Whole-Cell Biocatalysis in Seawater: New Halotolerant Yeast Strains for the Regio- and Stereoselectivity Reduction of 1-Phenylpropane-1,2-Dione in Saline-Rich Media

Article abstract of DOI:10.1002/cbic.202000023

The application of green chemistry concepts in catalysis has considerably increased in recent years, and the interest in using sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and Schwanniomyces for catalytic processes run in this medium. In the present work, we select other yeasts based on their halotolerance to widen the scope of this strategy. We consider them for the monoreduction of 1-phenylpropane-1,2-dione, a well-characterized reaction that produces acyloin intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this reaction is possible. The best ones were obtained for Saccharomyces cerevisiae FY86 and Kluyveromyces marxianus, for which acyloins with different stereochemistry were obtained with good to excellent enantiomeric excess.

Full text of DOI:10.1002/cbic.202000023

Accepted Article
Title: Comparison of the biocatalytic activity of some halotolerant
yeasts in freshwater and seawater
Authors: Cecilia Andreu and Marcel·lí del Olmo
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10.1002/cbic.202000023
ChemBioChem
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Comparison of the biocatalytic activity of some halotolerant
yeasts in freshwater and seawater
Cecilia Andreu*[a] and Marcel·lí del Olmo*[b]
Abstract: The application of Green Chemistry concepts in catalysis has considerably increased in recent years, and the interest in using
sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent
in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt
concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and
Schwannyomyces for catalytic processes run in this medium. In the present work we select other yeasts based on their halotolerance to
widen the scope of this strategy. We consider them for the monoreduction of 1-phenylpropane-1,2-dione, a well-characterized reaction that
produces acyloin intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this
reaction is possible. The best ones were obtained for S. cerevisiae FY86 and K. marxianus, for which acyloins with different stereochemistry
were obtained with good to excellent enantiomeric excess.
Introduction
biodegradable, and are employed under mild temperature,
At the beginning of the 1980´s, E.J. Ariëns established the
significance of stereochemistry in the therapeutic action of
drugs.[1] Since then, legislation on drug synthesis has been
more restrictive and has prompted the pharmaceutical industry
to manufacture enantiomerically pure compounds to both exploit
the practice of chiral change of already commercialized
racemates and develop enantiomerically pure de novo
compounds.[2] This, in turn, has stimulated the development of
cost-effective methods to enantiomerically manufacture pure
compounds, and a shift from the traditional process efficiency
concept has occurred to one that assigns economic value to
conserving energy and raw materials, eliminating waste, and
avoiding the use of toxic and hazardous chemicals.[3] Concepts
like environmental impact factor, atom and step economy, and
other Green Chemistry principles introduced by Anastas, are
now fundamental and must be considered in all organic
synthesis processes.[4] In this context, biocatalysis carried out by
either enzymes or whole cells, offers many advantages over
conventional chemical catalysis.[5] The good control that it
provides over chemo-, regio- and stereo-selectivity in reactions
of multifunctional molecules renders unnecessary the activation
pressure and pH conditions.[7] Biocatalytic reactions are also
performed in water. Many organic solvents are harmful, toxic,
and environmentally damaging, and sustainable solvents are a
topic of much interest for the chemical industry. Although a
range of greener or sustainable solvents, like ionic liquids,
supercritical fluids, liquid polymers, and others, have been
developed in the last few years,[8] water is the safest and the
most environmentally friendly solvent preferred by the
pharmaceutical industry for the synthesis of APIs thanks to its
low cost, and also because it is nontoxic, nonpolluting, and
nonflammable.[9]
However, the use of large quantities of water in industrial
processes does not benefit the environment and is another
important problem to investigate.[10] Seawater is an inexpensive
widely available resource that has been proposed as a bio-
sustainable alternative to using freshwater.[11-14] Oceans
constitute the most important reservoir of biodiversity in nature,
with microorganisms adapted to high salinity that can be used in
biotechnological applications to perform bioprocesses under
salinity conditions.[15,16] Recently, the ability of whole cells of
marine and other halotolerant yeasts to perform biocatalytic
or protection/deprotection steps often required in classic organic routes, processes in seawater has been described by us[17,18] and other
which results in shorter syntheses.[6] Besides, biocatalysts are
sustainable, renewable and
authors.[14,19,20] In our previous works, we used several
Debaryomyces and Schwanniomyces halotolerant yeast strains
for some biocatalytic processes using seawater as a solvent. We
demonstrated the capability of D. hansenii, D. hansenii var.
