PROCESS OF OMEGA-HYDROXYL FATTY ACID PRODUCTION

Abstract

Provided is a method of producing at least one omega-hydroxyl fatty acid, the method comprising: (a) contacting at least one alkane with at least one recombinant yeast cell in an aqueous medium, wherein the yeast cell is capable of oxidising the alkane to the corresponding omega-hydroxyl fatty acid and the yeast cell comprises a reduced fatty acid degradation capacity.

Claims

1: A method of producing at least one omega-hydroxyl fatty acid, the method comprising: contacting at least one alkane with at least one yeast cell in an aqueous medium, wherein the at least one yeast cell is capable of oxidizing the at least one alkane to a corresponding omega-hydroxyl fatty acid and the at least one yeast cell comprises a reduced fatty acid degradation capacity; the reduced fatty acid degradation capacity is a result of: (i) a decrease in expression relative to a wild type cell of at least one enzyme involved in a beta-oxidation pathway; and/or (ii) at least one loss-of-function mutation in at least one enzyme involved in the beta-oxidation pathway; and the aqueous medium comprises acetate as a co-substrate.

2: The method according to claim 1, wherein an acetate concentration is at least 20 mmol/L.

3: The method according to claim 1, wherein an acetate concentration is maintained between 80-800 mmol/L.

4: The method according to claim 1, wherein an acetate concentration is maintained between 160-500 mmol/L.

5: The method according to claim 1, wherein the aqueous medium further comprises a non-detectable concentration of glucose.

6: The method according to claim 1, wherein the acetate is selected from the group consisting of NaAc and HAc.

7: The method according to claim 1, wherein the at least one alkane comprises at least 6 carbon atoms.

8: The method according to claim 1, wherein the at least one alkane is selected from the group consisting of C.sub.7-C.sub.22 alkanes.

9: The method according to claim 1, wherein the at least one alkane is selected from the group consisting of C.sub.10-C.sub.12 alkanes.

10: The method according to claim 1, wherein the at least one yeast cell is selected from the group consisting of Candida, Yarrowia, Pichia, Torulopsis, Rhodotorula, and Wickerhamiella.

11: The method according to claim 1, wherein the at least one yeast cell is selected from the group consisting of Candida tropicalis and Yarrowia lipolytica.

12: The method according to claim 1, wherein the reduced fatty acid degradation capacity is a result of a decrease in expression relative to the wild type cell of the at least one enzyme selected from the group consisting of acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase.

13: A yeast cell, wherein the yeast cell is capable of oxidizing at least one alkane to a corresponding omega-hydroxyl fatty acid and the yeast cell comprises a reduced fatty acid degradation capacity; the reduced fatty acid degradation capacity is a result of: (i) a decrease in expression relative to an wild type cell of at least one enzyme involved in a beta-oxidation pathway; and/or (ii) at least one loss-of-function mutation in at least one enzyme involved in the beta-oxidation pathway; and the aqueous medium comprises acetate as a co-substrate.

14: The yeast cell according to claim 13, wherein an acetate concentration is maintained between 20-800 mmol/L.

15: The yeast cell according to claim 13, wherein the at least one alkane is selected from the group consisting of C.sub.7-C.sub.22 alkanes.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0074] FIG. 1 is a graph of the concentrations of 10-hydroxydecanoic acid and decanedioic acid over time when fed with glucose according to Comparative Example 1.

[0075] FIG. 2 is a graph of the concentrations of 10-hydroxydecanoic acid and decanedioic acid over time when fed with NaAc and HAc according to Example 1.

[0076] FIG. 3 is a graph of the concentrations of -hydroxyl fatty acids and dicarboxylic acids using different alkane substrates according to Example 2.

[0077] FIG. 4 is a graph of the concentrations of 10-hydroxydecanoic acid and decanedioic acid over time when fed with NaAc according to Example 3.

[0078] FIG. 5 is a graph of the concentrations of 10-hydroxydecanoic acid and decanedioic acid over time when fed with glucose according to Comparative Example 2.

EXAMPLES

[0079] The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Methods and Materials

[0080] In the examples, the identification of fermentation products was conducted by using GC-MS and the amount of the fermentation products were determined via Gas Chromatography-Flame Ionization Detection (GC-FID).

[0081] The samples of fermentation products were treated by TMS (trimethyl-silylation) derivatization before injection for GC-FID analysis.

[0082] The detailed procedure is as follows: sample was dissolved in ethyl acetate or acetonitrile (if necessary), then the solution was mixed with derivatization reagent (N,O-bis(trimethylsilyl)trifluoroacetamide, BSTFA) by 1:1 (v/v) in the 2 mL vial (80 uL: 80 uL in the 300 uL insert vial). Afterwards, the mixtures were heated to 80 C. in the oven for 30 min before injection to GC-FID analysis.

Instrument: Agilent 7890B;

[0083] Inlet conditions: Temperature: 260 C., Split ratio: 20:1; Injection Volume: 1 uL;
Column: Agilent 19091J-413L, HP5, 30 m320 um0.25 um;

Carrier gas: Helium;

[0084] Column flow: constant, 1 mL/min;
Oven Program: 60 C. (hold 1 min), 10 C./min to 300 C. (hold 5 min);
FID detector: [0085] Temperature: 280 C.; [0086] Air flow: 300 mL/min; [0087] H.sub.2 Fuel flow: 30 mL/min; [0088] Makeup flow: 25 mL/min.

