be distinguished from petroleum-derived organic compounds. 14Since carbon has a nuclear half-life of 5730 years, petroleum-based fuels containing "older" carbon can be distinguished from fatty diol compositions and bioproducts containing "newer" carbon (see, for example, Currie, "Source Apportionment of Atmospheric Particles, Characterization of Environmental Particles," J. Buffle and HP van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74, (1992)).

[0124] The basic premise of radiocarbon dating is that in the atmosphere 14 The constancy of C concentration in living organisms 14 This would result in the invariance of C. However, due to atmospheric nuclear tests since 1950 and the burning of fossil fuels since 1850, 14 C acquired a second geochemical time characteristic: in atmospheric CO2 (and therefore in the biosphere) 14 The C concentration approximately doubled during the peak of nuclear testing in the mid-1960s. Since then, with a relaxation "half-life" of roughly 7-10 years, the steady-state cosmic ray-derived (atmospheric) baseline isotope ratio has been approximately 1.2 × 10⁻¹². 14 C / 12 It is gradually returning to C). This latter half-life should not be interpreted literally; rather, it refers to the atmosphere and biosphere since the dawn of the nuclear age. 14 To track variations in C, a detailed atmospheric nuclear insertion / decay function must be used. It is this latter biosphere that underpins recent annual biosphere carbon dating. 14 This is the C-time characteristic. 14C can be measured by accelerator mass spectrometry (AMS), and the result is given in units of "modern carbon fraction" (fM). In this respect, fM has the same meaning as defined by the National Institute of Standards and Technology (NIST) Standard Materials (SRMs) 4990B and 4990C (known as oxalic acid standards HOxI and HOxII). The basic definition is HOxI 14 C / 12 This corresponds to 0.95 times the 1C isotope ratio (based on the year 1950). This is roughly equivalent to decay-corrected pre-industrial wood. For current biosphere (plant material), the fM is approximately 1.1. The fatty diol compositions and products described herein have at least about 1 fM 14 This includes bioproducts that may contain C. For example, the bioproducts of this disclosure contain at least about 1.01 fM 14 C, approximately 1 to 1.5 fM 14 C, approximately 1.04 to 1.18 fM 14 C, or approximately 1.111 to 1.124 fM 14 It may have C.

[0125] 14 Another measure of C is known as the modern carbon percentage (pMC). 14 For archaeologists or geologists using 14C dating, the year 1950 corresponds to "year 0." This also represents 100 pMC. In 1963, the peak of thermonuclear weapons activity, atmospheric "bomb carbon" reached nearly twice normal levels. Since its appearance, its distribution in the atmosphere has been estimated, and plants and animals that have survived since 1950 show values ​​above 100 pMC. This value has gradually decreased over time, and the current value is around 107.5 pMC. This is close to 107.5 pMC for fresh biomass materials such as corn. 14 This means that it is thought to give C properties. Petroleum-derived compounds are thought to have a pMC value of zero. When fossil carbon is mixed with modern carbon, dilution of the modern pMC content occurs. 107.5 pMC is the modern biomass material. 14 This represents the carbon content, with 0 pMC indicating petroleum-derived products. 14Assuming it represents the carbon content, the pMC value measured for a material will reflect the ratio of these two types of components. For example, a material derived 100% from modern soybeans will give a radiocarbon value close to 107.5 pMC. If this material is diluted by 50% with a petroleum-derived product, the radiocarbon value will be approximately 54 pMC. The bio-based carbon content is derived by assigning "100%" to 107.5 pMC and "0%" to 0 pMC. For example, a sample measured at 99 pMC gives a bio-based carbon content equivalent of 93%. This value is called the bio-based carbon result mean and assumes that all components of the material being analyzed originate from either modern biomaterials or petroleum-derived materials. Bioproducts containing one or more types of fatty diols as described herein may have at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 pMC. In other cases, the fatty diol compositions described herein may have pMCs of about 50 to about 100; about 60 to about 100; about 70 to about 100; about 80 to about 100; about 85 to about 100; about 87 to about 98; or about 90 to about 95. In yet other cases, the fatty diol compositions described herein may have pMCs of about 90, 91, 92, 93, 94, or 94.2.

[0126] Fatty diols such as 1,3-diols are useful and desirable molecules in many industrial applications. This disclosure describes how to produce such compounds through recombinant microorganisms, including in vivo, thereby creating a wide range of useful products. Such products include 1,3-diols and their compositions. Examples of 1,3-diols include C5 1,3-diol (1,3-pentanediol); C6 1,3-diol (1,3-hexanediol); C7 1,3-diol (1,3-heptanediol); C8 1,3-diol (1,3-octanediol); C9 1,3-diol (1,3-nonanediol); C 10 1,3-diol (1,3-decanediol); C 11 1,3-diol (1,3-undecanediol); C12 1,3-diol (1,3-dodecanediol); C 13 1,3-diol (1,3-tridecanediol); C 14 1,3-diol (1,3-tetradecanediol); C 15 1,3-diol (1,3-pentadecanediol); C 16 1,3-diol (1,3-hexadecanediol); C 17 1,3-diol (1,3-heptadecanediol); C 18 1,3-diol (1,3-octadecanediol); C 19 Examples include, but are not limited to, 1,3-diols (1,3-nonadecanediol). Although the majority described herein are even-chain 1,3-diols, odd-chain 1,3-diols, such as those having 7 to 21 carbon atoms, more preferably 5 to 19 carbon atoms, are also included.

[0127] The 1,3-diols of this disclosure have a variety of chain lengths and / or saturation and / or branching characteristics. In some embodiments, the 1,3-diol compositions include almost one type of 1,3-diol, for example, C51,3-diol (1,3-pentanediol); C61,3-diol (1,3-hexanediol); C71,3-diol (1,3-heptanediol); C81,3-diol (1,3-octanediol); C91,3-diol (1,3-nonanediol); C 10 1,3-diol (1,3-decanediol); C 11 1,3-diol (1,3-undecanediol); C 12 1,3-diol (1,3-dodecanediol); C 13 1,3-diol (1,3-tridecanediol); C 14 1,3-diol (1,3-tetradecanediol); C 15 1,3-diol (1,3-pentadecanediol); C 16 1,3-diol (1,3-hexadecanediol); C 17 1,3-diol (1,3-heptadecanediol); C 18 1,3-diol (1,3-octadecanediol); C19 Examples include 1,3-diols (1,3-nonadecanediol); and so on. In another embodiment, the 1,3-diol composition is mostly a mixture of specific ratios of specific 1,3-diols having a particular chain length. In yet another embodiment, the 1,3-diol composition is a combination of one or more 1,3-diols having a particular chain length, combined with other components or components for the purpose of producing detergents, surfactants, emulsifiers, softeners, solvents, plastics, and food additives.

[0128] In one embodiment, the fatty diol composition is a mixture of linear fatty alcohols containing C 12 It contains 1,3-diols. In another embodiment, the fatty diol composition contains C in a mixture of branched-chain fatty alcohols. 12 It contains 1,3-diols. In another embodiment, the fatty diol composition contains C in a mixture of linear and branched fatty alcohols. 12 Contains 1,3-diol. In another embodiment, the fatty diol composition is combined with additional components, such as a detergent or surfactant component, for example, C 12 It contains a 1,3-diol. In yet another embodiment, the fatty diol composition is combined with additional components, such as an emulsifier or solvent component, for example, C 12 It contains a 1,3-diol. In yet another embodiment, the fatty diol composition is combined with a polymer, C 12 Contains 1,3-diol. In this specification, fatty diol compositions can be used as components of plastics. In another embodiment, a fatty diol composition is added to a mixture of food components. 12 It contains 1,3-diols. In another embodiment, the fatty diol composition contains 1,3-diols as intermediates in the synthesis of surfactants or detergents, such as glucosides or ethoxides, or as basic chemical units that can be used in combination with fragrances or to synthesize other chemical substances.

[0129] In another embodiment, the fatty diol composition is a mixture of linear fatty alcohols containing C8- and C8- 10 -, and C 12 It contains 1,3-diols in a specific ratio. In another embodiment, the fatty diol composition contains a mixture of branched-chain fatty alcohols with C8- and C 10 -, and C 12 It contains 1,3-diols in a specific ratio. In another embodiment, the fatty diol composition is a mixture of linear and branched fatty alcohols containing C8- and C 10 -, and C 12 It contains 1,3-diols in a specific ratio. In another embodiment, the fatty diol composition is combined with additional components, such as detergent or surfactant components, to form C8-,C 10 -, and C 12 It contains 1,3-diols in a specific ratio. In yet another embodiment, the fatty diol composition is combined with additional components, such as emulsifiers or solvent components, to form C8-,C 10 -, and C 12 It contains 1,3-diols in a specific ratio. In yet another embodiment, the fatty diol composition is combined with a polymer, C8-,C 10 -, and C 12 It contains 1,3-diols in a specific ratio. In this specification, fatty diol compositions can be used as components of plastics. In another embodiment, the fatty diol composition contains C8-,C in a mixture of food components. 10 -, and C 12 It contains 1,3-diols in a specific ratio.

[0130] This disclosure further relates to the inclusion of C5-, C6-, C7-, C8-, C9-, C in a mixture of food-related ingredients. 10 -, and / or C 11This disclosure encompasses fatty diol compositions comprising 1,3-diol alone or in certain ratios. Such fatty diols may be useful as food stabilizers, food enhancers, food additives, or food substitutes. The fatty diol compounds and compositions of this disclosure can be formulated to produce desired products, including detergents, surfactants, emulsifiers, softeners, solvents, plastics, food additives, and the like. In one embodiment, C 10 -C 18 1,3-diols are intended for use as surfactants. In another embodiment, 1,3-diols are used directly or as intermediates in the synthesis of nutritional supplements, pharmaceuticals, pesticides, and other physiologically active molecules.

