Biosynthesis of glucose-d-13C6 from Hansenula polymorpha and methanol-13C

Article abstract of DOI:10.1002/jlcr.1935

A simple and effective method for synthesis of glucose-d- ¹³C₆ by fermentation using the methylotrophic yeast Hansenula polymorpha with 99% abundance methanol-¹³C is described. Using methanol-¹³C as a sole source of carbon, H. polymorpha can accumulate large amounts of α,α-trehalose-¹³C ₁₂ under unfavourable growth conditions; the trehalose can then be hydrolysed to give glucose-d-¹³C₆ with 98.5% abundance ¹³C.

Full text of DOI:10.1002/jlcr.1935

Technical Note
Received 18 August 2011,
Accepted 26 August 2011
Published online in 4 October 2011 Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1935
Biosynthesis of glucose-D-13C6 from Hansenula
polymorpha and methanol-13C
*
Zhan-feng Liu, Zheng Ren, Da-gang Xu, and Liang-jun Li
A simple and effective method for synthesis of glucose-D-13C6 by fermentation using the methylotrophic yeast Hansenula
polymorpha with 99% abundance methanol-13C is described. Using methanol-13C as a sole source of carbon, H. polymorpha
13
can accumulate large amounts of α,α-trehalose-
C
under unfavourable growth conditions; the trehalose can then be
12
hydrolysed to give glucose-D-13C6 with 98.5% abundance 13C.
Keywords: glucose-D-13C6; biosynthesis; methanol-13C; trehalose-13C12
Introduction
Experimental
Isotope-labelled D-glucose has been produced commercially by
biosynthesis for many years and has especially been used for
studying the in vivo metabolism of glucose.1,2 The first labelled
forms of D-glucose to be produced by biosynthesis contained the
radioactive isotopes 11C and 14C. The pioneering work in this area
was accomplished by Lifton and Welch, who prepared glucose-11C
using Swiss chard leaves and 11C-labelled carbon dioxide through
photosynthesis.3 Ishiwata et al. have modified the Lifton and Welch
method to achieve automation of the whole procedure.4 The use
of broad bean or algae has also been reported.5,6 Enzymatic synth-
esis is an alternative approach that has been used for preparing
isotope-labelled forms of D-glucose; for example, Sturani prepared
3-14C-labelled D-glucose and 4-14C-labelled D-glucose by the action
of ribulose diphosphate carboxylase on ribulose-1,5-diphosphate
and 14C-labelled carbon dioxide to give 3-phosphoglyceric
acid-1-14C followed by its conversion to D-glucose by reversing
glycolytic enzyme reactions.7 A number of biosynthetic methods
have been used to prepare 13C-labelled forms of D-glucose. Li
et al. developed a method for preparing glucose-D-13C6 by growth
of microalgae in a photobiological reactor with 13C-labelled carbon
dioxide as the carbon source.8 Luthra et al. prepared 13C-labelled
D-glucose (60 atom %) by photosynthesis using algae and
13C-labelled carbonate (90 atom %) as carbon source.9 Baxter
et al. synthesized D-glucose-3, 4-13C2 (60 atom % 13C2) via
a,a-trehalose-13C that was produced from sodium acetate-1-13C
(98 atom %) by sporulating yeast.10 A drawback in many of the
published methods is that the label becomes diluted and those
that utilize photosynthesis require complex apparatus to avoid
dilution or loss of the labelled carbon dioxide, and it is very difficult
to reproduce for scaling up the process. Following work by Jones
and Bellion, who investigated the major metabolic pathways of
methanol utilisation in the methylotrophic yeast Hansenula poly-
morpha (also known as Pichia angusta) by 13C NMR, 11 we recently
General
Methanol-13C (99 atom %) was prepared at the Shanghai Engi-
neering Research Center of Stable Isotope. H. polymorpha
(ATCC26012) was purchased from ATCC (Manassas, USA). The
labelled glucose and trehalose were analysed using a Waters
Quattro Premier XE VAB430 LCMS/MS (Waters Corporation,
Milford, MA, USA) with a Pinnacle II amino column and refractive
index detector. The mobile phase was a 30:70 v/v mixture of
water and acetonitrile at a flow rate of 1 ml/min. The 13C abun-
dance was measured using MAT 271 mass spectrometer (Pacific
Northwest National Laboratory, Richland, WA, USA). Fourier
Transform InfraRed spectra were obtained using a Nicolet 6700
instrument (Thermo Scientific, Rochester, NY, USA). NMR spectra
were recorded on a Bruker Avance 500 (Bruker BioSpin Corporation,
Billerica, MA, USA) instrument operating at 500 MHz for 1H NMR and
at 125 MHz for 13C NMR.
Fermentation conditions for 13C-labelling
Hansenula polymorpha was inoculated into the seed medium con-
taining (per litre): methanol-13C (10ml), (NH4)2SO4 (10 g), yeast
nitrogen base without amino acids and ammonium sulphate
(20 g) and biotin (0.4 mg) in 1 M K2HPO4-KH2PO4 buffer (pH 6.0).
The cultures were grown at 37 ꢀC and 150 rpm for 18 h, then 5 ml
was inoculated into 50 ml of the fermentation medium containing
(per litre): methanol-13C (10ml), H3PO4 (30 ml), CaSO4 (0.06 g),
K2SO4 (0.96 g), MgSO4 (3.26 g) and KOH (0.03 g) and trace elements
(2 ml) in deionized water with the pH adjusted to 5.0. The fer-
mentation was performed at 37 ꢀC and 150rpm for 24h; the
temperature was then raised to 42 ꢀC and held constant for 3 h.
Shanghai Research Institute of Chemical Industry, Shanghai Engineering Research
reported how fermentation with this organism using 13C-labelled Center of Stable Isotope, No.345 Yunling East Road, 200062 Shanghai, China
methanol as carbon source can be used to produce 13C- labelled
*Correspondence to: Zhan-feng Liu, Shanghai Research Institute of Chemical
a,a-trehalose.12 In the current work, we describe the full method
Industry, Shanghai Engineering Research Center of Stable Isotope, No.345 Yunling
East Road, 200062 Shanghai, China.
for preparing and isolating a,a-trehalose-13C12 followed by its che-
mical hydrolysis to give glucose-D-13C6 with 98.5% abundance 13C. E-mail: lzfyhy@163.com
J. Label Compd. Radiopharm 2012, 55 44–47
Copyright © 2011 John Wiley & Sons, Ltd.

