# Patent

## Title
Genetically modified microorganism for producing long-chain dicarboxylic acid and method of using thereof

## Abstract

Described herein are genetically-modified microorganisms for producing long-chain dicarboxylic acids and methods of using the microorganisms. The microorganisms contain a first nucleic acid encoding an Umbellularia californica lauroyl ACP-thioesterase (BTE) operably linked to a promoter or a second nucleic acid encoding a Cocos nucifera lauroyl ACP-thioesterase (FatB3) operably linked to a promoter.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 62/026,428, filed on Jul. 18, 2014, U.S. Provisional Application No. 62/032,956, filed on Aug. 4, 2014, and Taiwanese Patent Application No. 104121064, filed on Jun. 30, 2015. The contents of all three prior applications are hereby incorporated by reference herein in their entirety.


BACKGROUND
Long-carbon-chain nylon is a high-performing and high-value chemical material due to its unique thermal, physical, chemical and mechanical properties. Nylon 12 is a specialty resin designated for making fuel lines and brake systems. For high-grade Nylon 12, the price is usually more than 15 euros/kg. Analysis of a fatal explosion at a German plant of the world's largest Nylon 12 supplier, Evonik, revealed that explosion caused by contact between a highly active catalyst (Et2AlCl) and water is a major risk factor. Thus, there is a need for a safe process for producing long-chain dicarboxylic acids.
SUMMARY
In one aspect, described herein is a genetically modified microorganism that contains a first nucleic acid encoding an Umbellularia californica lauroyl ACP-thioesterase (BTE) operably linked to a promoter or a second nucleic acid encoding a Cocos nucifera lauroyl ACP-thioesterase (FatB3) operably linked to a promoter. The microorganism produces an increased amount of long-chain dicarboxylic acids as compared to the unmodified parent of the microorganism. The genetically modified microorganism can be Yarrowia lipolytica or Escherichia coli. 
The genetically modified microorganism can include one or more further modifications. In one embodiment, it contains one or more additional nucleic acids each operably linked to a promoter, each additional nucleic acid encoding a protein selected from the group consisting of an acetyl-CoA carboxylase (ACC), an acetyl-CoA carboxylase carboxyl transferase subunit α (AccA), an acetyl-CoA carboxylase biotin carboxyl carrier protein (AccB), an acetyl-CoA biotin carboxylase (AccC), an acetyl-CoA carboxylase transferase subunit β (AccD), a fatty acid synthase (FAS) subunit, a cytochrome P450 reductase (CPR), a long-chain alcohol oxidase (FAO1), a long-chain alcohol dehydrogenase (FADH), and an adenosine monophosphate-forming acetyl-coenzyme A synthetase (AceCS). Alternatively or additionally, the genetically modified microorganism can include a loss-of-function mutation in or expresses a lower level of one or more genes selected from the group consisting of a palmitoyl-acyl carrier protein (ACP) thioesterase gene, an acyl-coenzyme A oxidase gene, a citric synthetase (gltA) gene, or an acyl-coenzyme A synthetase (acs) gene.
In another aspect, described herein is a method of producing a long-chain dicarboxylic acid. The method includes culturing the genetically modified microorganism in a culture medium containing glucose or glycerol at pH 6 to 8 under conditions that allow production of a long-chain dicarboxylic acid, whereby the microorganism produces the long-chain dicarboxylic acid. The method can further include collecting the long-chain dicarboxylic acid, e.g., C10-C18 dicarboxylic acid.
The details of one or more embodiments are set forth in the accompanying drawing and the description below. Other features, objects, and advantages of the embodiments will be apparent from the description and drawing, and from the claims.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic representation showing a modified α,ω-dicarboxylic acid metabolic pathway.
FIG. 2 is a schematic diagram showing construction of a modified Y. lipolytica strain.
FIG. 3 is a graph showing HPLC analysis of dicarboxylic acid samples.
FIG. 4 is a graph showing the fatty acid contents of Δpox2 and Δpox5 mutants.
FIG. 5 is a schematic diagram showing construction of various modified Y. lipolytica strains.
FIG. 6 is a schematic diagram showing an expression cassette for expressing an Umbellularia californica lauroyl ACP-thioesterase (BTE) and a Cocos nucifera lauroyl ACP-thioesterase (FatB3).
FIG. 7 is a schematic diagram showing construction of an RNAi expression cassette for silencing expression of the palmitoyl-acyl carrier protein (ACP) thioesterase gene.
FIG. 8 is a set of graphs showing dicarboxylic acids produced by two modified Y. lipolytica strains.
FIG. 9 is a schematic diagram showing expression constructs for generating modified E. coli strain E1.
FIG. 10 is a graph showing HLPC analysis of dicarboxylic acid production of strain E1.
FIG. 11 is a schematic diagram showing expression constructs for generating modified E. coli strains E2 and E3.
FIG. 12 is a bar graph showing citrate and acetate productions of strains E2 and E3.
FIG. 13 is a schematic diagram showing expression constructs for generating modified E. coli strain E4.
FIG. 14 is a bar graph showing dicarboxylic acid productions of strains E2 and E4.
FIG. 15 is a schematic diagram showing construction of E. coli strain E5.
FIG. 16 is a schematic diagram showing construction of E. coli strain E6.
FIG. 17 is a bar graph showing dicarboxylic acid productions of strains E5 and E6.



