Method for aerobically producing alanine or a compound produced using alanine

Abstract

A process for producing alanine, including culturing a cell under aerobic conditions in an aqueous phase in the presence of an inorganic nitrogen source, and then contacting the aqueous phase and the cell cultured in the aqueous phase with a hydrophobic organic phase. The cell is a prokaryotic or a lower eukaryotic cell and expresses a recombinant alanine dehydrogenase.

Claims

1. A process for producing alanine comprising: culturing a cell that expresses a recombinant alanine dehydrogenase under aerobic conditions in an aqueous phase in the presence of an inorganic nitrogen source; and then contacting the aqueous phase and the cell cultured in the aqueous phase with a hydrophobic organic phase, wherein the cell is a prokaryotic or a lower eukaryotic cell.

2. The process of claim 1, wherein the cell comprises a nucleic acid comprising a sequence encoding an alkL polypeptide having the sequence motif of DXWAPAXQ(V/A)GXR (SEQ ID NO: 2) where X is a proteinogenic amino acid.

3. The process of claim 2, wherein the alkL polypeptide is heterologous.

4. The process of claim 1, wherein the contacting is performed by adding the hydrophobic organic phase to the aqueous phase and stirring the hydrophobic organic phase and the aqueous phase.

5. The process of claim 4, wherein the hydrophobic organic phase comprises a hydrophobic fatty acid ester.

6. The process of claim 4, wherein the hydrophobic organic phase comprises at least one selected from the group consisting of lauric acid, oleic acid, erucic acid, and methyl esters thereof.

7. The process of claim 1, wherein the hydrophobic organic phase comprises a hydrophobic fatty acid ester.

8. The process of claim 7, wherein the hydrophobic organic phase further comprises a hydrophobic fatty acid.

9. The process of claim 8, wherein the hydrophobic organic phase comprises at least one hydrophobic solvent selected from the group consisting of an alkane, a cycloalkane, an aryl, a heteroaryl, a dialkyl ether, a fatty alcohol, a triglyceride and a halohydrocarbon, each of which may be unsubstituted, substituted, branched or unbranched.

10. The process of claim 8, wherein the hydrophobic fatty acid is an unsaturated fatty acid.

11. The process of claim 1, wherein the cell comprises a heterologous transaminase that recognizes alanine as a substrate.

12. The process of claim 11, wherein the cell further comprises, a monooxygenase which, alone or in sequence, catalyzes an oxidation of a fatty acid or a fatty acid ester to produce a corresponding ω-oxo-fatty acid or ω-oxo-fatty acid ester.

13. The process of claim 12, wherein the monooxygenase is heterologous.

14. The process of claim 1, wherein the cell is a bacterial cell.

15. The process of claim 1, wherein the contacting is performed for at least 60 minutes.

16. The process of claim 1, wherein the aqueous phase comprises less than 10 mM alanine.

17. The process of claim 1, wherein the hydrophobic organic phase amounts to at least 5 percent by volume of the total of the volumes of the aqueous and the hydrophobic organic phase.

18. The process of claim 1, wherein the alanine dehydrogenase is an alanine dehydrogenase from Bacillus subtilus database code NP_391071 or an enzyme having at least 70% sequence identity to the alanine dehydrogenase and essentially the same enzymatic activity of the alanine dehydrogenase.

19. The process of claim 1, wherein the culturing of the cell is carried out while introducing air comprising oxygen into the aqueous phase by aeration.

20. The process of claim 1, wherein inorganic nitrogen source comprises at least one selected from the group consisting of ammonium chloride, ammonium nitrate, ammonium sulphate, ammonium hydroxide, ammonium phosphate, and ammonium carbonate such that an ammonium concentration in the aqueous medium is from 0.05 to 5 g/L.

Description

(1) In addition to, or instead of, a monooxygenase, the cell which is suitable for the process according to the invention may also include an alcohol dehydrogenase. In a preferred embodiment, the expression “alcohol dehydrogenase” as used in the present context is understood as meaning an enzyme which oxidizes an aldehyde or ketone to the corresponding primary or secondary alcohol, respectively. Examples comprising the alcohol dehydrogenases from Ralstonia eutropha (ACB78191.1), Lactobacillus brevis (YP_795183.1), Lactobacillus kefiri (ACF95832.1), from equine liver, from Paracoccus pantotrophus (ACB78182.1) and Sphingobium yanoikuyae (EU427523.1), and their respective variants.