fabryi, S. etchellsii and S. polymorphus var. polymorphus to
grow in the presence of NaCl concentrations up to at least
7.5% (w/v). These microorganisms were also more resistant to
organic solvents/reagents in seawater than in freshwater, and
their metabolic activity was also greater in the first medium.
These facts indicate the possibility of using such strains as
biocatalysts in seawater where, in addition, yeasts cells
displayed better performance to be efficiently recycled.[17,18]
The present work extends the possibility of using seawater as a
friendly reaction medium to other yeast strains. As a benchmark
we chose the reduction reaction of 1-phenylpropane-1,2-dione 1,
an activated ketone that can be used as the starting material of
[a]
[b]
Dr. C. Andreu
Departament de Química Orgànica
Universitat de València (UVEG)
Vicent Andrés Estellés s.n., E-46100, Burjassot, València, Spain
E-mail: cecilia.andreu@uv.es
Dr. M. del Olmo
Departament de Bioquímica i Biologia Molecular
Universitat de València (UVEG)
Dr. Moliner 50, E-46100, Burjassot, València, Spain
E-mail: m.del.olmo@uv.es
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/cbic.202000023
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different intermediates of pharmaceutical interest.[21-24]
1
was observed under any tested salt conditions. S. cerevisiae
FY86 and K. marxianus showed greater resistance than the
contains two ketone groups that can be regio- and
stereoselectively reduced, and it is possible to obtain up to eight
different compounds (Scheme 1).
Pichia strains, while
T delbrueckii and, particularly, M.
guilliermondii were the most halotolerant ones. These last two
strains were capable of growing in the presence of NaCl up to a
concentration of at least 7.5% (w/v).
2-hydroxyketones or acyloins (2 and 3) have been traditionally
obtained via a carboligation of two different aldehydes in a
cross-benzoin reaction catalyzed by thiamine diphosphate
dependent enzymes.[25] In fact (R)-Phenylacetylcarbinol ((R)-
PAC, (-)3), a chiral intermediate precursor used to produce
pharmaceutical ephedrine, has been synthesized in this way
since 1921. At that time Neuberg and Hirsch reported the
stereoselective condensation between benzaldehyde and
pyruvate catalyzed by the thiamine-dependent enzyme Pyruvate
decarboxylase (PDC) of fermenting brewer’s yeast.[26]
Other more recently described biocatalytic routes to chiral
acyloins are, among many others, the enantioselective -
hydroxylation of ketones catalyzed by cytochrome P450
monooxygenases (CYPs)[27] or the stereoselective oxidation of
1,2-diols mediated by different microorganisms.[28, 29]
Optically pure 1,2-diols are also interesting building blocks in
asymmetric synthesis. In fact, 1-phenylpropane-1,2-diol has
been used in neurodegenerative diseases as well as an
intermediate for the synthesis of cardiovascular and anti-
inflammatory drugs,[30] and different routes toward this kind of
compounds have been employed.[31] However, this work focused
on the monoreduction of 1 using several halotolerant yeast
strains in freshwater and seawater to study the influence of the
reaction medium on the regio- and enantioselecivity of these
biocatalysts.
Figure 1. Growth of the non-Debaryomyces/Schwanniomyces strains herein
considered on the YPD-derived plates supplemented with NaCl concentrations
of 3.5%, 5%, or 7.5% (w/v), or without salt (YPD). 106 cells from the overnight
cultures in YPD were spread on plates, which were then incubated for 48 h at
30°C. This figure shows the result of a representative experiment.