DETAILED DESCRIPTION
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.
Described below are genetically-modified microorganisms for producing long-chain dicarboxylic acids and methods of using the microorganisms.
To improve production of dicarboxylic acids, one or more modifications can be introduced into the α,ω-dicarboxylic acid metabolic pathway of a microorganism. See FIG. 1. Such modifications can include, for example, increasing free fatty acid contents, enhancing substrate specificity of ω-oxidation enzymes, increasing expression of key proteins in fatty acid synthesis (e.g., acetyl-CoA carboxylase and fatty acid synthase), knocking out a gene upstream of β-oxidation (e.g., pox2, pox5, or fadD), decreasing fatty acid degradation, knocking out a, citric synthetase gene (e.g., gltA), increasing fatty acid synthesis, knocking out an acyl-coenzyme A synthetase gene (e.g., acs), decreasing triglyceride accumulation, increasing expression of an adenosine monophosphate-forming acetyl-coenzyme A synthetase (AceCS), enhancing expression of a lauroyl-ACP thioesterases (e.g., Cocos nucifera FatB3 or Umbellularia californica BTE), decreasing or silencing expression of a palmitoyl-acyl carrier protein (ACP) thioesterase, and expressing w-oxidation metabolic pathway genes (e.g., cpr, fao1, and fadH).
Accordingly, a genetically-modified microorganism can contain a nucleic acid encoding an Umbellularia californica lauroyl ACP-thioesterase (BTE). It can alternatively or further include a nucleic acid encoding a Cocos nucifera lauroyl ACP-thioesterase (FatB3).
The genetically-modified microorganism can also have a nucleic acid that encodes an acetyl-CoA carboxylase (ACC), a fatty acid synthase (FAS) subunit, a cytochrome P450 reductase (CPR), a long-chain alcohol oxidase (e.g., FAO1), or a long-chain alcohol dehydrogenase (e.g., FADH).
Each of the above-described nucleic acid is operably linked to a suitable promoter for gene expression in the genetically-modified microorganism. If appropriate or necessary, the sequence of the nucleic acid can also be codon-optimized for expression in the genetically-modified microorganism.
Expression of one or more genes or proteins can also be decreased in the genetically-modified microorganism. For example, the expression of an ACP thioesterase gene, an acyl-coenzyme A oxidase gene (e.g., pox2, pox5, or fadD), a citric synthetase gene (gltA), or an acyl-coenzyme A synthetase gene (acs) can be decreased or silenced in the genetically-modified microorganism. Such a microorganism can have a loss-of-function mutation (e.g., deletion) in the gene or an expression construct that expresses an RNAi molecule targeting the gene.
As used herein, the term “promoter” refers to a nucleotide sequence containing elements that initiate the transcription of an operably linked nucleic acid sequence in a desired host cell. At a minimum, a promoter contains an RNA polymerase binding site. It can further contain one or more enhancer elements which, by definition, enhance transcription, or one or more regulatory elements that control the on/off status of the promoter. A promoter can be an inducible or constitutive promoter. Exemplary promoters include glyceraldehyde-3-phosphate dehydrogenase (GAP), fructose 1,6-bisphosphate aldolase intron (FBAin), beta-lactamase (bla, conferring ampicillin resistance), lac operon, T7, and SP6 promoters.
An expression cassette for expressing any of the genes described above can be introduced into a suitable host cell to produce a genetically modified microorganism using methods known in the art or described herein. Methods known in the art and described below can be used to knock-out a gene or decrease expression of a gene in a host cell to construct the genetically-modified microorganism.
Suitable host cells include, but are not limited to, Candida tropicalis, Candida cloaceae, Escherichia coli, and Yarrowia lipolytica. 
The modified microorganism can then be cultured in a medium suitable for long chain dicarboxylic acid production. For example, the medium can contain glucose or glycerol as a carbon source. After a sufficient culturing period, dicarboxylic acids, in particular DCA12, can be isolated from the medium.
A computer readable file containing a sequence listing is electronically co-filed with this application via EFS-Web. The computer readable file, submitted under 37 CFR §1.821(e), also serves as the copy required by 37 CFR §1.821(c). The file (filename “28X9833.TXT”) was created on Jun. 30, 2015 and has a size of 204,809 bytes. The content of the computer readable file is hereby incorporated by reference herein in its entirety.