Z.-F. Liu et al.
Isolation of trehalose-13C12
H2a, b; H3a, b; H4a, b; H5a, b; H6a, b). 13C NMR (125 MHz, D2O)
d (ppm): 95.4 (d, J = 46 Hz, C1b), 91.6 (d, J = 40 Hz, C1a), 75.4
Cells were harvested from 100 ml broth by centrifugation (m, C5b), 75.3 (m, C3b), 73.6 (ddd, J=45Hz, 39Hz and 2.5Hz, C2b),
(4000 Â g, 20 min, 0 ꢀC), washed twice with distilled water 72.3 (dd, J=38Hz and 38Hz, C3a), 70.9 (m, C2a, C5a), 69.1 (m, C4a,
(20 ml) and extracted with 40% (v/v) ethanol (20 ml). After C4b), 60.3 (d, J=43Hz, C6b), 60.0 (d, J=43Hz, C6a).
30 min at 90 ꢀC, the material was centrifuged (4000 Â g, 20 min,
0 ꢀC), 10% ZnSO4 (1 ml) and saturated BaOH solution (2 ml) was
Results and discussion
added to the supernatant liquor, protein was removed by centri-
fugation (4000 Â g, 20 min, 0 ꢀC), the soluble fraction being
The methylotrophic yeast H. polymorpha uses methanol as a sole
source of carbon and energy; a,a-trehalose can be accumulated at
high levels when the cells are exposed to unfavourable growth
conditions such as high temperature, high osmolarity and nutrient
limitations.15 The work by Jones and Bellion11 showed that dilu-
tion of 13C-labelled a,a-trehalose is obtained when the cells are
cultivated in fermentation medium with methanol-13C as the
source of carbon, and our recent work has shown how this can
be used to synthesize and isolate almost no dilution of 13C-
labelled a,a-trehalose on a hundreds of milligrammes scale when
the cells are cultivated in the seed and fermentation medium with
methanol-13C as the sole source of carbon. 12 In natural substrates,
a,a-trehalose is hydrolysed by the enzyme trehalase to give
equimolar quantities of a-D-glucose and b-D-glucose;16 we there-
fore developed a method for producing 13C-labelled D-glucose
adjusted to pH 6.0 with 1 M NaOH and passed through a mixed
column of D201 and D101 resin (2:1) eluted with water. The elu-
ate was concentrated under vacuum on a rotary evaporator, and
the residue was crystallised from aqueous alcohol affording the
dihydrate of a,a-trehalose-13C12 (85 mg, 0.22mmol). The conver-
sion ratio of the 13C atoms from the labelled methanol into tre-
halose is around 11%. The 13C atoms in the thalli and unreacted
methanol-13C were recycled; the recovery rate of the 13C atoms
was 75%. Mp 95–97 ꢀC (Lit. 97.0ꢀC);13 IR (KBr): 3416, 2933, 1637,
1458, 1355, 1241, 1149, 1100, 1030, 996, 955, 910, 811, 803, 537;
MS(m/z): 353, 185, 167, 131.
Synthesis of glucose-D-13C6
13C-enriched a,a-trehalose dihydrate (50 mg, 0.13mmol) was dis- by combining the biosynthesis of a,a-trehalose-13C12 from
solved in aqueous 2 M H2SO4 (2 ml) and heated in a sealed tube H. polymorpha and methanol-13C followed by its hydrolysis.
at 100 ꢀC for 6 h. The solution was neutralised with saturated
Scheme 1 shows the biosynthetic pathway to 13C-labelled
aqueous BaCO3 and filtered. The pH of the filtrate was adjusted a,a-trehalose from methanol-13C in H. polymorpha and its subse-
to 6.0 with 1 M NaOH and passed through a column of SK1B quent hydrolysis to glucose-D-13C6, which we chose to perform using
resin (H+), and the effluent was subsequently passed through a a chemical method. From the cultures of H. polymorpha grown with