Table 1. Differences in the capacity of several unconventional yeast to grow
on YPD plates containing NaCl.
Strain
0[a]
3.5[a]
++
5[a]
+
7.5[a]
FY86
+++
+++
+++
+++
+++
+++
-
-
-
P. fermentans
P. glucozyma
K. marxianus
M. guilliermondii
-
-
-
-
+++
+++
+++
++
+++
+++
++
T. delbrueckii
+++
Scheme 1. Model reaction for screening the biocatalytic ability of the yeasts
used in this work.
[a] NaCl (%)
Additional growth experiments with the four strains showing
halotolerant behavior were carried out in the liquid culture
media. For this purpose, overnight cultures prepared with
freshwater (YPD) or seawater (SW-YPD) were inoculated in the
corresponding fresh media and growth was monitored for 48 h
by measuring its OD600 (Figure 1S). The results were consistent
with those described before. Thus all the strains were capable of
growing in SW-YPD, although optical density was always lower
than in YPD for S. cerevisiae FY86 and K. marxianus. With the
most tolerant strains, namely M. guilliermondii and T.
delbrueckii, growth was similar or slightly higher in seawater
after 48 h.
Results
Growth of several yeast strains in the presence of salt
Encouraged by our previous results by using seawater as a
solvent in reactions biocatalyzed by Debaryomyces and
Schwanniomyces species,[17,18] we checked the ability of six
yeast strains (Saccharomyces cerevisiae FY86, Pichia
fermentans, Pichia glucozyma, Kluyveromyces marxianus,
Meyerozyma guilliermondii and Torulaspora delbrueckii) to grow
in the presence of NaCl, the main component of seawater.
Plates with the rich medium usually employed for yeast growth
(YPD, see the Experimental section), which contained salt
concentrations between 0 and 7.5% (w/v) were used for this
purpose. Figure 1 and Table 1 show the results found after 48 h
of incubation at 28°C. Two of these strains (P. fermentans and P.
glucozyma) were sensitive to the presence of salt and no growth
All these results make these strains interesting candidates for
catalytic studies in hypersaline media, such as seawater.
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10.1002/cbic.202000023
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Ability to catalyze the monoreduction of 1-phenylpropane-
1,2-dione (1) by halotolerant yeast strains in freshwater and
seawater medium at 30°C
phenyl-1-propanone) was the preferred regioisomer by S.
etchellsii, D. fabryi, S. cerevisiae FY86, D. hansenii and M.
guilliermondii. However, when S. polymorphus, K. marxianus
and T. delbrueckii were used, acyloin 3 (1-Hydroxy-1-phenyl-2-
propanone) was preferred. According to the rs value (ratio
between the majority and minority isomers, 2:3 or 3:2) shown on
the top of each series of bars in Figure 2, S. cerevisiae FY86
and S. polymorphus were the most regioselective strains when
considering both freshwater and seawater media, and each
displayed a different regio preference. Regioselectivity was
similar in both media for S. etchellsii, FY86 and T. delbrueckii,
but some differences in it were
The four found halotolerant strains were used to catalyze the
reduction of 1-phenylpropane-1,2-dione (1) at 30°C and for 2 h
using seawater and freshwater as reaction media in the
presence of glucose (2%, w/v). The Debaryomyces (D. fabryi
and D. hansenii) and Schwanniomyces (S. etchellsii and S.
polymorphus) species used by our group in previous studies
done in seawater[18] were also employed under these standard
conditions. The results shown in Figure 2 indicate that all the
strains were capable of catalyzing the reaction in both media
and of showing some regioselectivity. Acyloin 2 (2-Hydroxy-1-
Figure 2. Comparison of the results obtained in the reduction reaction of 1 biocatalyzed by several yeast strains in freshwater and seawater at 30°C for 2 h. The
ratio between the percentage of the majority and minority isomers, 2/3 or 3/2 (rs value), is shown over the bars. The average value in three replicates is shown.