Exemplary nucleic acid and amino acid sequences of the proteins described herein are provided in the sequence listing: Y. lipolytica acetyl-CoA carboxylase (ACC) (SEQ ID NOs:1 and 2), E. coli acetyl-CoA carboxylase carboxyl transferase subunit α (AccA) (SEQ ID NOs: 3 and 4), E. coli acetyl-CoA carboxylase biotin carboxyl carrier protein (AccB/BCCP) (SEQ ID NOs:5 and 6), E. coli acetyl-CoA biotin carboxylase (AccC) (SEQ ID NOs:7 and 8), E. coli acetyl-CoA carboxylase transferase subunit β (AccD) (SEQ ID NOs:9 and 10), Y. lipolytica fatty acid synthase subunit α (FASA) (SEQ ID NOs:11 and 12), Y. lipolytica fatty acid synthase subunit β (FASB) (SEQ ID NOs:13 and 14), Y. lipolytica acetyl-CoA carboxylase transferase subunit β (AccD) (SEQ ID NOs:15 and 16), Y. lipolytica fatty acid synthase subunit alpha-active site 1 (FASA-1) (SEQ ID NOs:17 and 18), Y. lipolytica codon-optimized Umbellularia californica lauroyl ACP-thioesterase (BTE) (SEQ ID NOs:19 and 20), E. coli codon-optimized BTEΔNC (SEQ ID NOs:21 and 22), Y. lipolytica codon-optimized Cocos nucifera lauroyl palmitoyl-acyl carrier protein (ACP) thioesterase (FatB3) (SEQ ID NOs:23 and 24), E. coli codon-optimized FatB3ΔNC (SEQ ID NOs:25 and 26), Y. lipolytica ACP thioesterase (SEQ ID NOs:27 and 28), Candida tropicalis cytochrome P450 reductase (CPR/CTP 00485) (SEQ ID NOs:29 and 30), E. coli codon-optimized Candida tropicalis CPR nucleic acid sequence (SEQ ID NO: 31), Candida albicans fatty alcohol oxgenase (FAO1) (SEQ ID NOs:32 and 33), E. coli codon-optimized Candida albicans FAO1 nucleic acid sequence (SEQ ID NO:34), Candida albicans fatty aldehyde hydrogenase (FADH) (SEQ ID Nos:35 and 36), E. coli codon-optimized Candida albicans FADH (SEQ ID NO:37), Y. lipolytica acyl-coenzyme A oxidase (PDX2) (SEQ ID NOs:38 and 39), Y. lipolytica acyl-coenzyme A oxidase (PDX5) (SEQ ID NOs:40 and 41), E. coli acyl-coenzyme A oxidase (FadD) (SEQ ID NOs:42 and 43), E. coli adenosine monophosphate-forming acetyl-CoA synthetase (AceCS) (SEQ ID NOs:44 and 45), E. coli acyl-CoA synthetase (ACS) (SEQ ID NOs:46 and 47), and E. coli citric synthetase (gltA) (SEQ ID NOs:48 and 49).
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent.
Modified Y. lipolytica Strains
Since Yarrowia and Pichia expression systems are similar, the Yarrowia expression system used in this study was design based on a Pichia pastoris expression system. A construct for single/double-crossover homologous recombination was designed to insert a co-oxidation gene into the acyl coenzyme A oxidase gene (pox1-5) of a Yarrowia strain in order to knock out the β-oxidation activity of the strain. Geneticin was used as the selectable marker. A schematic diagram of the construct is shown in FIG. 2.
Splice overlap extension (SOE) polymerase chain reaction (PCR) was used to generate a fusion construct containing pox2 or pox5 and a selectable marker (Kan::G418). See FIG. 2. The fusion construct was cloned into the pUC19 vector. The fusion construct was used to generate pox2- and pox5-deficient strains. Our analysis showed that this strategy significantly reduced unnecessary strain replication and DNA purification steps. The PCR product was used directly to efficiently transform a strain.
The electroporation method was used to introduce constructs into cells. First, Y. lipolytica cells were incubated in TE/LiAc/H2O for 30 minutes, and then washed with Sorbitol to obtain competent Y. lipolytica cells. Constructs were then introduced into the cells via electroporation. 50-500 μg/mL Geneticin was used to select for antibiotic-resistant transformants.
Y. lipolytica cells were cultured under various conditions in different media, i.e., YNB medium (0.17% YNB without amino acid, 0.5% ammonium sulphate, glucose or glycerol, 0.15% Yeast extract, 0.5% NH4Cl, 0.01% Uracil, 2% Casamino acids, and 0.02% Tween-80) and NL medium (10% Glucose, 0.85% Yeast extract, and 0.3% Peptone). The cells and culturing media were collected for analysis using gas chromatography (GC) or high-performance liquid chromatography (HPLC).
For GC, a 5 mL culture sample was adjusted to pH 10.0 and then centrifuged. The supernatant was collected and the pH was adjusted to pH 2.0. The pellet was also collected. 14% BF3-Methanol (0.1 mL) and 0.2 mL Hexane was added to the sample and heated at 80-90° C. for 60 minutes. 0.2 mL of saline solution was added, and then 0.5 mL Hexane was added. GC analysis was then performed on the sample.
For HPLC, 5 mL of ethyl acetate was added to 5 mL of culture. The culture was then subjected to a Beatbeader sonicator for about one minute to break the cells and then centrifuged at 6000 rpm. The supernatant was collected. The solvent was allowed to evaporate from the supernatant. 1 mL of 99.5% ethanol was added to dissolve the extract. The sample was then analyzed by HPLC. See FIG. 3.