column of WA30 resin (OH-) and eluted with water. The eluate methanol-13C as the sole carbon source, we isolated 85 mg of the
was concentrated under vacuum on a rotary evaporator afford- dihydrate of a,a-trehalose-13C12 from a cell volume of 100 ml.
ing glucose-D-13 C6 (42 mg, 0.23mmol, 89%) with 98.5% abun- Eighty-five milligrammes of the dihydrate of a,a-trehalose-13C12
13
dance C. Mp 149–152 ꢀC (Lit. 150–152 ꢀC 14); IR (KBr): 3415, needed 86 mg of the labelled methanol in theory. The conversion
2932, 1637, 1369, 1120, 1083, 1053, 1008, 894, 824, 627; MS ratio of the 13C atoms from the labelled methanol into trehalose is
1
(m/z): 185, 154, 123, 92; H NMR (500 MHz, D2O) d (ppm): 5.16 around 11%. We also recycled 13C atoms from the thalli and
(d, J = 170 Hz, H1a), 4.55 (d, J = 152 Hz, H1b), 4.0–3.0 (6H, m, unreacted methanol-13C with a recovery rate of 75%. Chemical
CH3OH
1
6,7
8
HCHO
HCOOH
CO2
CH2OH
CO
CH2OH
2
CHOH
CHOH
CH2OPO3
CO
CHOH
CHOH
CH2OPO3
xylulose 5-phosphate-13C5
2-
CH2OH
CO
CHO
2-
CHOH
CH2OH
CH2OH
(A)
ATP
(B)
3
CO2
ADP
CH2OH
2-
CHO
CH2OPO3
4
CO
CHOH
CO
HO
O
2-
2-
OH
6
OH
CH2OPO3
CH2OPO3
O
O
CHOH
CHOH
CHOH
6
5
(C)
(D)
5
OH
O
O
5
4
4
HO
HO
HO
+
HO
HO
HO
HO
OH
2O
2O
3
3
1
1
H
H
HO
HO
OH
HO
2-
CH2OPO3
Glucose-β
-D-13C6
Fructose 1,6-bisphosphate-13C6
α,α-trehalose-13C12
Glucose-
α
-D-13C6
Scheme 1. Biosynthetic route to a,a-trehalose-13C12 from methanol-13C in Hansenula polymorpha and its chemical hydrolysis to glucose-D-13C6. Bold asterisks indicate 13C-labelling
sites. Key to enzymes: 1, alcohol oxidase; 2, dihydroxyacetone synthase; 3, dihydroxyacetone kinase; 4, triose-phosphate isomerase; 5, aldolase; 6, formaldehyde dehydrogenase;
7, formylglutathione hydrolase; 8, formate dehydrogenase. Major metabolic routes depicted are: (A) re-arrangements via transaldolase and transketolase; (B) pentose phosphate
13
pathway; (C) a,a-trehalose-
C synthesis; (D) chemical hydrolysis.
12
J. Label Compd. Radiopharm 2012, 55 44–47
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

Z.-F. Liu et al.
Figure 1. 1H NMR spectrum of glucose-13C6.
hydrolysis of 50 mg a,a-trehalose-13C12 led to isolation of 42 mg have the expected splitting patterns for the uniformly 13C-labelled
glucose-D-13C6 with a yield of 89% and 98.5% abundance 13C. The compound. Figure 3 shows HPLC spectra of glucose-D-13C6. By
yield for conversion of 13C atoms from methanol-13C into glucose- combining the biosynthesis of a,a-trehalose-13C12 by H. polymorpha
1
D-13C6 is therefore 10%. H and 13C NMR spectra for the isolated with methanol-13C followed by its chemical hydrolysis using
glucose-D-13C6 are shown in Figures 1 and 2, respectively; both the methods described here, approximately 0.7g of highly pure
Figure 2. 13C NMR spectrum of glucose-13C6.
www.jlcr.org
Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 44–47

Z.-F. Liu et al.
Figure 3. HPLC spectrum of glucose-D-13C6.
glucose-D-13C6 with high abundance 13C is obtainable per litre of
cell culture.
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nology Commission (No. 08DZ2200200) for financial support.
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