Standard deviation was lower than 5% in all cases.
found for other strains. The results were better in the freshwater
medium for D. fabryi and M. guilliermondii, and in the seawater
one for D. hansenii, K. marxianus and S. polymorphus.
recovered. Differences between both media were less significant
in this study than in the previous one[18] because shorter
reaction times were used (2 hours) due to the high reactivity of 1.
Some particularly relevant situations were found in this sense for
the D. hansenii (compare entries 23-24), S. polymorphus
(entries 43-44) and T. delbrueckii (entries 49-50). Additional
reactions were performed at 50°C using K. marxianus, whose
thermotolerance is well known.[32] In this case, bigger differences
were found, like the above-mentioned ones (see entries 31 and
32). Conversely, although all the strains were capable of
catalyzing the reduction of 1 at 4°C, reactivity at this temperature
was lower and longer times were necessary. In this case,
conversion was also dependent on the medium, and it was
better in freshwater or seawater depending on the strain.
Regioselectivity
and
stereoselectivity
in
freshwater/seawater at different temperatures
A broader analysis was carried out using all these biocatalysts at
different temperatures in freshwater and seawater (Table 2 and
Figures 2S-3S). Our previous study found that halotolerant
species Debaryomyces and Schwanniomyces showed better
thermotolerance in the second medium.[18] This study confirms a
similar behavior for all the strains considered when reactions
were performed at 45°C. In overall, greater activity was
observed for these strains in seawater, and some amount of
dialcohols 4 and 5, and none of starting diketone 1, were
Regarding regioselectivity (Figure 2S and Table 2), we found
that S. cerevisiae FY86 (entries 13-18), and particularly S.
polymorphus (entries 39-44), were the most regioselective
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Table 2. Reduction of 1-phenylpropane-1,2-dione (1) by the halotolerant yeast strains considered in this work in freshwater and seawater medium at 4°C, 30°C
and 45°C
Strain
T(°C)/t (h)
Entry[a]
1-conv[b]
2-conv/ee[b]
3-conv/ee[b]
2 + 5 conv [b]
rs[c]
1 / FW
2 / SW
3 / FW
4 / SW
5 / FW
6 / SW
-
15
-
-
-
72 / 98-S
62 / 96-S
57 / 97-S
49 / 99-S
51 / 88-S
30 / 98- S
21 / 3-S
23 / 7-S
36 / 27-R
35 / 36-R
41 / 15-R
41 / 46-R
7
-
7
16
8
29
3.4
2.7
1.6
1.4
1.2
1.4
4 / 22
30 / 2
45 / 2
S. etchellsii
-
7 / FW
8 / SW
9 / FW
10 / SW
11 / FW
12 / SW
-
37
-
-
5
-
70/ 95-S
41 / 92-S
63 / 89-S
48 / 90-S
55 / 94-S
50 / 92- S
28 / 14-S
20 / 4-S
33 / 14-R
44 / 19-R
35 / 30-R
39 / 41-R
2
2
4
8
6
2.5
2
1.9
1.1
1.6
1.3
4 / 22
30 / 2
45 / 2
D. fabryi
11
13 / FW
14 / SW
15 / FW
16 / SW
17 / FW
18 / SW
5
1
12
9
4
-
65/ 99-S
61 / 99-S
57 / 99-S
60 / 99-S
61 / 99-S
63 / 99- S
29 / 82-R
36 / 91-R