Instrument:
Shimadzu 20ALC


Column:
Vercogel 120-5 C8, 5 um, 4.6 × 250 mm



(Vercopak no. 15835)


Eluent:
A: 0.1% TFA in H2O



B: AeCN













Gradient:
Time
% A
% B






0
70
30



20
0
100



22
70
30








Flow rate:
1.0 ml/min


Column oven:
30° C.


Detection:
UV 220 nm


Samples:
Citric acid (CA), Sebacic acid (C10 DCA),



Dodecanedioic acid (C12 DCA), Tetradecanedioic



acid (C14 DCA), Hexadecanedioic acid (C16 DCA),



Octanedecanedioic acid (C18 DCA)


Injection:
10 μl









Our data showed that a pox2-deficient Y. lipolytica mutant accumulated more fatty acids than a pox5-deficient Y. lipolytica mutant. There was a 20% increase as compared to the wild-type. See FIG. 4.
Wild-type Y. lipolytica was cultured in YPD medium for one day, and then inoculated into 250 ml of YNB medium (10% glucose or glycerol) at an initial pH of 6.18 or 6.42. The cells were then cultured in a shaker bottle for 5 days without controlling the pH. Dicarboxylic acid production was measured. See Table 1.













TABLE 1








C10DCA
C12DCA
C14DCA
C16DCA


Day
pH

(g/L)
(g/L)
(g/L)
(g/L)









Residual








glu (g/L)


D5
3.38
0
0.40
—
0.25
—




Residual




gly (g/L)


D5
3.19
0
0.47
—
—
—









Four Y. lipolytica strains (ω1, ω2, ω3, and ω4) were constructed using the targeted gene knockout method described above. See FIG. 5.
Strain ω4 was cultured in YPD medium for one day, inoculated into 500 ml of NL medium at an initial pH of 5.0, and then cultured in a fermenter without controlling the pH. Dicarboxylic acid production was measured. See Table 2.