(2) FIG. 1 shows a comparison of the alanine production by four E. coli strains which differ in that two strains express alkL, in the presence and absence of organic phase as described in Example 1.

(3) The present invention is furthermore illustrated by the following figures and nonlimiting examples, from which further features, embodiments, aspects and advantages of the present invention may be seen.

EXAMPLE

Production of Alanine by E. coli Whole-cell Catalyst with or without AlkL Expression, Compared in the Presence and Absence of an Organic Phase

(4) The increased production of alanine under the specific conditions of the present invention has been studied on strains W3110 [alaDH_Bs] and W3110 [alaDH_Bs-TA-alkL] in a parallel fermentation system with 8 bioreactors from DASGIP. W3110 [alaDH_Bs] is a strain of E. coli W3110 which comprises a pJ294-based plasmid, originally from DNA2.0, with the gene of the Bacillus subtilis alanine dehydrogenase. W3110 [alaDH_Bs-TA-alkL] is a strain which contains a pJ281-based plasmid, originally also from DNA2.0, with the genes of the abovementioned alanine dehydrogenase and a transaminase, and a further pJ294-based plasmid with the gene of porin alkL (PCT/EP2011/053834, DE102011110945) described.

(5) 1 l reactors were used for the fermentation. The pH probes were calibrated by means of a two-point calibration with standard solutions of pH 4.0 and pH 7.0. The reactors were filled with 300 ml of drinking water and autoclaved for 20 min at 121° C. to ensure sterility. Thereafter, the pO.sub.2 probes were polarized overnight (at least for 6 h) in the DASGIP system. On the next morning, the water was removed in a clean bench and replaced by 300 ml of high-cell-density medium supplemented with 100 mg/l ampicillin. Thereafter, the pO.sub.2 probes were calibrated with a one-point calibration (stirrer: 400 rpm/gassing: 10 sl/h air), and the feed, modificator and inducer paths were cleaned by means of clean-in-place. To this end, the tubes were rinsed with 70% ethanol, then with 1 M NaOH, then with sterile fully-demineralized water and finally filled with the respective media.

(6) Starting from the respective cryo cultures, alanine-producing E. coli strains were first grown overnight in LB medium (25 ml in a 100 ml baffled flask) supplemented with 100 mg/l ampicillin at 37° C. and 200 rpm for approximately 18 h. Thereafter, in each case 2 ml of the cultures were inoculated into high-cell-density medium (glucose 15 g/l (30 ml/l of a separately autoclaved 500 g/l stock solution supplemented with 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl), (NH.sub.4).sub.2SO.sub.4 1.76 g/l, K.sub.2HPO.sub.4 19.08 g/l, KH.sub.2PO.sub.4 12.5 g/l, yeast extract 6.66 g/l, trisodium citrate dihydrate 2.24 g/l, ammonium iron citrate solution 17 ml/l of a separately autoclaved 1% stock solution, trace element solution 5 ml/l separately autoclaved stock solution (HCl (37%) 36.50 g/l, MnCl.sub.2*4H.sub.2O 1.91 g/l, ZnSO.sub.4*7H.sub.2O 1.87 g/l, ethylenediaminetetraacetic acid dihydrate 0.84 g/l, H.sub.3BO.sub.3 0.30 g/l. Na.sub.2MoO.sub.4*2H.sub.2O 0.25 g/l, CaCl.sub.2*2H.sub.2O 4.70 g/l, FeSO.sub.4*7H.sub.2O 17.80 g/l, CuCl.sub.2*2H.sub.2O 0.15 g/l)) (per strain 25 ml in a 100 ml baffled flask) supplemented with 100 mg/l ampicillin and incubated for a further 5.5 h at 37° C./200 rpm.

(7) The optical density of the cultures at 600 nm was determined. To inoculate the reactors with an optical density of 0.1, suitable amounts of the preculture were taken up in a 5 ml syringe under sterile conditions and the reactors were inoculated by means of a cannula through a septum covered with a layer of 70% ethanol.