[0131] A study on chiral 1,3-diols. [Examples]

[0132] The following embodiments further illustrate the present disclosure but shall not be deemed to limit the scope of the present disclosure in any way.

[0133] Example 1: Culture of recombinant Escherichia coli strain for 1,3-diol production All experiments were initiated from single colonies or from frozen stocks of specific microbial strains. For each strain, a high-treatment (HTP) protocol was performed in quads in 96-well plates as follows: 40 μL of Luria-Bertani (LB) culture (obtained from LB cultures growing in 96-well plates) was seeded into 360 μL of LB medium, and then incubated at 32°C with shaking for 3–4 hours. 40 μL of LB strain was seeded into 360 μL of Nlim medium (see below). After growing at 32°C for 2 hours and then at 30–35°C, the cultures were induced with IPTG (final concentration 1 mM). The cultures were then incubated at 30–35°C with shaking for 20 hours, unless otherwise specified, and then extracted according to the standard extraction protocol detailed below. The protocol for the shaking flask was followed similarly, except that the medium volume was scaled up so that the final production medium volume was 15 ml instead of 400 μl. The shaking flask medium also contained 0.25% (v / v) Triton X100. Appropriate antibiotics were added at all stages depending on the microbial strain. TIFF2026069552000010.tif70128

[0134] The baseline process in the bioreactor was as follows: Cells in cell bank vials were cultured at 32°C in LB shaking flasks containing antibiotics until the culture OD reading was greater than 1. These cultures were transferred to minimal seeding medium (containing ammonium chloride, sodium chloride, monobasic potassium phosphate, magnesium sulfate, calcium chloride, glucose, trace element solution, iron(III) citrate tribasic monohydrate, buffer, and antibiotics) at 5% v / v and cultured overnight at 32°C. These seed cultures were then seeded into the prepared production bioreactor.

[0135] The initial bioreactor medium for this process contained the same components as the seeding medium in various concentrations, as well as trace amounts of vitamin solutions and, optionally, small amounts of complex medium components such as casamino acids, corn steep powder, or yeast extracts. After sterilization, heat-unstable vitamins or amino acids, glucose, and antibiotics were optionally added to the bioreactor.

[0136] Before sowing, the bioreactor parameters were stabilized and the controller was turned on (dissolved oxygen setting: 10-50%; temperature setting: 27-37°C; aeration setting: 0.25-1vvm; pH setting: 6.5-7.5). Seed cultures at 5% v / v were sown into the bioreactor, and induction was performed with 1 mM IPTB once the culture density reached the desired setting.

[0137] A supply solution, consisting of glucose, sucrose, fructose, xylose, or glycerol, along with other possible media components, was added to the bioreactor basal medium. This supply solution was then supplied to the culture at a maximum rate of 1–50 g / L / hour of glucose (relative to the nominal culture volume), and a DO or pH trigger was used to indicate to the controller when the medium had exhausted its carbon source and when the next supply solution should be added. The bioreactors were harvested at 48–96 hours.

[0138] Example 2: Analysis of 1,3-diols Extraction was performed on a fermentation broth sample produced by a recombinant E. coli strain using the following procedure: 1. Vortex the broth at 3000 rpm for 30 seconds, then weigh it. 2. Immediately after vortexing with Vortex Genie, collect 500 μL of broth. 3. Add 5 mL of butyl acetate containing 500 mg / L (1-undecanol) as an internal standard. 4. Extract the broth using a vortex apparatus (DVX-2500 multi-tube vortex apparatus, VWR) at 2500 rpm for 20 minutes. 5. Centrifuge the extract at room temperature for 10 minutes (at 4750 rpm). 6. Pipette 100 μL of the supernatant from the top layer into a GC vial with an insert. 7. At room temperature, add 100 μL of (BSTFA + 1% TMCS) to the GC vial and derivatize. 8. Mix the extract and BSTFA reagent for 30 seconds, then inject it into the GC / MS as described below: Specific device settings Initial temperature: 60℃ Initial time: 5 minutes Equilibriumization time: 1 minute Program speed: 25°C / min Final temperature: 300℃ Final time: 1.6 minutes Detector: MSD Inlet temperature: 300℃ Transfer line temperature: 300℃ MS source: 230℃ MS quadrupole: 150℃ Split ratio: 20:1 Column flow: 1 mL / min Sample volume: 1 μL

[0139] Example 3: 1,3-diol production using a pathway containing TE derived from Anelococcus tetrasius or Lactobacillus plantarum and carB This example demonstrates unexpected 1,3-diol production in recombinant Escherichia coli using a metabolic pathway that includes a microbial thioesterase from Anaerococcus tetradius (TE_EEI82564, genbank accession number WP_004837416) or a microbial thioesterase from Lactobacillus plantarum (TE_CAD63310, genbank accession number WP_003640969), and a mutant of carboxylic acid reductase from Mycobacterium smegmatis, CarB (genbank accession number YP_889972).

[0140] The genes encoding carB2 (SEQ ID NO: 6) and TE_EEI82564 were cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that their transcription was controlled by an IPTG-inducible Ptrc promoter, and they formed an operon with genes encoding alcohol dehydrogenase alrA, as well as variants of 3-ketoacyl-ACP synthase (fabB) and a transcription regulator (fadR). alrA is not required for the production of fatty alcohols or fatty diols, but it enhances their production rate. The plasmid was named pVA369 (see Table 5). The gene TE_CAD63310 from L. plantarum was cloned in the same manner together with the gene carB12 (SEQ ID NO: 4), and the resulting plasmid was named pJP2 (see Table 5 below).

[0141] The base strains used for plasmid transformation were V668 and DJ81. Briefly, the genomes of the base strains were modified as follows: In V668, the fadE (acyl-CoA dehydrogenase) gene was deleted, and the synthetic fatty acid biosynthesis operon and phosphopantetheinyltransferase (entD) were overexpressed. Briefly, the genome of the base strain DJ81 was modified as follows: the acyl-CoA dehydrogenase (fadE) gene was deleted, and the synthetic fatty acid biosynthesis operon, phosphopantetheinyltransferase (entD), and mutant thioesterase (tesA) were overexpressed.

[0142] Plasmids pVA369 and pJP2 were introduced into base strains D848 and V668, respectively, to obtain strains VA370 and JP-11 (see Table 6 below). The strains were then cultured as described in Examples 1 and 2, and their ability to produce fatty alcohols was analyzed. Surprisingly, both strains yielded several unknown peaks.

[0143] Figure 4 shows the GC-MS chromatograph of an extract from the VA370 strain expressing TE_EEI82564. Two peaks in the GC-MS chromatograph at RT=8.199 min and RT=9.094 min did not correspond to the expected retention times of fatty alcohols and fatty acids, e.g., dodecanol and tetradecanol or dodecanoic acid and tetradecanoic acid. Peak 1 eluted before dodecanol, and peak 2 eluted after tetradecanol and before tetradecanoic acid. The ion fragmentation patterns of peaks 1 and 2 (see Figure 5) suggested that these two peaks were 1,3-trimethylsiloxyoctane and 1,3-trimethylsiloxydecane, which are BSTFA-derived products of 1,3-octanediol and 1,3-decanediol (see Example 2), respectively. For illustrative purposes, Figure 6 shows a schematic diagram of the ion fragment of 1,3-trimethylsiloxidecane, as observed at peak 1 in Figure 5. Derivatized 1,3-dodecanediol and 1,3-tetradecanediol were also observed in trace amounts.

[0144] Similarly, extracts from the JP-11 strain expressing TE_CAD63310 contained novel peaks, which were identified as 1,3-octanediol, 1,3-decanediol, 1,3-tetradecanol, and 1,3-tetradecenol, based on their ion fragmentation patterns and retention times, as described above. In the HTP fermentation protocol, JP-11 produced a total of 1.9 ± 0.05 g / L of 1,3-diols, as well as fatty alcohols such as octanol, decanol, dodecanol, dodecenol, tetradecanol, and tetradecenol. The product distribution of the JP-11 strain is shown in Figure 7.

[0145] The production of 1,3-diol was surprising for two reasons: (i) This production requires a 3-OH fatty acid as an intermediate, and it is most likely that the 3-OH fatty acid was derived from 3-OH acyl-ACP by the action of thioesterase (see Figure 2). Both thioesterases used in this example have already been expressed in E. coli and have been reported to produce only fatty acids (Jing et al. BMC Biochemistry 2011, 12:44), suggesting that they are not suitable for the production of 3-OH fatty acids and therefore 1,3-diol. (ii) This production requires the reduction of the 3-OH fatty acid intermediate by carboxylic acid reductase CarB to a 3-OH fatty aldehyde, which is then further reduced by alcohol dehydrogenase (ADH) to 1,3-diol. While ADH is known to be rather indiscriminate, CarB has not previously been shown to convert 3-OH fatty acids to 3-OH fatty aldehydes. Therefore, the applicants discovered that certain microbial thioesterases, such as TE_EEI82564 and TE_CAD63310, overproduce 1,3-diols when co-expressed with CarB in E. coli host cells. Analysis of the 1,3-diols shows that they are very rich in (R) enantiomers, demonstrating the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0146] (Table 5) Plasmids used for 1,3-diol production TIFF2026069552000011.tif57128

[0147] (Table 6) Strains used for 1,3-diol production TIFF2026069552000012.tif52128

[0148] Example 4A: Production of 1,3-diols using a pathway containing fatB1 and carB derived from Umberularia californica This example demonstrates 1,3-diol production in recombinant Escherichia coli using a metabolic pathway that includes fatB1 (Genbank accession number Q41635), a plant thioesterase derived from Umbellularia californica, and CarB, a mutant carboxylic acid reductase derived from Mycobacterium smegmatis.