TABLE 2








C10DCA
C12DCA
C14DCA
C16DCA


Day
pH

(g/L)
(g/L)
(g/L)
(g/L)









Residual








glu (g/L)


D2
4.22
84.3
0.28
0.37
1.02
—


D3
3.19
45.0
0.45
0.40
1.02
—


D4
3.34
0
0.37
0.49
0.88
—


D5
4.91
0
0.43
0.52
1.09
—




Residual




gly (g/L)


D2
3.45
94
0.24
0.08
1.06
—


D3
2.37
37
0.23
0.57
0.85
—









We constructed three additional Y. lipolytica strains, each expressing a lauroyl ACP-thioesterase (BTE, from Umbellularia californica), a lauroyl ACP-thioesterase (FatB3, from Cocos nucifera), or both. See FIG. 6.









TABLE 3









Relative fatty acid content (%)
















Sample
C8:0
C10:0
C12:0
C14:0
16:0
18:0
18:1
18:2
18:3



















Wild-type1
—
7.1
6.3
5.9
19.2
28.6
23.1
5.6
2.1


WT-B1
—
7.1
6.8
7.5
19.5
26.9
22.3
5.8
2.5


WT-F1
—
6.9
7.1
7.8
18.5
27.1
21.8
6.4
2.4


WT-B/F1
—
7
13.5
6.8
16.5
23.7
19.5
5.4
3.2


Wild-type2
—
6.5
8.1
5.8
21.2
26.5
22.1
5.1
3.2


WT-B2
—
7.6
10.25
8.1
17.5
23.5
21.5
6.1
3.7


WT-F2
—
7.2
15.2
7.8
15.6
20.1
20.3
7.1
2.4


WT-B/F2
—
6.9
24.6
6.8
13.5
18.5
19.2
5.7
3.3


Wild-type3
—
6.6
10.2
5.9
20.5
25.6
21.1
4.9
3.9


WT-B3
—
6.3
16.5
8.9
16.2
20.5
21.5
5.6
3.7


WT-F3
—
7.5
21.5
7.5
13.4
17.6
20.7
6.7
3.2


WT-B/F3
—
7.1
29.5
5.9
10.2
15.9
20.3
7.1
3.3





WT-B: expresses BTE,


WT-F: expresses FatB3,


WT-B/F: expresses BTE and FatB3


1NL medium;


2BMGY medium at day 3;


3BMGY medium at day 7






The strains were cultured in YPD medium for one day, inoculated into 250 ml of NL or BMGY medium (2% Peptone, 1% yeast extract, 100 mM potassium phosphate pH 6.0, 1.34% yeast nitrogen base (w/o amino acids), 0.4 μg/mL biotin, and 1% glycerol), and then cultured in a shaker bottle for 7 days. Free fatty acid production was then measured. See Table 3.
We also constructed strain ω5 (deposited at the Bioresource Collection and Research Center in Taiwan on Dec. 10, 2014 as BCRC 920096) by introducing the BTE and FatB3 genes into strain ω4. Strain ω5 was cultured in YNB medium at an initial pH of 6.18, and then cultured in a fermenter for 6 days without controlling the pH. Dicarboxylic acid production was then measured. See Table 4.













TABLE 4







Residual
C10DCA
C12DCA
C14DCA
C16DCA


Day
pH
glu (g/L)
(g/L)
(g/L)
(g/L)
(g/L)





















D2
6.16
94.7
0.50
—
0.86
—


D3
3.17
11.8
0.30
—
0.73
—


D4
3.71
0
0.36
0.31
0.68
—


D5
3.75
0
0.31
0.27
0.51
—


D6
3.38
0
0.64
0.53
0.99
—









In order to decrease DCA12 degradation, we constructed strain ω6 (ω5::Δ palmitoyl ACP-thioesterase; deposited at the Bioresource Collection and Research Center in Taiwan on Dec. 10, 2014 as BCRC 920097) using RNA interference. See FIG. 7.