(8) The following standard programme was used:

(9) TABLE-US-00001 DO control system pH control system Preset 0% Preset 0 ml/h P 0.1 P 5 Ti 300 s Ti 200 s Min 0% Min 0 ml/h Max 100%  Max 40 ml/h  XO2 N (Gas (Rotation) From To mixture) From To F (Gas flow) From To During the 0% 30% During the  0% 100% During the 15% 80% entire 400 rpm 1500 rpm entire 21%  21% entire 6 sl/h 72 sl/h process process process Script Firing of the trigger 31% DO (1/60 h) IPTG induction 2 h after feed start Feed trigger 50% DO Feed rate 3 [ml/h]

(10) The experiment being performed can be divided into two phases, namely the growing phase, during which the cells are to reach a certain optical density, and the subsequent alanine production phase, in which alanine is to be produced by enzymes formed during expression, after the organic phase consisting of 25% (w/w) methyl laurate and 75% (w/w) oleic acid has been added. Corresponding experiments without organic phase were used as controls. The pH values were adjusted unilaterally to pH 6.8, using ammonia (12.5%). During the growing and biotransformation phases, the dissolved oxygen (DO, dissolved oxygen) in the culture was regulated at 30% via the stirrer speed and the gassing rate.

(11) The fermentation was carried out as a feed batch, the feed start, 5 g/lh glucose feed (500 g/l glucose supplemented with 1% MgSO.sub.4*7H.sub.2O and 2.2% NH.sub.4Cl) having been triggered by a DO peak. At the time of the feed start, the temperature, too, was lowered from previously 37° C. to 30° C. The expression of alanine dehydrogenase (and of the other recombinantly introduced proteins) was induced 2 h after the feed start by the automatic addition of IPTG (final concentration 1 mM). Before the start of the alanine production which was induced by the organic phase, the optical density of the culture broths was determined.

(12) The production of alanine was started 14 h after the feed start. To this end, 150 ml of a mixture of methyl laurate and oleic acid (technical-grade purity, 90%) were added as a batch to the fermentation broth. To have sufficient ammonium ions available for the production of alanine, 5 ml of a 3 M ammonium sulphate solution were added to the fermentation broth half an hour before the organics were added. For sampling, 2 ml of fermentation broth were removed from the reactor and immediately mixed with the quenching solution (40% (v/v) ethanol; 0.8% (w/v) NaCl; −20° C.). Thereafter, the samples were centrifuged for 10 min at 0° C./5100 rpm. The supernatant was discarded and the cell pellet was resuspended in 2 ml of methanol (−20° C.). The alanine concentration within the cells was determined with the aid of these samples.

(13) Alanine was determined by means of HPLC/UV measurement following derivatization by means of ortho-phthalic aldehyde. The methanolic supernatant was measured. The most important chromatographic parameters are compiled in the table hereinbelow.

(14) TABLE-US-00002 Column Luna 5 u C 8, 100 A, 150 × 4.60 mm, Phenomenex HPLC system Agilent 1200 Solvent A 2.5 ml acetic acid (100%) per 1 l double-distilled water, pH adjustment with sodium hydroxide solution to pH 6.0 Solvent B Methanol Column temp. 40° C. Flow rate 1 ml/min Gradient 0.0-1 min: 30.0% B, 1.0-17.0 min: 90.0% B, 17-19.5 min: 90.0% B, 19.6-20.5 min: 30.0% B Detector DAD, 334 nm Derivatization/ Automatic derivatization by means of injector injection programme, 1 μl of sample is reacted with 9 μl of volume derivatizing reagent; composition of the derivatizing reagent: 10 g/l o-phthalic aldehyde dissolved in borate buffer (0.4 mol/l), with addition of mercaptoethanol (5 ml/l) and methanol (100 ml/l) Calibration External calibration, measurement range 50-2000 mg/l, 5-point calibration, calibration before and after the sample series, average over the two calibration series, quadratic regression

(15) Samples were taken from all reactors 15 minutes before the addition of the organics and 2 h, 3.5 h, 20.5 h and 22.5 h after the addition of the organics. The conversion rates for oxygen (OTR=oxygen transfer rate) and carbon (CTR=carbon dioxide transfer rate) were determined during the fermentation via the offgas analysis at the DASGIP systems. The fermentation was terminated 23 h after the beginning of the biotransformation. The stirrer, the gassing unit, the temperature control and the pH control were stopped, and the reactors were left to stand quietly for 5-10 minutes.

(16) Results:

(17) After the addition of the organic phase, both study cases revealed a pronounced increase in the alanine concentration in the used cells, with or without AlkL expression. In each of the comparative experiments without addition of organics, markedly lower alanine concentrations are measured, and these concentrations show no tendency to increase (see FIG. 1).