[0149] The genes encoding carB8 (SEQ ID NO: 8) and fatB1 were cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that their transcription was controlled by an IPTG-inducible Ptrc promoter and they formed an operon with the gene encoding the alcohol dehydrogenase alrA. The plasmid was named pNH330 (see Table 5).

[0150] The base strain used for plasmid transformation was strain D178. Briefly, the genome of strain D178 was modified as follows: the fadE (acyl-CoA dehydrogenase) gene was deleted and phosphopantetheinyltransferase (entD) was overexpressed. Plasmid pNH330 was introduced into D178 to obtain strain stNH1371 (see Table 6). The strain was then cultured as described in Examples 1 and 2, and its ability to produce fatty alcohols and 1,3-diols was analyzed. The 1,3-diol peak was identified as described in Example 2.

[0151] The stNH1371 strain produced 39.5 ± 3.2 mg / L of 1,3-diols in the HTP fermentation protocol. 1,3-Dodecanediol was one of the 1,3-diols produced. In addition to 1,3-diols, fatty alcohols such as dodecanol were also detected. Analysis of 1,3-diols may indicate that they are very rich in (R) enantiomers, demonstrating the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0152] Example 4B: Production of 1,3-diols using a pathway involving phaG and carB derived from Pseudomonas putida. This example demonstrates 1,3-diol production in recombinant Escherichia coli using a metabolic pathway that includes phaG (Genbank accession number AAN67031), a thioesterase / transacylase derived from Pseudomonas putida, and CarB, a mutant carboxylic acid reductase derived from Mycobacterium smegmatis.

[0153] The genes encoding carB8 (see the sequence listing attached to this specification below) and phaG were cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that their transcription was controlled by an IPTG-inducible Ptrc promoter and they formed an operon with the gene encoding the alcohol dehydrogenase alrA. The plasmid was named pNH328 (see Table 5).

[0154] The base strain used for plasmid transformation was strain D178. Briefly, the genome of strain D178 was modified as follows: the fadE (acyl-CoA dehydrogenase) gene was deleted and phosphopantetheinyltransferase (entD) was overexpressed. Plasmid pNH328 was introduced into D178 to obtain strain stNH1369 (see Table 6 above). The strain was then cultured as described in Examples 1 and 2, and its ability to produce fatty alcohols and 1,3-diols was analyzed. The 1,3-diol peak was identified as described in Example 2.

[0155] The stNH1369 strain produced 600 ± 27 mg / L of 1,3 - diol in the HTP fermentation protocol. The 1,3 - diol produced was 1,3 - octanediol, 1,3 - decanediol, 1,3 - dodecanediol, and 1,3 - tetradecanediol. In addition to the 1,3 - diol, trace amounts of fatty acids were detected. Analysis of the 1,3 - diol indicates that they may contain a very rich (R) enantiomer, demonstrating the enantioselectivity of 3 - ketoacyl - ACP reductase (FabG) of the native Escherichia coli fatty acid biosynthetic mechanism.

[0156] Example 5: Production of 1,3 - diol using a pathway containing AAR from Synechococcus elongatus This example shows the production of 1,3 - diol in recombinant Escherichia coli using a metabolic pathway containing a mutant acyl - ACP reductase AAR (genbank accession number YP_400611; wild - type) from Synechococcus elongatus. For the mutant AAR sequence, refer to the sequence listing attached hereto (below).

[0157] The gene encoding the AAR mutant (SEQ ID NO: 2) was cloned into the pCL1920 derivative vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of the gene was controlled by the IPTG - inducible Ptrc promoter and the gene formed an operon with the gene encoding alcohol dehydrogenase alrA. The plasmid was named pNT16 (see Table 5).

[0158] The base strain used for plasmid transformation was DV2. Briefly, the genome of the DV2 strain was engineered by deleting the fadE (acyl-CoA dehydrogenase) gene. Plasmid pNT16 was introduced into the DV2 base strain by transformation to obtain the Becos247 strain (see Table 6). Then, Becos247 was cultured as described in Examples 1 and 2, and its ability to produce fatty alcohols was analyzed. Surprisingly, this strain produced 1,3-diol. The peak of 1,3-diol was identified as described in Example 2.

[0159] In 5 L fermentation, the Becos247 strain produced 0.57 g / L of 1,3-diol, which accounted for 9.1% of all the fatty acid species produced. The 1,3-diol produced was 1,3-dodecanediol, 1,3-tetradecenediol, and 1,3-tetradecanediol. In addition, fatty alcohols such as decanol, dodecenol, dodecanol, tetradecenol, tetradecanol, hexadecenol, hexadecanol, and octadecanol, as well as a small amount of fatty acids, were produced.

[0160] The production of 1,3-diol via 3-OH fatty aldehyde as an intermediate in this experiment is surprising (see Figure 3). This is because the acyl-ACP reductase, a wild-type AAR derived from Synechococcus elongatus used in this example, has been previously expressed in Escherichia coli and has been reported to produce only fatty alcohols from acyl-ACP and not 1,3-diol from 3-OH acyl-ACP (Schirmer et al. (2010) Science 329, 559). Analysis of 1,3-diol may show that they contain a very rich (R) enantiomer, demonstrating the enantioselectivity of 3-ketoacyl-ACP reductase (FabG) of the native Escherichia coli fatty acid biosynthetic mechanism.

[0161] Example 6A: Production of 1,3-diol using a pathway containing fatB1 and carB from Umbellularia californica This example demonstrates how to demonstrate 1,3-diol production in recombinant Escherichia coli using a metabolic pathway that includes the plant thioesterase fatB1 from Umberularia californica and the carboxylic acid reductase CarB from Mycobacterium smegmatis.

[0162] The genes encoding wild-type carB and fatB1 are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of these genes is controlled by an IPTG-inducible Ptrc promoter and that these genes form an operon with the gene encoding alcohol dehydrogenase alrA. The plasmid is then transformed into a base strain such as DV2 (see Example 5).

[0163] Next, the resulting strains are cultured as described in Examples 1 and 2 and analyzed for their ability to produce fatty alcohols. The strains are expected to produce 1,3-diols. Analysis of the 1,3-diols will show that they are very rich in (R) enantiomers, demonstrating the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0164] Example 6B: Production of 1,3-diols using a simplified pathway containing fatB1 and carB from Umberularia californica. This example demonstrates how 1,3-diol production in recombinant E. coli is demonstrated using a simplified metabolic pathway that includes the plant thioesterase fatB1 from Umberularia californica and the carboxylic acid reductase CarB from Mycobacterium smegmatis.

[0165] The genes encoding wild-type carB and fatB1 are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) so that the transcription of these genes is controlled by an IPTG-inducible Ptrc promoter. The plasmid is then transformed into a base strain such as the DV2 strain (see Example 5).

[0166] Next, the resulting strains are cultured as described in Examples 1 and 2 and analyzed for their ability to produce fatty alcohols and diols. The strains are expected to produce 1,3-diols, demonstrating that thioesterases and carboxylic acid reductases are sufficient to enable microbial cells to produce 1,3-diols. Analysis of the 1,3-diols will show that they are very rich in (R) enantiomers, demonstrating the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0167] Example 7: Production of 1,3-diols using a pathway containing tesA and carB derived from E. coli. This example demonstrates how to demonstrate 1,3-diol production in recombinant E. coli using a metabolic pathway involving thioesterase tesA and carboxylic acid reductase CarB derived from Mycobacterium smegmatis.

[0168] The genes encoding wild-type carB and wild-type tesA are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of these genes is controlled by an IPTG-inducible Ptrc promoter and that these genes form an operon with the gene encoding alcohol dehydrogenase alrA. The plasmid is then transformed into a base strain such as the DV2 strain (see Example 5).

[0169] Next, the resulting strains are cultured as described in Examples 1 and 2 and analyzed for their ability to produce fatty alcohols and diols. The strains are expected to produce 1,3-diols, demonstrating that thioesterases and carboxylic acid reductases are sufficient to enable microbial cells to produce 1,3-diols. Analysis of the 1,3-diols will show that they are very rich in (R) enantiomers, demonstrating the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0170] Example 8: Production of 1,3-diols using a simplified pathway including wild-type AAR from Synechococcus elongatus This example demonstrates how to demonstrate 1,3-diol production in recombinant Escherichia coli using a metabolic pathway involving acyl-ACP reductase AAR derived from Synechococcus elongatas.

[0171] The gene encoding wild-type AAR is cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) so that the transcription of the gene is controlled by an IPTG-inducible Ptrc promoter. The plasmid is then transformed into a base strain such as the DV2 strain (see Example 5).

[0172] Next, the resulting strains are cultured as described in Examples 1 and 2 and analyzed for their ability to produce fatty alcohols and diols. The strains are expected to produce 1,3-diols, which demonstrates that heterologous production of AARs is sufficient to enable microbial cells to produce 1,3-diols. Analysis of the 1,3-diols will show that they are very rich in (R) enantiomers, which will demonstrate the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0173] Example 9: Production of 1,3-diols using a pathway containing fatB and carB derived from Cinnamomum camphora. This example demonstrates how to demonstrate 1,3-diol production in recombinant E. coli using a metabolic pathway that includes the plant thioesterase fatB from Cinnamomum camphora and the carboxylic acid reductase carB from Mycobacterium smegmatis.

[0174] The genes encoding wild-type carB and fatB derived from Cinnamomum camphora are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of these genes is controlled by an IPTG-inducible Ptrc promoter and that these genes form an operon with the gene encoding alcohol dehydrogenase alrA. The plasmid is then transformed into a base strain such as the DV2 strain (see Example 5).

[0175] Next, the resulting strains are cultured as described in Examples 1 and 2 and analyzed for their ability to produce fatty alcohols and diols. The strains are expected to produce 1,3-diols, demonstrating that thioesterases and carboxylic acid reductases are sufficient to enable microbial cells to produce 1,3-diols. Analysis of the 1,3-diols will show that they are very rich in (R) enantiomers, demonstrating the enantioselectivity of 3-ketoacyl ACP reductase (FabG) in the natural E. coli fatty acid biosynthesis mechanism.