TABLE 5







Residual
C10DCA
C12DCA
C14DCA
C16DCA


Day
pH
glu (g/L)
(g/L)
(g/L)
(g/L)
(g/L)





















D2
4.24
58
1.90
—
—
—


D3
3.35
0.2
0.50
—
—
—


D4
2.85
0
0.52
—
—
—


D5
3.85
0
0.71
0.58
—
—









Strain ω6 was cultured in YNB medium at an initial pH of 6.18, and then cultured in a shaker bottle for 5 days without controlling the pH. Dicarboxylic acid production was measured. See Table 5.
Strains ω5 and ω6 were cultured in YPD medium for one day, and then inoculated into 250 ml of YNB medium (2% glucose) in a shaker bottle. The pH was maintained at 6.0 for two days. Additional 2% glucose was then added every 6 hours to maintain the pH at 7.5 for 5 days. For strain ω5, production of DCA12 increased from 12.9% to 51.2% (1.23 g/L) as compared to strain ω4. For strain ω6, DCA12 production was increased to 59.8% (2.35 g/L). See FIG. 8 and Table 6.













TABLE 6







Strain
C10DCA
C12DCA
C14DCA
C16DCA






















ω5
9.2
51.2
23.7
15.9



ω6
11.1
59.8
10.9
18.2










We constructed strain ω7 (ω6::AccD::FASA-1; deposited at the Bioresource Collection and Research Center in Taiwan on Dec. 10, 2014 as BCRC 920098). Strain ω7 was cultured in YNB medium for 6 days without controlling the pH. Dicarboxylic acid production was measured. See Table 7.













TABLE 7







Residual
C10DCA
C12DCA
C14DCA
C16DCA


Day
pH
glu (g/L)
(g/L)
(g/L)
(g/L)
(g/L)





















D2
4.24
93
1.69
—
0.29
0.27


D3
3.35
21
0.15
—
0.41
0.73


D4
2.85
0
0.49
0.46
0.62
0.48


D5
3.85
0
0.53
0.47
0.62
0.48


D6
5.47
0
0.52
0.58
0.62
0.46









Strain ω7 was cultured in YNB medium in a fermentor under 1 vvm aeration and at 300 rpm. The pH was maintained at 6.0 for the first two days, and then every 6 hours, additional 2% glucose was added to maintain the pH at 6.0 for additional 5 days. Dicarboxylic acid production was measured. See Table 8.












TABLE 8






C10DCA
C12DCA
C14DCA
C16DCA
C18DCA


Day
(g/L)
(g/L)
(g/L)
(g/L)
(g/L)







D 0
0.24
0.21
0.15
0.17
0.10


D 1
0.45
0.38
0.18
0.26
0.18


D 2
0.77
0.45
0.27
0.35
0.16


D 3
0.53
0.72
0.32
0.39
0.17


D 4
0.48
1.11
0.41
0.43
0.15


D 5
0.32
1.19
0.49
0.45
0.21









Strain ω7 was cultured in YNB medium in a fermentor under 1 vvm aeration and at 300 rpm. The pH was maintained at 6.0 for the first two days, and then every 6 hours, additional 2% glucose was added to maintain the pH at 7.5 for additional 5 days. Dicarboxylic acid production was measured. See Table 9.












TABLE 9






C10DCA
C12DCA
C14DCA
C16DCA
C18DCA


Day
(g/L)
(g/L)
(g/L)
(g/L)
(g/L)