Example 1

Seed Culture Phase:

[0089] Colonies on agar plate of Candida tropicalis ATCC20962 strain were precultured in 100 mL seed medium and shaken at 200 rpm, 30 C. for 20 h in 1 L baffled shaking flask.

[0090] Seed medium contains (per liter): glucose, 30 g; corn steep liquor (Sigma), 9 g; KH.sub.2PO.sub.4, 2 g; K.sub.2HPO.sub.4, 1 g; MgSO4, 1.0 g; CaCl.sub.2, 0.1 g; NaCl, 0.1 g; urea, 3.6 g; and 1 mL/L of a trace elements solution (per liter: H.sub.3BO.sub.3, 0.5 g; CuSO.sub.4.5H.sub.2O, 0.04 g; KI, 0.1 g; FeCl.sub.3.6H.sub.2O, 0.6 g; MnSO.sub.4.H.sub.2O, 0.4 g; Na.sub.2MoO.sub.4.2H.sub.2O, 0.2 g; ZnSO.sub.4.7H.sub.2O, 0.4 g).

Growth Phase:

[0091] The cells were inoculated at 10% (v/v) into 400 mL of growth medium in a 1.4 L DASGIP fermenter (DASGIP Parallel Bioreactor Systems for Microbial Research and Development, 1 L working volume). 30 g/L decane was added to induce the expression of related enzymes such as cytochrome P450 (He, F., (2005), Yeast, 22:481-491; Van Beillen, J. B., (2006), App and Env. Microbiology:59-65 and Kogure T. (2007), FEMS Microbiol Lett: 106-111). The culture was grown at 30 C. at an aeration rate of 1.0 wm for 30 hrs as growth phase. The pH was maintained at 5.80 by automatic addition of 4 mol/L NaOH or 5 mol/L HCl solutions. Dissolved oxygen was maintained at 25% saturation by agitation and O.sub.2-cascade control mode. After 6 h growth, glucose (600 g/L) was fed exponentially based on the equation below, wherein =0.05 h.sup.1 and Y.sub.X/S=0.35 g DCW (dry cell weight)/g glucose and m.sub.s=0.04 g/g DCW*h. C.sub.s0 is substrate concentration, C.sub.X0 is DCW at the beginning of the feeding.

[00001]$F_f\left(\mathrm{Feedrate}\right)=\left(\frac{}{Y_{X/S}}+m_S\right).Math.\frac{c_{X.Math..Math.0}.Math.V_0.Math.e^{.Math.t}}{c_{S.Math..Math.0}}$

[0092] Growth medium contains (per liter): glucose, 30 g; (NH.sub.4).sub.2SO.sub.4, 8 g; corn steep liquor (Sigma), 9 g; KH.sub.2PO.sub.4, 2 g; K.sub.2HPO.sub.4, 1 g; MgSO4, 1.0 g; CaCl.sub.2, 0.1 g; NaCl, 0.1 g; antifoam 204 (Sigma Lot #: MKBP4191V), 1 mL and 1 mL/L of a trace elements solution (per liter: H.sub.3BO.sub.3, 0.5 g; CuSO.sub.4.5H.sub.2O, 0.04 g; KI, 0.1 g; FeCl.sub.3.6H.sub.2O, 0.6 g; MnSO.sub.4.H.sub.2O, 0.4 g; Na.sub.2MoO.sub.4.2H.sub.2O, 0.2 g; ZnSO.sub.4.7H.sub.2O, 0.4 g).

Conversion Phase:

[0093] Conversion of decane started after 30 hrs of growth phase. pH was raised to 7.5 gradually in 2 hrs with 4 mol/L NaOH solution. Decane was fed in a rate of 0.4 mL/h and NaAc (300 g/L, pH7.5) was fed in a rate of 1.0-2.0 mL/h (the concentration of Ac.sup. was from 10 g/L to 30 g/L). Then pH was maintained at 7.5 by automatic addition of 75% (v/v) HAc. Conversion phase lasted for 130 hrs. Broth samples were taken at intervals to determine cell density, residual glucose, Ac.sup. concentrations and products concentrations.

[0094] The concentration of residual glucose was detected enzymatically (glucose oxidase) and the concentration was not detectable.

[0095] The concentration of acetic group (Ac.sup.) was quantified by H-NMR method which calculated samples against the internal standard (TMSP, 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt) with known concentration. The concentration of Ac was from 10 g/L to 30 g/L.

[0096] The concentrations of 10-hydroxydecanoic acid and decanedioic acid over time when fed with NaAc and HAc are shown in FIG. 2.

Sample Extraction:

[0097] 200 uL sample broth was acidified to pH 1-2 by adding 5M HCl, then 8-fold (1.6 mL) ethyl acetate (EA) was added. After shaking vigorously, EA phase was taken by centrifugation for 2 min under 25 C., 13000 rpm. Then 1 mL of EA phase was used for GC-FID analysis to determine the oxidized products (10-hydroxydecanoic acid and decanedioic acid) in broth.