[0176] Example 10: Production of 1,3-diols using a pathway containing acr1 derived from Acinetobacter baillii This example demonstrates how to demonstrate 1,3-diol production in recombinant E. coli using a metabolic pathway involving the fatty acyl-CoA reductase acr1 (Genbank accession number AAC45217) derived from Acinetobacter baylyi.

[0177] The gene encoding acr1 is cloned into the pCL1920-derived vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of the gene is controlled by the IPTG-inducible Ptrc promoter and the gene forms an operon with the genes encoding acyl-CoA synthetase (fadD) and thioesterase. The plasmid is introduced by transformation into a parental strain such as the DV2 strain (see Example 5).

[0178] Next, as described in Examples 1 and 2, the resulting strain is cultured and analyzed for its ability to produce fatty alcohols and diols. The strain is expected to produce 1,3-diol.

[0179] Example 11: Production of 1,3-diol using a pathway containing FAR from Marinobacter aquaeolei This example demonstrates how to demonstrate the production of 1,3-diol in recombinant Escherichia coli using a metabolic pathway containing the fatty acyl-ACP reductase FAR (genbank accession number YP_959486) from Marinobacter aquaeolei.

[0180] The gene encoding the wild-type FAR is cloned into the pCL1920-derived vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of the gene is controlled by the IPTG-inducible Ptrc promoter. The plasmid is introduced by transformation into a parental strain such as the DV2 strain (see Example 5).

[0181] Next, as described in Examples 1 and 2, the resulting strain is cultured and analyzed for its ability to produce fatty alcohols and diols. The strain is expected to produce 1,3-diol, demonstrating that the heterologously produced FAR is sufficient to enable the cells to produce 1,3-diol.

[0182] Example 12: Production of 1,3-diols using a pathway containing a FAR complex derived from photorhabdus luminescence. This example demonstrates how to demonstrate 1,3-diol production in recombinant E. coli using a metabolic pathway involving the fatty acyl-ACP reductase FAR complex (genbank accession numbers AHH25015~17) derived from Photorhabdus luminescens, containing LuxC, LuxD, and LuxE.

[0183] The genes encoding LuxC, LuxD, and LuxE are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that their transcription is controlled by an IPTG-inducible Ptrc promoter and they form an operon with the gene encoding the alcohol dehydrogenase alrA. The plasmid is then transformed into a base strain such as the DV2 strain (see Example 5).

[0184] Next, the resulting strains are cultured as described in Examples 1 and 2, and their ability to produce fatty alcohols and diols is analyzed. The strains are expected to produce 1,3-diols.

[0185] Example 13: Production of 3-(S)-lipid diols using fadB(His450Gln) This example demonstrates how to demonstrate 3-(S)-lipiddiol production in recombinant Escherichia coli using a metabolic pathway involving a 3-hydroxyacyl-ACP acyl-CoA transacylase or thioesterase fadB(His450Gln) (Genbank accession number AAC45217) derived from Acinetobacter bailey, which retains enoyl-CoA hydratase activity but lacks dehydrogenase activity and expresses the lipoacyl-CoA reductase acr1.

[0186] The genes encoding TesA, FadD, FadB (His450Gln), and Acr1 are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of these genes is controlled by an IPTG-inducible Ptrc promoter and that these genes complete an operon sufficient for the synthesis of fatty alcohols. The plasmid is then transformed into a base strain such as the MG1655 strain (see Example 5), but the strain has an additional gene encoding FadE introduced into its genome under the control of the IPTG-inducible Ptrc promoter.

[0187] Next, the resulting strains are cultured as described in Examples 1 and 2, and their ability to produce fatty alcohols and diols is analyzed. The strains are expected to produce 3-(S)-fatty diols.

[0188] Example 14. Production of 3-(S)-lipid diols using fadB(Glu119Gln) This example demonstrates how to demonstrate 3-(S)-lipiddiol production in recombinant E. coli using a metabolic pathway involving a 3-hydroxy-acyl-ACP-acyl-CoA transacylase or thioesterase fadB(Glu119Gln) (Genbank accession number AAC45217) derived from Acinetobacter bailey, which retains dehydrogenase activity but lacks dehydratase activity and expresses the lipoacyl-CoA reductase acr1.

[0189] The genes encoding TesA, FadD, FadB (Glu119Gln), and Acr1 are cloned into a pCL1920-inducible vector (SC101 replicon, spectinomycin resistance marker) such that the transcription of these genes is controlled by an IPTG-inducible Ptrc promoter and that these genes complete an operon sufficient for the synthesis of fatty alcohols. The plasmid is then transformed into a base strain such as the MG1655 strain (see Example 5), but the strain has an additional gene encoding FadA introduced into its genome under the control of the IPTG-inducible Ptrc promoter.

[0190] Next, the resulting strains are cultured as described in Examples 1 and 2, and their ability to produce fatty alcohols and diols is analyzed. The strains are expected to produce 3-(S)-fatty diols.

[0191] The above aspects and embodiments can be modified in various ways without departing from the spirit and scope of this disclosure. Such modifications and alterations are within the scope of this disclosure.