D 0
0.27
0.26
0.12
0.18
0.14


D 1
0.30
0.49
0.23
0.15
0.12


D 2
0.76
1.01
0.53
0.14
0.22


D 3
0.84
1.51
0.74
0.21
0.32


D 4
0.67
2.23
1.26
0.27
0.34


D 5
0.60
2.78
1.47
0.43
0.41









Modified E. coli Strains

Modified E. coli strains were constructed using expression vectors to express certain proteins. To eliminate the β-oxidation activity of the strains, the fadD gene was deleted. The ΔfadD strain was used as the host strain to construct strains E1, E2, E3, E4, E5, and E6.
We constructed strain E1 (ΔfadD::BTEΔNC::FatB3::CPR::FAO::FADH). The BTEΔNC and FatB3 genes were inserted into the Acc65I/SalI and HindIII/BamHI sites in the pHS vector, respectively. The CPR, FAO, and FADH genes were inserted into the BamHI/EcoRI, SalI/HindIII, and XhoI sites in the pHR vector, respectively. The resulting expression constructs (pHS-B+F and pHR-CFF) were introduced into host E. coli cells to generate strain E1. See FIG. 9.
Strain E1 was cultured in YNB or LB medium in a fermentor under 1 vvm aeration and at 300 rpm. The pH was maintained at 6.0 for 1 day. Every 6 hours thereafter, additional 1% glucose was added to maintain the pH at 7.5 for two days. Dicarboxylic acid production was measured. See FIG. 10. As shown in FIG. 10, production reached 0.2 g.
We also constructed strains E2 (ΔfadD::BTEΔNC::CPR::FAO::FADH) and E3 (ΔfadD::BTEΔNC::AceCS::CPR::FAO::FADH). See FIG. 11. As shown in FIG. 11, construct pHS-B was generated by inserting the BTEΔNC gene into the pHS vector at the Acc65I/BamHI sites. Construct pHS-B+AceCS was generated by inserting the BTEΔNC and AceCS genes into the Acc65I/SalI and HindIII/BamHI sites of the pHS vector, respectively. pHS-B and pHR-CRR were introduced into host E. coli cells to generate strain E2 and pHS-B+AceCS and pHR-CFF were introduced into host E. coli cells to generate strain E3.
Strains E2 and E3 were cultured in LB medium in a fermentor under 1 vvm aeration and at 300 rpm. The pH was maintained at 6.0 for 1 day. Every 6 hours thereafter, additional 1% glucose was added to maintain the pH at 7.5 for two days. Acetate production was measured. As shown in FIG. 12, strain E3 produced less acetate than strain E2.
Strain E4 (ΔfadD::ACC::BTEΔNC:: FatB3::CPR::FAO::FADH) was constructed. See FIG. 13. Construct pHSR-ACC was generated by inserting the AccA, AccBC, and AccD genes into the NdeI/SpeI, SpeI/EagI, and EagI/XhoI sites of the pHSR vector, respectively. Constructs pHSR-ACC, pHS-B+F, and pHR-CFF were introduced into host E. coli cells to generate strain E4.
Strain E2 and E4 were cultured in LB medium in a fermentor under 1 vvm aeration and at 300 rpm. The pH was maintained at 6.0 for 1 day. Every 6 hours thereafter, additional 1% glucose was added to maintain the pH at 7.5 for two days. Dicarboxylic acid production was measured. As shown in FIG. 14, C12DCA production reached 0.36 g/L.
Strain E5 (ΔfadD::Δacs::CPR::FAO::FADH) was constructed. See FIG. 15. As shown in FIG. 15, a cassette (for knocking out the acs gene) including FRT sites flanked by homology arms were created using PCR using acs forward primer (SEQ ID NO:50) and reverse primer (SEQ ID NO:51). E. coli cells were transformed with pRedET followed by induction and transformation with the linear cassette. The linear cassette was then inserted into the target locus. Construct pHR-CFF was introduced into the Δacs strain.
Strain E6 (ΔfadD::ΔgltA::CPR::FAO::FADH) was constructed using the same method. See FIG. 16. The functional cassette was generated using PCR using gltA forward primer (SEQ ID NO:52) and gltA reverse primer (SEQ ID NO:53). Construct pHR-CFF was introduced into the ΔgltA strain.
Strains E5 and E6 were cultured in LB medium in a fermentor under 1 vvm aeration and at 300 rpm. The pH was maintained at 6.5 for 1 day. Every 6 hours thereafter, additional 1% glucose was added to maintain the pH at 7.5 for two days. Dicarboxylic acid production was measured. As shown in FIG. 17, C12DCA production reached 0.02 g/L and 0.05 g/L.
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the described embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims and their equivalents