Comparative Example 1

[0098] Seed culture phase and growth phase were performed as those in Example 1. In conversion phase, conversion of decane started after 30 hrs of growth phase. pH was raised to 7.5 gradually in 2 hrs with 4 mol/L NaOH solution. Decane was fed in a rate of 0.4 mL/h and glucose (600 g/L) was fed in a rate that kept the concentration of glucose at around 10 g/L. Then pH was maintained at 7.5 by automatic addition of 4 mol/L NaOH solution. Conversion phase lasted for 130 hrs. Broth samples were taken at intervals to determine cell density, residual glucose, and products concentrations.

[0099] As shown in FIG. 1, when glucose was fed in conversion phase according to comparative example 1, the main product was decanedioic acid, whose concentration was 13.88 g/L at 160 h, and nearly no 10-hydroxydecanoic acid was found (0.15 g/L). The selectivity of 10-hydroxydecanoic acid was 1.1%.

[0100] However, as shown in FIG. 2, when NaAc and HAc was fed in conversion phase according to Example 1, the dominant product was 10-hydroxydecanoic acid rather than decanedioic acid (7.56 g/L 10-hydroxydecanoic acid vs 0.96 g/L decanedioic acid at 160 h). The selectivity of 10-hydroxydecanoic acid was 88.7%.

Example 2

[0101] Seed culture phase and growth phase were performed as those in Example 1. Then the induced cells were collected by centrifugation for 4 min at 4500 rpm, 4 C. Further, the collected cells were washed by 10 mM PBS (phosphate buffered saline, pH7.5) to obtain resting cells.

[0102] An aliquot of the resting cells was suspended in medium containing NaAc (28 g/L) and alkane substrate (10 g/L) in 25 mL 10 mM PBS (pH7.5) to make a cell suspension of OD10. This cell suspension was used in conversion phase. Three alkanes: decane, undecane and dodecane, were tested as substrate, respectively. The conversion phase was conducted in 250 mL Schott baffled shaking flasks, in which air was replaced by pure oxygen, for 24 hrs under 30 C. and 200 rpm. The products in broth was extracted with equal amount of ethyl acetate after acidification. The quantification of concentration of each product was determined by GC-FID. After 24 hr of conversion, the residual NaAc concentration was about 2-3 g/L.

[0103] The results in FIG. 3 showed that for all these three substrates, the main products were all -hydroxyl fatty acids. The concentration of 10-hydroxydecanoic acid and decanedioic acid was 0.452 g/L and 0.039 g/L, respectively, and the selectivity of 10-hydroxydecanoic acid was 92.0%. The concentration of 11-hydroxyundecanoic acid and undecanedioic acid was 0.469 g/L and 0.098 g/L, respectively, and the selectivity of 11-hydroxyundecanoic acid was 82.7%. The concentration of 12-hydroxydodecanoic acid and dodecanedioic acid was 0.502 g/L and 0.204 g/L, respectively, and the selectivity of 12-hydroxydodecanoic acid was 71.1%.

Example 3

Seed Culture Phase:

[0104] Colonies on agar plate of Yarrowia lipolytica C122 strain with accession number CGMCC 14251 (deposited on 19 Jun. 2017 with China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China) were precultured in 50 mL seed medium and shaken at 200 rpm, 30 C. for 24 h in 500 mL baffled shaking flask.

[0105] Seed medium contains (per liter): glucose, 10 g; peptone, 20 g; yeast extract, 10 g; decane, 3 g.

[0106] Then the induced cells were collected by centrifugation for 4 min at 4500 rpm, 4 C. Further, the collected cells were washed by 10 mM PBS (phosphate buffered saline, pH7.5) to obtain resting cells.

[0107] An aliquot of the resting cells was suspended in medium containing NaAc (28 g/L) and alkane substrate (40 g/L) in 25 mL 10 mM PBS (pH7.5) to make a cell suspension of OD20. This cell suspension was used in conversion phase. Decane was tested as alkane substrate.

Conversion Phase:

[0108] The conversion phase was conducted in 250 mL Schott baffled shaking flasks, in which air was replaced by pure oxygen, for 24 hrs under 30 C. and 200 rpm. The products in broth were extracted with equal amount of ethyl acetate after acidification. The quantification of concentration of each product was determined by GC-FID.

[0109] FIG. 4 is a graph of the concentrations of 10-hydroxydecanoic acid and decanedioic acid over time when fed with NaAc according to Example 3. The results in FIG. 4 showed that the main product was w-hydroxyl fatty acid. The concentration of 10-hydroxydecanoic acid was 0.260 g/L, and the selectivity of 10-hydroxydecanoic acid was 58.3%.

Comparative Example 3

[0110] Seed culture phase and conversion phase were performed as those in Example 3, except that in conversion phase, NaAc was replaced by glucose (20 g/L).

[0111] The results in FIG. 5 showed that the main product was decanedioic acid. The concentration of decanedioic acid was 0.205 g/L, and the selectivity of 10-hydroxydecanoic acid was 4.0%.