[0192] Sequence information SEQUENCE LISTING <110> GENOMATICA, INC. <120> MICROBIAL PRODUCTION OF FATTY DIOLS <150> US 62 / 026,573 <151> 2014-07-18 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 342 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic AAR Variant polypeptide" <400> 1 Met Ala Phe Gly Leu Ile Gly His Ala Thr Ser Leu Glu Gln Ala Arg 1 5 10 15 Asp Val Trp Arg Arg Leu Gly Tyr Asp Glu Tyr Ala Asp Gln Gly Leu 20 25 30 Glu Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val 35 40 45 Thr Ser Ala Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Gly 50 55 60 Phe Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg 65 70 75 80 Lys Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile 85 90 95 Ser Ala Leu Gly Gly Phe Thr Ser Ile Ile Phe Glu Asn Phe Asp Leu 100 105 110 Ala Lys Leu Arg Gln Val Arg Asp Thr Thr Leu Glu Phe Glu Arg Phe 115 120 125 Thr Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg Gln Val Glu 130 135 140 Ala Ala Ala Lys Thr Leu Gly Ile Asp Ile Ala Gln Ala Thr Val Ala 145 150 155 160 Val Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu 165 170 175 Asp Leu Lys Leu Gly Val Gly Asp Leu Ile Leu Thr Ala Arg Asn Gln 180 185 190 Glu Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu 195 200 205 Pro Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala 210 215 220 Ser Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln Pro 225 230 235 240 Cys Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Gly Ser Lys Val 245 250 255 Gln Gly Glu Gly Ile Tyr Val Leu Asn Gly Gly Val Val Glu His Cys 260 265 270 Phe Asp Ile Asp Trp Gln Ile Met Ser Leu Ala Glu Met Ala Arg Pro 275 280 285 Glu Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe 290 295 300 Glu Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile 305 310 315 320 Glu Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln 325 330 335 Pro Leu Ala Leu Ala Ile 340 <210> 2 <211> 1029 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic AAR Variant polynucleotide" <400> 2 atggcattcg gtcttatcgg tcatgcaacc agtttggagc aggcccgcga cgtttggcgc 60 aggctgggct acgacgaata cgccgatcaa ggattggagt tttggagtag cgctcctcct 120 caaatcgttg atgaaatcac agtcaccagt gccacaggca aggtgattca cggtcgctac 180 atcgaatcgg ggttcttgcc ggaaatgctg gcggcgcgcc gcttcaaaac agcaacgcgc 240 aaagttctca atgccatgtc ccatgcccaa aaacacggca tcgacatctc ggccttgggg 300 ggctttacct cgattatttt cgagaatttc gatttggcca agttgcggca agtgcgcgac 360 actaccttgg agtttgaacg gttcaccacc ggcaatactc acacggccta cgtaatctgt 420 agacaggtgg aagccgctgc taaaacgctg ggcatcgaca ttgcgcaagc gacagtagcg 480 gttgtcggcg cgactggcga tatcggtagc gctgtctgcc gctggctcga cctcaaactg 540 ggtgtcggtg atttgatcct gacggcgcgc aatcaggagc gtttggataa cctgcaggct 600 gaactcggcc ggggcaagat tctgcccttg gaagccgctc tgccggaagc tgactttatc 660 gtgtgggtcg ccagtatgcc tcagggcgta gtgatcgacc cagcaaccct gaagcaaccc 720 tgcgtcctaa tcgacggggg ctaccccaaa aacttgggca gcaaagtcca aggtgagggc 780 atctatgtcc tcaatggcgg ggtagttgaa cattgcttcg acatcgactg gcagatcatg 840 tccttggcag agatggcgcg gcccgagcgc cagatgtttg cctgctttgc cgaggcgatg 900 ctcttggaat ttgaaggctg gcatactaac ttctcctggg gccgcaacca aatcacgatc 960 gagaagatgg aagcgatcgg tgaggcatcg gtgcgccacg gcttccaacc cttggcattg 1020 gcaatttga 1029 <210> 3 <211> 1174 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic CAR Variant polypeptide" <400> 3 Met Gly Thr Ser Asp Val His Asp Ala Thr Asp Gly Val Thr Glu Thr 1 5 10 15 Ala Leu Arg Asp Arg Gln Arg Thr Arg Arg Ile Ala Glu Leu Tyr Ala 20 25 30 Thr Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp 35 40 45 Ala Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu 50 55 60 Phe Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu 65 70 75 80 Leu Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg 85 90 95 Phe Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala Val 100 105 110 Ala Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp 115 120 125 Ala Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu Thr Leu Asp 130 135 140 Leu Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn 145 150 155 160 Ala Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro Arg 165 170 175 Ile Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val Glu Ser Val 180 185 190 Arg Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp His His Pro 195 200 205 Glu Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu 210 215 220 Ala Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile Ala Asp Glu 225 230 235 240 Gly Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln 245 250 255 Arg Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys 260 265 270 Gly Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser 275 280 285 Gly Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro 290 295 300 Leu Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala Val Gln Asn 305 310 315 320 Gly Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe 325 330 335 Glu Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg 340 345 350 Val Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val Asp Arg Leu 355 360 365 Val Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala 370 375 380 Glu Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val 385 390 395 400 Ser Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr 405 410 415 Leu Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala 420 425 430 Val Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile Asp Tyr Lys 435 440 445 Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr 450 455 460 Pro Arg Gly Glu Leu Leu Val Arg Ser Ile Thr Leu Thr Pro Gly Tyr 465 470 475 480 Tyr Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr 485 490 495 Tyr His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val 500 505 510 Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe 515 520 525 Val Ala Val Ala Asn Leu Glu Ser Val Phe Ser Gly Ala Ala Leu Val 530 535 540 Arg Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala 545 550 555 560 Val Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp Pro Ala Ala 565 570 575 Leu Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala 580 585 590 Glu Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val Glu Thr Glu 595 600 605 Pro Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly Lys Leu Leu 610 615 620 Arg Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr 625 630 635 640 Ala Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg 645 650 655 Ala Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala Ala 660 665 670 Thr Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr 675 680 685 Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser Asn Leu Leu 690 695 700 Ser Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro 705 710 715 720 Ala Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala Gln Arg Thr 725 730 735 Ala Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala 740 745 750 Thr Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala 755 760 765 Glu Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro 770 775 780 Arg Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu 785 790 795 800 Thr Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile 805 810 815 Thr Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Cys Ala Arg Leu Thr 820 825 830 Gln Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu Leu 835 840 845 Ala Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly Asp Gln Asn 850 855 860 Leu Gly Leu Thr Pro Glu Leu Trp His Arg Leu Ala Ala Glu Val Asp 865 870 875 880 Leu Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg 885 890 895 Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val Ile Lys Leu 900 905 910 Ala Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser Thr Ala Lys 915 920 925 Val Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr 930 935 940 Val Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn Gly Tyr Gly 945 950 955 960 Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu 965 970 975 Cys Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His 980 985 990 Pro Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu 995 1000 1005 Leu Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr 1010 1015 1020 Ile Gly Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly Leu Thr 1025 1030 1035 Val Asp Phe Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln 1040 1045 1050 Arg Glu Gly Tyr Val Ser Tyr Asp Val Met Asn Pro His Asp Asp 1055 1060 1065 Gly Ile Ser Leu Asp Val Phe Val Asp Trp Leu Ile Arg Ala Gly 1070 1075 1080 His Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp Trp Val Arg Arg 1085 1090 1095 Phe Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys Arg Arg Ala Gln 1100 1105 1110 Thr Val Leu Pro Leu Leu His Ala Phe Arg Ala Pro Gln Ala Pro 1115 1120 1125 Leu Arg Gly Ala Pro Glu Pro Thr Glu Val Phe His Ala Ala Val 1130 1135 1140 Arg Thr Ala Lys Val Gly Pro Gly Asp Ile Pro His Leu Asp Glu 1145 1150 1155 Ala Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg Glu Phe Gly Leu 1160 1165 1170 Ile <210> 4 <211> 3525 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic CAR Variant (carB12) polynucleotide" <400> 4 atgggcacga gcgatgttca cgacgcgacc gacggcgtta ccgagactgc actgcgtgat 60 cgccagcgca ctcgtcgtat tgcagaactg tacgcaacgg acccagagtt cgcagcagca 120 gctcctctgc cggccgttgt cgatgcggcg cacaaaccgg gcctgcgtct ggcggaaatc 180 ctgcagaccc tgttcaccgg ctacggcgat cgtccggcgc tgggctatcg tgcacgtgag 240 ctggcgacgg acgaaggcgg tcgtacggtc acgcgtctgc tgccgcgctt cgataccctg 300 acctatgcac aggtgtggag ccgtgttcaa gcagtggctg cagcgttgcg tcacaatttc 360 gcacaaccga tttacccggg cgacgcggtc gcgactatcg gctttgcgag cccggactat 420 ttgacgctgg atctggtgtg cgcgtatctg ggcctggtca gcgttccttt gcagcataac 480 gctccggtgt ctcgcctggc cccgattctg gccgaggtgg aaccgcgtat tctgacggtg 540 agcgcagaat acctggacct ggcggttgaa tccgtccgtg atgtgaactc cgtcagccag 600 ctggttgttt tcgaccatca tccggaagtg gacgatcacc gtgacgcact ggctcgcgca 660 cgcgagcagc tggccggcaa aggtatcgca gttacgaccc tggatgcgat cgcagacgaa 720 ggcgcaggtt tgccggctga gccgatttac acggcggatc acgatcagcg tctggccatg 780 attctgtata ccagcggctc tacgggtgct ccgaaaggcg cgatgtacac cgaagcgatg 840 gtggctcgcc tgtggactat gagcgggatc acgggcgacc cgaccccggt tatcaacgtg 900 aacttcatgc cgctgaacca tctgggcggt cgtatcccga ttagcaccgc cgtgcagaat 960 ggcggtacca gctacttcgt tccggaaagc gacatgagca cgctgtttga ggatctggcc 1020 ctggtccgcc ctaccgaact gggtctggtg ccgcgtgttg cggacatgct gtaccagcat 1080 catctggcga ccgtggatcg cctggtgacc cagggcgcgg acgaactgac tgcggaaaag 1140 caggccggtg cggaactgcg tgaacaggtc ttgggcggtc gtgttatcac cggttttgtt 1200 tccaccgcgc cgttggcggc agagatgcgt gcttttctgg atatcacctt gggtgcacac 1260 atcgttgacg gttacggtct gaccgaaacc ggtgcggtca cccgtgatgg tgtgattgtt 1320 cgtcctccgg tcattgatta caagctgatc gatgtgccgg agctgggtta cttctccacc 1380 gacaaaccgt acccgcgtgg cgagctgctg gttcgtagca tcacgttgac tccgggttac 1440 tacaagcgcc cagaagtcac cgcgtccgtt ttcgatcgcg acggctatta ccacaccggc 1500 gacgtgatgg cagaaaccgc gccagaccac ctggtgtatg tggaccgccg caacaatgtt 1560 ctgaagctgg cgcaaggtga atttgtcgcc gtggctaacc tggagtccgt tttcagcggc 1620 gctgctctgg tccgccagat tttcgtgtat ggtaacagcg agcgcagctt tctgttggct 1680 gttgttgtcc ctaccccgga ggcgctggag caatacgacc ctgccgcatt