## Claims

1. A genetically modified microorganism, comprising a first nucleic acid encoding an Umbellularia californica lauroyl acyl carrier protein (ACP) thioesterase (BTE) operably linked to a promoter, a second nucleic acid encoding a Cocos nucifera lauroyl ACP-thioesterase (FatB3) operably linked to a promoter, and one or more exogenous nucleic acids each operably linked to a promoter, each exogenous nucleic acid encoding a protein selected from the group consisting of an acetyl-CoA carboxylase (ACC), an acetyl-CoA carboxylase carboxyl transferase subunit a (AccA), an acetyl-CoA carboxylase biotin carboxyl carrier protein (AccB), an acetyl-CoA biotin carboxylase (AccC), an acetyl-CoA carboxylase transferase subunit β (AccD), a fatty acid synthase (FAS) subunit, a cytochrome P450 reductase (CPR), a long-chain alcohol oxidase (FAQ1), a long-chain alcohol dehydrogenase (FADH), and an adenosine monophosphate-forming acetyl-coenzyme A synthetase (AceCS), wherein the microorganism is Escherichia coli and produces an increased amount of long-chain dicarboxylic acids as compared to the unmodified parent of the microorganism, wherein the genetically modified microorganism further comprising a loss-of-function mutation in or expressing a lower level of one or more genes selected from the group consisting of a palmitoyl-acyl carrier protein (ACP) thioesterase gene, an acyl-coenzyme A oxidase gene, a citric synthetase (gltA) gene, or an acyl-coenzyme A synthetase (acs) gene.

2. A genetically modified microorganism comprising a first nucleic acid encoding an Umbellularia californica lauroyl ACP thioesterase (BTE) operably linked to a promoter, and a second nucleic acid encoding a Cocos nucifera lauroyl ACP thioesterase (FatB3) operably linked to a promoter, and further contains a loss-of-function mutation in or expresses a lower level of one or more genes selected from the group consisting of a palmitoyl-ACP thioesterase gene, an acyl-coenzyme A oxidase gene, a citric synthetase (gltA) gene, or an acyl-coenzyme A synthetase (acs) gene, wherein the microorganism is Yarrowia lipolytica and produces an increased amount of long-chain dicarboxylic acids as compared to the unmodified parent of the microorganism, wherein the microorganism (1) contains a loss-of-function mutation in or expresses a lower level of an acyl-coenzyme A oxidase gene and (2) further contains three exogenous nucleic acids each operably linked to a promoter and encoding a CPR, a FAO1, and a FADH, respectively.

3. The genetically modified microorganism of claim 2, wherein the microorganism contains a loss-of-function mutation in or expresses a lower level of an ACP thioesterase gene.

4. The genetically modified microorganism of claim 3, wherein the microorganism contains additional exogenous nucleic acids each operably linked to a promoter and encoding an AccD and a FAS subunit, respectively.

5. The genetically modified microorganism of claim 1, wherein the one or more exogenous nucleic acids include three nucleic acids each encoding a CPR, a FAO1, and a FADH, respectively.

6. The genetically modified microorganism of claim 5, wherein the one or more exogenous nucleic acids further include nucleic acids each encoding an AccA, an AccB, and an AccD.

7. The genetically modified microorganism of claim 5, further comprising a loss-of-function mutation in or expressing a lower level of an acs gene or a gltA gene.

8. The genetically modified microorganism of claim 5, wherein the BTE is BTEΔNC containing the sequence of SEQ ID NO:22 and the acyl-coenzyme A oxidase gene is fadD.

9. The genetically modified microorganism of claim 6, wherein the BTE is BTEΔNC containing the sequence of SEQ ID NO:22 and the acyl-coenzyme A oxidase gene is fadD.

10. The genetically modified organism of claim 2, wherein the acyl-coenzyme A oxidase gene is pox2 or pox5.

11. The genetically modified organism of claim 3, wherein the acyl-coenzyme A oxidase gene is pox2 or pox5.

12. The genetically modified organism of claim 4, wherein the acyl-coenzyme A oxidase gene is pox2 or pox5.

13. A method of producing a long-chain dicarboxylic acid, the method comprising:
providing the genetically-modified microorganism of claim 1; and
culturing the microorganism in a culture medium containing glucose or glycerol at pH 6 to 8 under conditions that allow production of a long-chain dicarboxylic acid; whereby the microorganism produces the long-chain dicarboxylic acid.

14. The method of claim 13, further comprising collecting the long-chain dicarboxylic acid.

15. The method of claim 14, wherein the long-chain dicarboxylic acid is C10-C18 dicarboxylic acid.

16. The method of claim 15, wherein the long-chain dicarboxylic acid is C12 dicarboxylic acid.