29 / 76-R
29 / 85-R
32 / 73-R
24 / 76-R
1
2
2
2
3
2.2
1.7
1.9
2
1.9
2.6
4 / 22
30 / 2
45 / 2
FY 86
13
19 / FW
20 / SW
21 / FW
22 / SW
23 / FW
24 / SW
11
-
-
-
48
-
50/ 76-S
56 / 83-S
56/ 87-S
55 / 87-S
20 / 75-S
54 / 89- S
38 / 58-S
35 / 41-S
40 / 36-S
30 / 53-S
32 / 43-S
35 / 16-S
1
9
4
15
-
11
1.3
1.6
1.4
1.8
1.6
1.5
4 / 22
30 / 2
45 / 2
D. hansenii
25 / FW
26 / SW
27 / FW
28/ SW
29 / FW
30 / SW
31 / FW
32 / SW
-
-
3
-
-
-
40/ 98-S
36 / 98-S
43/ 97-S
31 / 96-S
62 / 87-S
47 / 99- S
35 / 94-S
27 / 96- S
52 / 58-S
59 / 80-S
49 / 25-S
62 / 65-S
33 / 19-S
37 / 77-S
26 / 13-S
34/ 77-S
8
5
5
7
5
16
-
38
1.3
1.6
1.1
2
1.9
1.3
1.4
1.3
4 / 22
30 / 2
45 / 2
50 / 2
K. marxianus
39
-
33 / FW
34 / SW
35 / FW
36/ SW
37 / FW
38/ SW
-
5
-
-
1
-
71/ 98-S
67 / 96-S
61/ 92-S
48 / 88-S
52 / 94-S
33 / 91- S
26 / 4-S
26 / 5-S
29 / 8-R
42 / 0
37 / 26-R
32 / 15-R
3
2
10
10
11
35
2.7
2.6
2.1
1.1
1.4
1
4 / 22
30 / 2
45 / 2
M. guilliermondii
39 / FW
40 / SW
41 / FW
42/ SW
43 / FW
44/ SW
-
19
-
-
49
-
17/ 64-S
14 / 28-S
27/ 75-S
24 / 55-S
17 / 56-S
29 / 65- S
78 / 24-R
63/ 25-S
67 / 25-R
70 / 4-R
34 / 0
5
4
6
6
-
4.6
4.5
2.5
2.9
2
4 / 22
30 / 2
45 / 2
S. polymorphus
64 / 5-S
7
2.2
45 / FW
46 / SW
47 / FW
48/ SW
49 / FW
50/ SW
-
-
-
-
20
-
44/ 99-S
55 / 99-S
45/ 97-S
46 / 97-S
33 / 98-S
43 / 98- S
40 / 80-R
39 / 74-R
52 / 53-R
51 / 58-R
47 / 35-R
50 / 33-R
16
6
3
3
-
1.1
1.4
1.2
1.1
1.4
1.2
4 / 22
30 / 2
45 / 2
T. delbrueckii
7
[a] Reaction medium: freshwater (FW) or seawater (SW) containing glucose (2%, w/v). [b] Conversion (%) (determined by 1HNMR)/enantiomeric excess (%)
(determined by chiral HPLC)-absolute configuration (determined by comparing tR with those described in the literature under identical conditions,[33,17] and by
measuring the optical rotation of the enantiomerically enriched purified samples).[34,35] [c] rs: regioselectivity determined as the ratio between the majority and
minority isomer (2/3 or 3/2)
biocatalysts when considering the three checked temperatures
etchellsii, D. fabryi, and M. guilliermondii), it was high only in the
on the whole (rs > 2 in almost all cases). For the other strains (S.
reactions carried out at 4°C.
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Among the most regioselective strains, enantioselectivity (see
Table 2 and Fig 3S) was bad in both regioisomers 2 and 3 in the
reactions biocatalyzed by S. polymorphus (entries 39-44). A
change in the stereopreference of 3 at 4°C depending on the
reaction medium was striking. The enantioselectivity for FY86
was excellent in 2 and good to excellent in 3 (entries 13-18). It is
worth mentioning that the reaction medium also influenced this
aspect, especially with monoalcohol 3, as stereoselectivity in
seawater was slightly better. In all cases, the preferred
enantiomer for acyloins 2 and 3 was that with an S and R
configuration respectively.