gaaagcagcc 1740 ctggcggatt cgctgcagcg tacggcgcgt gatgccgagc tgcagagcta tgaagtgccg 1800 gcggacttca ttgttgagac tgagcctttt agcgctgcga acggtctgct gagcggtgtt 1860 ggcaagttgc tgcgtccgaa tttgaaggat cgctacggtc agcgtttgga gcagatgtac 1920 gcggacatcg cggctacgca ggcgaaccaa ttgcgtgagc tgcgtcgcgc tgcggctact 1980 caaccggtga tcgacacgct gacgcaagct gcggcgacca tcctgggtac cggcagcgag 2040 gttgcaagcg acgcacactt tactgatttg ggcggtgatt ctctgagcgc gctgacgttg 2100 agcaacttgc tgtctgactt ctttggcttt gaagtcccgg ttggcacgat tgttaaccca 2160 gcgactaatc tggcacagct ggcgcaacat atcgaggcgc agcgcacggc gggtgaccgc 2220 cgtccatcct ttacgacggt ccacggtgcg gatgctacgg aaatccgtgc aagcgaactg 2280 actctggaca aattcatcga cgctgagact ctgcgcgcag cacctggttt gccgaaggtt 2340 acgactgagc cgcgtacggt cctgttgagc ggtgccaatg gttggttggg ccgcttcctg 2400 accctgcagt ggctggaacg tttggcaccg gttggcggta ccctgatcac cattgtgcgc 2460 ggtcgtgacg atgcagcggc ctgtgcacgc ttgactcagg cttacgatac ggacccagag 2520 ctgtcccgcc gcttcgctga gttggcggat cgccacttgc gtgtggtggc aggtgatatc 2580 ggcgatcaga atctgggcct gaccccggag ctgtggcacc gtctggcagc agaggtcgat 2640 ctggtcgttc atccagcggc cctggtcaac cacgtcctgc cgtaccgcca gctgtttggt 2700 ccgaatgttg ttggcaccgc cgaagttatc aagttggctc tgaccgagcg catcaagcct 2760 gttacctacc tgtccacggc gaaggtcgcg atgggtattc ctgattttga ggaggacggt 2820 gacattcgta ccgtcagccc ggttcgtccg ctggatggtg gctatgcaaa tggctatggc 2880 aacagcaagt gggctggcga ggtgctgctg cgcgaggcac atgacctgtg tggcctgccg 2940 gttgcgacgt ttcgtagcga catgattctg gcccacccgc gctaccgtgg ccaagtgaat 3000 gtgccggaca tgttcacccg tctgctgctg tccctgctga tcacgggtgt ggcaccgcgt 3060 tccttctaca ttggtgatgg cgagcgtccg cgtgcacact acccgggcct gaccgtcgat 3120 tttgttgcgg aagcggttac taccctgggt gctcagcaac gtgagggtta tgtctcgtat 3180 gacgttatga atccgcacga tgacggtatt agcttggatg tctttgtgga ctggctgatt 3240 cgtgcgggcc acccaattga ccgtgttgac gactatgatg actgggtgcg tcgttttgaa 3300 accgcgttga ccgccttgcc ggagaaacgt cgtgcgcaga ccgttctgcc gctgctgcat 3360 gcctttcgcg cgccacaggc gccgttgcgt ggcgcccctg aaccgaccga agtgtttcat 3420 gcagcggtgc gtaccgctaa agtcggtccg ggtgatattc cgcacctgga tgaagccctg 3480 atcgacaagt acatccgtga cctgcgcgag ttcggtctga tttag 3525 <210> 5 <211> 1173 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic CAR Variant (carB2) polypeptide" <400> 5 Met Thr Ser Asp Val His Asp Ala Thr Asp Gly Val Thr Glu Thr Ala 1 5 10 15 Leu Asp Asp Arg Gln Ser Thr Arg Arg Ile Ala Glu Leu Tyr Ala Thr 20 25 30 Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp Ala 35 40 45 Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu Phe 50 55 60 Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu Leu 65 70 75 80 Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg Phe 85 90 95 Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala Val Ala 100 105 110 Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp Ala 115 120 125 Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu Thr Leu Asp Leu 130 135 140 Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn Ala 145 150 155 160 Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro Arg Ile 165 170 175 Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val Glu Ser Val Arg 180 185 190 Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp His His Pro Glu 195 200 205 Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu Ala 210 215 220 Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile Ala Asp Glu Gly 225 230 235 240 Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln Arg 245 250 255 Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly 260 265 270 Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser Gly 275 280 285 Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro Leu 290 295 300 Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala Val Gln Asn Gly 305 310 315 320 Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe Glu 325 330 335 Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg Val 340 345 350 Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val Asp Arg Leu Val 355 360 365 Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala Glu 370 375 380 Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val Ser 385 390 395 400 Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr Leu 405 410 415 Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala Val 420 425 430 Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile Asp Tyr Lys Leu 435 440 445 Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr Pro 450 455 460 Arg Gly Glu Leu Leu Val Arg Ser Ile Thr Leu Thr Pro Gly Tyr Tyr 465 470 475 480 Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr Tyr 485 490 495 His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val Tyr 500 505 510 Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val 515 520 525 Ala Val Ala Asn Leu Glu Ser Val Phe Ser Gly Ala Ala Leu Val Arg 530 535 540 Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val 545 550 555 560 Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp Pro Ala Ala Leu 565 570 575 Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala Glu 580 585 590 Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val Glu Thr Glu Pro 595 600 605 Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly Lys Leu Leu Arg 610 615 620 Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr Ala 625 630 635 640 Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg Ala 645 650 655 Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala Ala Thr 660 665 670 Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr Asp 675 680 685 Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser Asn Leu Leu Ser 690 695 700 Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro Ala 705 710 715 720 Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala Gln Arg Thr Ala 725 730 735 Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala Thr 740 745 750 Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala Glu 755 760 765 Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro Arg 770 775 780 Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu Thr 785 790 795 800 Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile Thr 805 810 815 Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala Arg Leu Thr Gln 820 825 830 Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu Leu Ala 835 840 845 Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly Asp Pro Asn Leu 850 855 860 Gly Leu Thr Pro Glu Ile Trp His Arg Leu Ala Ala Glu Val Asp Leu 865 870 875 880 Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg Gln 885 890 895 Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val Ile Lys Leu Ala 900 905 910 Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ser Val 915 920 925 Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr Val 930 935 940 Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn Gly Tyr Gly Asn 945 950 955 960 Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys 965 970 975 Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His Pro 980 985 990 Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu Leu 995 1000 1005 Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr Ile 1010 1015 1020 Gly Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly Leu Thr Val 1025 1030 1035 Asp Phe Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln Arg 1040 1045 1050 Glu Gly Tyr Val Ser Tyr Asp Val Met Asn Pro His Asp Asp Gly 1055 1060 1065 Ile Ser Leu Asp Val Phe Val Asp Trp Leu Ile Arg Ala Gly His 1070 1075 1080 Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe 1085 1090 1095 Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys Arg Arg Ala Gln Thr 1100 1105 1110 Val Leu Pro Leu Leu His Ala Phe Arg Ala Pro Gln Ala Pro Leu 1115 1120 1125 Arg Gly Ala Pro Glu Pro Thr Glu Val Phe His Ala Ala Val Arg 1130 1135 1140 Thr Ala Lys Val Gly Pro Gly Asp Ile Pro His Leu Asp Glu Ala 1145 1150 1155 Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg Glu Phe Gly Leu Ile 1160 1165 1170 <210> 6 <211> 3525 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic CAR Variant (carB2) polynucleotide" <400> 6 atgggcacga gcgatgttca cgacgcgacc gacggcgtta ccgagactgc actggatgat 60 cgccagagca ctcgtcgtat tgcagaactg tacgcaacgg acccagagtt cgcagcagca 120 gctcctctgc cggccgttgt cgatgcggcg cacaaaccgg gcctgcgtct ggcggaaatc 180 ctgcagaccc tgttcaccgg ctacggcgat cgtccggcgc tgggctatcg tgcacgtgag 240 ctggcgacgg acgaaggcgg tcgtacggtc acgcgtctgc tgccgcgctt cgataccctg 300 acctatgcac aggtgtggag ccgtgttcaa gcagtggctg cagcgttgcg tcacaatttc 360 gcacaaccga tttacccggg cgacgcggtc gcgactatcg gctttgcgag cccggactat 420 ttgacgctgg atctggtgtg cgcgtatctg ggcctggtca gcgttccttt gcagcataac 480 gctccggtgt ctcgcctggc cccgattctg gccgaggtgg aaccgcgtat tctgacggtg 540 agcgcagaat acctggacct ggcggttgaa tccgtccgtg atgtgaactc cgtcagccag 600 ctggttgttt tcgaccatca tccggaagtg gacgatcacc gtgacgcact ggctcgcgca 660 cgcgagcagc tggccggcaa aggtatcgca gttacgaccc tggatgcgat cgcagacgaa 720 ggcgcaggtt tgccggctga gccgatttac acggcggatc acgatcagcg tctggccatg 780 attctgtata ccagcggctc tacgggtgct ccgaaaggcg cgatgtacac cgaagcgatg 840 gtggctcgcc tgtggactat gagcgggatc acgggcgacc cgaccccggt tatcaacgtg 900 aacttcatgc cgctgaacca tctgggcggt cgtatcccga ttagcaccgc cgtgcagaat 960 ggcggtacca gctacttcgt tccggaaagc gacatgagca cgctgtttga ggatctggcc 1020 ctggtccgcc ctaccgaact gggtctggtg ccgcgtgttg cggacatgct gtaccagcat 1080 catctggcga ccgtggatcg cctggtgacc cagggcgcgg acgaactgac tgcggaaaag 1140 caggccggtg cggaactgcg tgaacaggtc ttgggcggtc gtgttatcac cggttttgtt 1200 tccaccgcgc cgttggcggc agagatgcgt gcttttctgg atatcacctt gggtgcacac 1260 atcgttgacg gttacggtct gaccgaaacc ggtgcggtca cccgtgatgg tgtgattgtt 1320 cgtcctccgg tcattgatta caagctgatc gatgtgccgg agctgggtta cttctccacc 1380 gacaaaccgt acccgcgtgg cgagctgctg gttcgtagca tcacgttgac tccgggttac 1440 tacaagcgcc cagaagtcac cgcgtccgtt ttcgatcgcg acggctatta ccacaccggc 1500 gacgtgatgg cagaaaccgc gccagaccac ctggtgtatg tggaccgccg caacaatgtt 1560 ctgaagctgg cgcaaggtga atttgtcgcc gtggctaacc tggagtccgt tttcagcggc 1620 gctgctctgg tccgccagat tttcgtgtat ggtaacagcg agcgcagctt tctgttggct 1680 gttgttgtcc ctaccccgga ggcgctggag caatacgacc ctgccgcatt gaaagcagcc 1740 ctggcggatt cgctgcagcg tacggcgcgt gatgccgagc tgcagagcta tgaagtgccg 1800 gcggacttca ttgttgagac tgagcctttt agcgctgcga acggtctgct gagcggtgtt 1860 ggcaagttgc tgcgtccgaa tttgaaggat cgctacggtc agcgtttgga gcagatgtac 1920 gcggacatcg cggctacgca ggcgaaccaa ttgcgtgagc tgcgtcgcgc tgcggctact 1980 caaccggtga tcgacacgct gacgcaagct gcggcgacca tcctgggtac cggcagcgag 2040 gttgcaagcg acgcacactt tactgatttg ggcggtgatt ctctgagcgc gctgacgttg 2100 agcaacttgc tgtctgactt ctttggcttt gaagtcccgg ttggcacgat tgttaaccca 2160 gcgactaatc tggcacagct ggcgcaacat atcgaggcgc agcgcacggc gggtgaccgc 2220 cgtccatcct ttacgacggt ccacggtgcg gatgctacgg aaatccgtgc aagcgaactg 2280 actctggaca aattcatcga cgctgagact ctgcgcgcag cacctggttt gccgaaggtt 2340 acgactgagc cgcgtacggt cctgttgagc ggtgccaatg gttggttggg ccgcttcctg 2400 accctgcagt ggctggaacg tttggcaccg gttggcggta ccctgatcac cattgtgcgc 2460 ggtcgtgacg atgcagcggc ccgcgcacgc ttgactcagg cttacgatac ggacccagag 2520 ctgtcccgcc gcttcgctga gttggcggat cgccacttgc gtgtggtggc aggtgatatc 2580 ggcgatccga atctgggcct gaccccggag atttggcacc gtctggcagc agaggtcgat 2640 ctggtcgttc atccagcggc cctggtcaac cacgtcctgc cgtaccgcca gctgtttggt 2700 ccgaatgttg ttggcaccgc cgaagttatc aagttggctc tgaccgagcg catcaagcct 2760 gttacctacc tgtccacggt tagcgtcgcg atgggtattc ctgattttga ggaggacggt 2820 gacattcgta ccgtcagccc ggttcgtccg ctggatggtg gctatgcaaa tggctatggc 2880 aacagcaagt gggctggcga ggtgctgctg cgcgaggcac atgacctgtg tggcctgccg 2940 gttgcgacgt ttcgtagcga catgattctg gcccacccgc gctaccgtgg ccaagtgaat 3000 gtgccggaca tgttcacccg tctgctgctg tccctgctga tcacgggtgt ggcaccgcgt 3060 tccttctaca ttggtgatgg cgagcgtccg cgtgcacact acccgggcct gaccgtcgat 3120 tttgttgcgg aagcggttac taccctgggt gctcagcaac gtgagggtta tgtctcgtat 3180 gacgttatga atccgcacga tgacggtatt agcttggatg tctttgtgga ctggctgatt 3240 cgtgcgggcc acccaattga ccgtgttgac gactatgatg actgggtgcg tcgttttgaa 3300 accgcgttga ccgccttgcc ggagaaacgt cgtgcgcaga ccgttctgcc gctgctgcat 3360 gcctttcgcg cgccacaggc gccgttgcgt ggcgcccctg aaccgaccga agtgtttcat 3420 gcagcggtgc gtaccgctaa agtcggtccg ggtgatattc cgcacctgga tgaagccctg 3480 atcgacaagt acatccgtga cctgcgcgag ttcggtctga tttag 3525 <210> 7 <211> 1174 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic CAR Variant (carB8) polypeptide" <400> 7 Met Gly Thr Ser Asp Val His Asp Ala Thr Asp Gly Val Thr Glu Thr 1 5 10 15 Ala Leu Asp Asp Arg Gln Arg Thr Arg Arg Ile Ala Glu Leu Tyr Ala 20 25 30 Thr Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp 35 40 45 Ala Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu 50 55 60 Phe Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu 65 70 75 80 Leu Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg 85 90 95 Phe Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala Val 100 105 110 Ala Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp 115 120 125 Ala Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu Thr Leu Asp 130 135 140 Leu Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn 145 150 155 160 Ala Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro Arg 165 170 175 Ile Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val Glu Ser Val 180 185 190 Arg Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp His His Pro 195 200 205 Glu Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu 210 215 220 Ala Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile Ala Asp Glu 225 230 235 240 Gly Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln 245 250 255 Arg Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys 260 265 270 Gly Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser 275 280 285 Gly Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro 290 295 300 Leu Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala Val Gln Asn 305 310 315 320 Gly Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe 325 330 335 Glu Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg 340 345 350 Val Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val Asp Arg Leu 355 360 365 Val Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala 370 375 380 Glu Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val 385 390 395 400 Ser Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr 405 410 415 Leu Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala 420 425 430 Val Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile Asp Tyr Lys 435 440 445 Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr 450 455 460 Pro Arg Gly Glu Leu Leu Val Arg Ser His Thr Leu Thr Pro Gly Tyr 465 470 475 480 Tyr Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr 485 490 495 Tyr His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val 500 505 510 Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe 515 520 525 Val Ala Val Ala Asn Leu Glu Ser Val Phe Ser Gly Ala Ala Leu Val 530 535 540 Arg Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala 545 550 555 560 Val Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp Pro Ala Ala 565 570 575 Leu Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala 580 585 590 Glu Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val Glu Thr Glu 595 600 605 Pro Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly Lys Leu Leu 610 615 620 Arg Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr 625 630 635 640 Ala Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg 645 650 655 Ala Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala Ala 660 665 670 Thr Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr 675 680 685 Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser Asn Leu Leu 690 695 700 Ser Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro 705 710 715 720 Ala Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala Gln Arg Thr 725 730 735 Ala Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala 740 745 750 Thr Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala 755 760 765 Glu Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro 770 775 780 Arg Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu 785 790 795 800 Thr Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile 805 810 815 Thr Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala Arg Leu Thr 820 825 830 Gln Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu Leu 835 840 845 Ala Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly Asp Pro Asn 850 855 860 Leu Gly Leu Thr Pro Glu Ile Trp His Ser Leu Ala Ala Glu Val Asp 865 870 875 880 Leu Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg 885 890 895 Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val Ile Lys Leu 900 905 910 Ala Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Gly 915 920 925 Val Ala Arg Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr 930 935 940 Val Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn Gly Tyr Gly 945 950 955 960 Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu 965 970 975 Cys Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His 980 985 990 Pro Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu 995 1000 1005 Leu Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr 1010 1015 1020 Ile Gly Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly Leu Thr 1025 1030 1035 Val Asp Phe Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln 1040 1045 1050 Arg Glu Gly Tyr Val Ser Tyr Asp Val Met Asn Pro His Asp Asp 1055 1060 1065 Gly Ile Ser Leu Asp Val Phe Val Asp Trp Leu Ile Arg Ala Gly 1070 1075 1080 His Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp Trp Val Arg Arg 1085 1090 1095 Phe Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys Arg Arg Ala Gln 1100 1105 1110 Thr Val Leu Pro Leu Leu His Ala Phe Arg Ala Pro Gln Ala Pro 1115 1120 1125 Trp Arg Gly Ala Pro Glu Pro Thr Glu Val Phe His Ala Ala Val 1130 1135 1140 Arg Thr Ala Lys Val Gly Pro Gly Asp Ile Pro His Leu Asp Glu 1145 1150 1155 Ala Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg Glu Phe Gly Leu 1160 1165 1170 Ile <210> 8 <211> 3525 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic CAR Variant (carB8) polynucleotide" <400> 8 atgggcacga gcgatgttca cgacgcgacc gacggcgtta ccgagactgc actggatgat 60 cgccagagga ctcgtcgtat tgcagaactg tacgcaacgg acccagagtt cgcagcagca 120 gctcctctgc cggccgttgt cgatgcggcg cacaaaccgg gcctgcgtct ggcggaaatc 180 ctgcagaccc tgttcaccgg ctacggcgat cgtccggcgc tgggctatcg tgcacgtgag 240 ctggcgacgg acgaaggcgg tcgtacggtc acgcgtctgc tgccgcgctt cgataccctg 300 acctatgcac aggtgtggag ccgtgttcaa gcagtggctg cagcgttgcg tcacaatttc 360 gcacaaccga tttacccggg cgacgcggtc gcgactatcg gctttgcgag cccggactat 420 ttgacgctgg atctggtgtg cgcgtatctg ggcctggtca gcgttccttt gcagcataac 480 gctccggtgt ctcgcctggc cccgattctg gccgaggtgg aaccgcgtat tctgacggtg 540 agcgcagaat acctggacct ggcggttgaa tccgtccgtg atgtgaactc cgtcagccag 600 ctggttgttt tcgaccatca tccggaagtg gacgatcacc gtgacgcact ggctcgcgca 660 cgcgagcagc tggccggcaa aggtatcgca gttacgaccc tggatgcgat cgcagacgaa 720 ggcgcaggtt tgccggctga gccgatttac acggcggatc acgatcagcg tctggccatg 780 attctgtata ccagcggctc tacgggtgct ccgaaaggcg cgatgtacac cgaagcgatg 840 gtggctcgcc tgtggactat gagcgggatc acgggcgacc cgaccccggt tatcaacgtg 900 aacttcatgc cgctgaacca tctgggcggt cgtatcccga ttagcaccgc cgtgcagaat 960 ggcggtacca gctacttcgt tccggaaagc gacatgagca cgctgtttga ggatctggcc 1020 ctggtccgcc ctaccgaact gggtctggtg ccgcgtgttg cggacatgct gtaccagcat 1080 catctggcga ccgtggatcg cctggtgacc cagggcgcgg acgaactgac tgcggaaaag 1140 caggccggtg cggaactgcg tgaacaggtc ttgggcggtc gtgttatcac cggttttgtt 1200 tccaccgcgc cgttggcggc agagatgcgt gcttttctgg atatcacctt gggtgcacac 1260 atcgttgacg gttacggtct gaccgaaacc ggtgcggtca cccgtgatgg tgtgattgtt 1320 cgtcctccgg tcattgatta caagctgatc gatgtgccgg agctgggtta cttctccacc 1380 gacaaaccgt acccgcgtgg cgagctgctg gttcgtagcc acacgttgac tccgggttac 1440 tacaagcgcc cagaagtcac cgcgtccgtt ttcgatcgcg acggctatta ccacaccggc 1500 gacgtgatgg cagaaaccgc gccagaccac ctggtgtatg tggaccgccg caacaatgtt 1560 ctgaagctgg cgcaaggtga atttgtcgcc gtggctaacc tggagtccgt tttcagcggc 1620 gctgctctgg tccgccagat tttcgtgtat ggtaacagcg agcgcagctt tctgttggct 1680 gttgttgtcc ctaccccgga ggcgctggag caatacgacc ctgccgcatt gaaagcagcc 1740 ctggcggatt cgctgcagcg tacggcgcgt gatgccgagc tgcagagcta tgaagtgccg 1800 gcggacttca ttgttgagac tgagcctttt agcgctgcga acggtctgct gagcggtgtt 1860 ggcaagttgc tgcgtccgaa tttgaaggat cgctacggtc agcgtttgga gcagatgtac 1920 gcggacatcg cggctacgca ggcgaaccaa ttgcgtgagc tgcgtcgcgc tgcggctact 1980 caaccggtga tcgacacgct gacgcaagct gcggcgacca tcctgggtac cggcagcgag 2040 gttgcaagcg acgcacactt tactgatttg ggcggtgatt ctctgagcgc gctgacgttg 2100 agcaacttgc tgtctgactt ctttggcttt gaagtcccgg ttggcacgat tgttaaccca 2160 gcgactaatc tggcacagct ggcgcaacat atcgaggcgc agcgcacggc gggtgaccgc 2220 cgtccatcct ttacgacggt ccacggtgcg gatgctacgg aaatccgtgc aagcgaactg 2280 actctggaca aattcatcga cgctgagact ctgcgcgcag cacctggttt gccgaaggtt 2340 acgactgagc cgcgtacggt cctgttgagc ggtgccaatg gttggttggg ccgcttcctg 2400 accctgcagt ggctggaacg tttggcaccg gttggcggta ccctgatcac cattgtgcgc 2460 ggtcgtgacg atgcagcggc ccgcgcacgc ttgactcagg cttacgatac ggacccagag 2520 ctgtcccgcc gcttcgctga gttggcggat cgccacttgc gtgtggtggc aggtgatatc 2580 ggcgatccga atctgggcct gaccccggag atttggcaca gtctggcagc agaggtcgat 2640 ctggtcgttc atccagcggc cctggtcaac cacgtcctgc cgtaccgcca gctgtttggt 2700 ccgaatgttg ttggcaccgc cgaagttatc aagttggctc tgaccgagcg catcaagcct 2760 gttacctacc tgtccacggt tggggtcgcg aggggtattc ctgattttga ggaggacggt 2820 gacattcgta ccgtcagccc ggttcgtccg ctggatggtg gctatgcaaa tggctatggc 2880 aacagcaagt gggctggcga ggtgctgctg cgcgaggcac atgacctgtg tggcctgccg 2940 gttgcgacgt ttcgtagcga catgattctg gcccacccgc gctaccgtgg ccaagtgaat 3000 gtgccggaca tgttcacccg tctgctgctg tccctgctga tcacgggtgt ggcaccgcgt 3060 tccttctaca ttggtgatgg cgagcgtccg cgtgcacact acccgggcct gaccgtcgat 3120 tttgttgcgg aagcggttac taccctgggt gctcagcaac gtgagggtta tgtctcgtat 3180 gacgttatga atccgcacga tgacggtatt agcttggatg tctttgtgga ctggctgatt 3240 cgtgcgggcc acccaattga ccgtgttgac gactatgatg actgggtgcg tcgttttgaa 3300 accgcgttga ccgccttgcc ggagaaacgt cgtgcgcaga ccgttctgcc gctgctgcat 3360 gcctttcgcg cgccacaggc gccgtggcgt ggcgcccctg aaccgaccga agtgtttcat 3420 gcagcggtgc gtaccgctaa agtcggtccg ggtgatattc cgcacctgga tgaagccctg 3480 atcgacaagt acatccgtga cctgcgcgag ttcggtctga tttag 3525