greener solvent, water, as the reaction medium.[7] However, the
low solubility of hydrophobic substrates in water entails using
large quantities of this liquid, which is another problem in
industrial processes, and an important one to analyze and
solve.[10] Seawater has been proposed as a bio-sustainable
alternative to using freshwater. [11-14,17,18,36] Seawater is a widely
available inexpensive resource; in fact, 71% of the Earth's
surface is covered by oceans. Recent reports have
demonstrated the possibility of using seawater as an
alternative to freshwater in biocatalytic processes. [14,17-20,36]
The stereoselectivity in obtaining acyloin 3 was generally poor in
the reactions biocatalyzed by S. etchelsii (entries 1-6), D. fabryi
(entries 7-12), M. guilliermondii (entries 33-38) and T.
delbrueckii (entries 45-51). The enantiomer with the
R
configuration was also the majority and, once again, the
influence of the reaction medium was notorious. So the two
former strains showed more enantioselectivity in seawater for
almost all the tested conditions, while the two last ones did so in
freshwater. Acyloin (S)-2 was always obtained with high
stereoselectivity. Finally, D. hansenii and K. marxianus were
more selective in acyloins
2 and 3, both having an S
configuration. The results obtained with the first strain were poor,
but with K. marxianus, good to excellent stereoselectivities were
found for both 3 and 2 respectively, especially when the
reactions were carried out in seawater.
Biocatalyst reuse in freshwater and seawater
In previous works in which the whole cells of halotolerant
yeasts were used as biocatalysts, we found that using
seawater in the medium reaction could help to cut the costs of
the process because it allowed yeast cells to be reutilized
18]
several times.[17,
Based on these results, in this case we
also explored the possibility of reusing cells in several cycles by
comparing both freshwater and seawater media. According to
the above-described results, and by especially considering
enantioselectivity to obtain the three chiral acyloins, we chose
strains S. cerevisiae FY86 and K. marxianus to explore this
possibility.
Figure 3 shows that both strains displayed different behaviors.
Reusing FY86 was more effective in freshwater than in seawater,
and better at 30°C than at 4°C. Cell viability determined by the
trypan blue exclusion assay after the third round (shown at the
top of the bar in Figure 3) was similar at 30°C, but considerably
higher at 4°C in the first medium. In contrast, it was possible to
more effectively recycle K. marxianus in seawater than in
freshwater, and also at 30°C than at 4°C. Cell viability was also
higher in the first medium at both temperatures. It is worth
mentioning that the stereoselectivity in both obtained acyloins
remained unchanged after the three rounds.
Figure 3. Conversion after the three rounds of recycling cells of S. cerevisiae
FY86 and K. marxianus of substrate 1 in freshwater (gray bars) and seawater
(blue bars). Reactions were carried out at 30°C and 4°C, and for 2 h in the first
case and for 24 h in the second one. Cell viability, as determined by the trypan
blue exclusion assay after the third cycle, is shown at the top of the bar. The
above figure shows the average value in three replicates. Standard
deviation was lower than 5% in all cases.
In this context, it is important to know the scope of strains and
reactions for which the use of seawater can represent an
interesting alternative to freshwater. In this work, we
demonstrate the capacity of several yeast strains to grow in
saline media. We used them to carry out the regio- and
stereoselective monoreduction of 1-phenylpropane-1,2-dione 1,
an interesting precursor of different intermediates of
pharmaceutical interest, by employing freshwater and seawater
as reaction media.[21-24]
The eight screened halotolerant yeast strains showed higher
thermotolerance when the employed medium was prepared in
seawater. This was shown by the bigger conversions in the
reactions run in this medium at 45°C (see Table 2). At lower
Discussion
Biocatalysis has proven to be an excellent alternative to other
catalytic processes that employ conventional chemicals, due to
not only its good control of chemo- regio- and
stereoselectivity[6] but also to its sustainability. Reactions are
carried out at mild temperatures and pressure using the
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10.1002/cbic.202000023
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temperatures however (4°C or 30°C), behavior depended on the
strain, and conversions were sometimes better in one medium
or in another.
the different activity of halotolerant yeast strains for organic
transformations in seawater under some conditions.