Claims

1. Recombinant microorganisms that produce 1,3-fatty diols when grown in fermentation broth containing a simple carbon source, (a) Thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity; and (b) Carboxylic acid reductase (EC 6.2.1.3 or EC 1.2.1.42) activity It is being manipulated to express a nucleic acid sequence encoding a polypeptide containing, The recombinant microorganisms mentioned above.

2. The recombinant microorganism according to claim 1, wherein the 1,3-lipid diol is produced in vivo.

3. The 1,3-fatty diol is C ５ 1,3-fatty diol, C ６ 1,3-fatty diol, C ７ 1,3-fatty diol, C ８ 1,3-fatty diol, C ９ 1,3-fatty diol, C １０ 1,3-fatty diol, C １１ 1,3-fatty diol, C １２ 1,3-fatty diol, C １３ 1,3-fatty diol, C １４ 1,3-fatty diol, C １５ 1,3-fatty diol, C １６ 1,3-fatty diol, C １７ 1,3-fatty diol, C １８ 1,3-fatty diol, and C １９ The recombinant microorganism according to claim 1, which is selected from the group consisting of 1,3-fatty diol, and C

4. The recombinant microorganism according to claim 1, further expressing a nucleic acid sequence encoding a polypeptide containing alcohol dehydrogenase (EC1.1.1.-) activity.

5. The recombinant microorganism according to claim 1, wherein the simple carbon source is derived from renewable raw materials.

6. The recombinant microorganism according to claim 1, wherein the thioesterase is selected from the group consisting of fatB1, TE_EEI82564, TE_CAD63310, and phG.

7. The recombinant microorganism according to claim 1, wherein the carboxylic acid reductase is carB.

8. The recombinant microorganism according to claim 4, wherein the alcohol dehydrogenase is alrA.

9. A cell culture comprising the microorganism described in any one of claims 1 to 8.

10. A cell culture according to claim 9, which produces 1,3-lipid diols.

11. The above 1,3 fatty diols are C ５ 1,3-lipid diol, C ６ 1,3-lipid diol, C ７ 1,3-lipid diol, C ８ 1,3-lipid diol, C ９ 1,3-lipid diol, C １０ 1,3-lipid diol, C １１ 1,3-lipid diol, C １２ 1,3-lipid diol, C １３ 1,3-lipid diol, C １４ 1,3-lipid diol, C １５ 1,3-lipid diol, C １６ 1,3-lipid diol, C １７ 1,3-lipid diol, C １８ 1,3-lipid diol, and C １９ A cell culture according to claim 10, selected from the group consisting of 1,3-lipid diols.

12. A method for producing 1,3-lipid diols, comprising the microorganism described in claim 1.

13. (a) A step of preparing recombinant microorganisms in a fermentation broth, wherein the microorganisms express a nucleic acid sequence encoding a polypeptide comprising thioesterase (EC3.1.2.-, EC3.1.1.5, or EC3.1.2.14) activity; carboxylic acid reductase (EC6.2.1.3 or EC1.2.1.42) activity; and optionally alcohol dehydrogenase (EC1.1.1.) activity; and (b) A step of isolating 1,3 fatty diols from the fermentation broth, wherein the fermentation broth contains a simple carbon source. A method for producing 1,3-lipid diols, including [the specified substance].

14. The above 1,3 fatty diols are C ５ 1,3-lipid diol, C ６ 1,3-lipid diol, C ７ 1,3-lipid diol, C ８ 1,3-lipid diol, C ９ 1,3-lipid diol, C １０ 1,3-lipid diol, C １１ 1,3-lipid diol, C １２ 1,3-lipid diol, C １３ 1,3-lipid diol, C １４ 1,3-lipid diol, C １５ 1,3-lipid diol, C １６ 1,3-lipid diol, C １７ 1,3-lipid diol, C １８ 1,3-lipid diol, and C １９ The method according to claim 13, selected from the group consisting of 1,3-lipid diols.

15. A recombinant microorganism for producing 1,3-lipid diols when grown in a fermentation broth containing a simple carbon source, wherein the recombinant microorganism is engineered to express a nucleic acid sequence encoding a polypeptide containing acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity.

16. The recombinant microorganism according to claim 15, further expressing a nucleic acid sequence encoding a polypeptide containing alcohol dehydrogenase (EC1.1.1.-) activity.

17. The recombinant microorganism according to claim 15, wherein the 1,3-lipid diol is produced in vivo.

18. The above 1,3 fatty diols are C ５ 1,3-lipid diol, C ６ 1,3-lipid diol, C ７ 1,3-lipid diol, C ８ 1,3-lipid diol, C ９ 1,3-lipid diol, C １０ 1,3-lipid diol, C １１ 1,3-lipid diol, C １２ 1,3-lipid diol, C １３ 1,3-lipid diol, C １４ 1,3-lipid diol, C １５ 1,3-lipid diol, C １６ 1,3-lipid diol, C １７ 1,3-lipid diol, C １８ 1,3-lipid diol, and C １９ A recombinant microorganism according to claim 17, selected from the group consisting of 1,3-lipid diols.

19. The above 1,3 fatty diols are C １２ The recombinant microorganism according to claim 17, wherein the microorganism is a 1,3-fatty diol.

20. The recombinant microorganism according to claim 15, wherein the simple carbon source is derived from renewable raw materials.

21. A cell culture comprising the microorganism described in any one of claims 15 to 20.

22. The cell culture according to claim 21, which produces 1,3-lipid diols.

23. The above 1,3 fatty diols are C ５ 1,3-lipid diol, C ６ 1,3-lipid diol, C ７ 1,3-lipid diol, C ８ 1,3-lipid diol, C ９ 1,3-lipid diol, C １０ 1,3-lipid diol, C １１ 1,3-lipid diol, C １２ 1,3-lipid diol, C １３ 1,3-lipid diol, C １４ 1,3-lipid diol, C １５ 1,3-lipid diol, C １６ 1,3-lipid diol, C １７ 1,3-lipid diol, C １８ 1,3-lipid diol, and C １９ A cell culture according to claim 22, selected from the group consisting of 1,3-lipid diols.

24. A method for producing 1,3-lipid diols, comprising the microorganism described in claim 15.

25. (a) a step of preparing recombinant microorganisms in a fermentation broth, wherein the microorganisms are operated to express a nucleic acid sequence encoding a polypeptide having acyl-ACP reductase (EC1.2.1.80 or EC1.2.1.42) activity; and (b) A step of isolating 1,3 fatty diols from the fermentation broth, wherein the fermentation broth contains a simple carbon source. A method for producing 1,3-lipid diols, including [the specified substance].

26. The method according to claim 25, further comprising expressing a nucleic acid sequence encoding a polypeptide containing alcohol dehydrogenase (EC1.1.1.-) activity.

27. The 1,3-fatty diol is C ５ 1,3-fatty diol, C ６ 1,3-fatty diol, C ７ 1,3-fatty diol, C ８ 1,3-fatty diol, C ９ 1,3-fatty diol, C １０ 1,3-fatty diol, C １１ 1,3-fatty diol, C １２ 1,3-fatty diol, C １３ 1,3-fatty diol, C １４ 1,3-fatty diol, C １５ 1,3-fatty diol, C １６ 1,3-fatty diol, C １７ 1,3-fatty diol, C １８ 1,3-fatty diol, and C １９ The method according to claim 25, selected from the group consisting of 1,3-fatty diol and C

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