All the strains showed some regioselectivity for either acyloins 2
or 3, although it was not absolute in any case. The preferred Experimental Section
acyloin depended on the strain. It was also possible to note
some influence of the reaction medium nature, namely
freshwater or seawater (see Figure 2S). Stereoselectivity was
also determined by both the strain and the reaction medium
(Figure 3S). Acyloin 2 S with excellent enantioselectivity was
obtained in almost all cases. Conversely, the results were more
variable for acyloin 3, which could be obtained with absolute R
or S configuration. The enantiomeric excess in 3 was poor in
most cases, except in the reactions biocatalyzed by S.
cerevisiae FY 86 and K. marxianus. With these two strains,
stereoselectivity was better when reactions were carried out in
seawater at the lowest temperature (4°C), although long reaction
times were required. Variations in the medium pH were also
studied, but no improvement in the biocatalytic behavior of the
strains considered herein was observed (data not shown).
The possibility of reusing a catalyst is an important factor to take
into account to improve all chemical processes. For this reason,
we checked the possibility of reusing cells for several rounds by
employing the two most enantioselective strains for both
acyloins at the temperatures at which they were more
stereoselective (Figure 3). The reactions carried out in
freshwater and seawater as reaction media were compared.
Once again, we found different behaviors depending on the
strain. Actually, S. cerevisiae FY86 was more effectively
recycled in freshwater, while seawater provided the best results
in this sense for K. marxianus. For both strains, reuse at 30°C
was more successful than at 4°C.
General
The commercially available reagents were purchased from
Sigma-Aldrich. Product purification by flash chromatography was
performed on Merck silica gel 60 (particle size: 0.040-0.063 mm).
Thin-layer chromatography (TLC) was carried out on Merck
silica plates 60 F254. The seawater used in the experiments
came from the Mediterranean Sea (El Perelló beach, València,
Spain). It was sterilized by autoclaving under standard
conditions and was maintained at 18°C. The water salinity for
this area, determined by weighing the solid residue after
lyophilization, was around 4% (w/v). The sodium content,
analyzed by Flame Emission Spectroscopy (589.0 nm, Thermo
Scientific model iCE 3000 Series), was 12.46±0.17 g L-1, and pH
ranged from 7.5 to 8.5, depending on the batch.
Products were characterized by NMR spectra recorded in a
Bruker DRX 300 spectrometer using deuterated chloroform as a
solvent. Chemical shifts are reported in ppm in relation to the
residual solvent peak. Enantiomeric excess was established by
High-Performance Liquid Chromatography (HPLC) with a Merck
Hitachi Lachrom system employing Chiralcel ODH column and
using racemic alcohols 2 (commercially available) and 3 as
standards.[17] The assignment of the absolute configuration was
made by comparing the tR with those described in the literature
under identical conditions,[17,33] and by the measure of the optical
rotation of the enantiomerically enriched purified samples in a
Perkin Elmer 241 Polarimeter at λ=589 nm.[34,35]
The results presented in this work widen the scope of yeasts
potentially useful for biocatalysis in seawater, and are the basis
for future studies to carry out a proof of concept in a pilot plant. It
should be essential to evaluate in them the most appropriate
reaction conditions (amount of cells, time…) to scale the process
at industrial level, allowing a reduction in the ratio seawater
volume/substrate load. Also the substitution of the extraction
solvent methylene chloride by other green solvents, as
cyclopentyl methyl ether, should be considered for this purpose.
Strains and culture conditions
The yeast strains used for this work were most of them obtained
from the Colección Española de Cultivos Tipo (CECT), Servei
Central de Suport a la Investigació, Universitat de València:
Schwanniomyces etchellsii (CECT 11406), Debaryomyces
hansenii var fabryi (CECT 10099), Kluyveromyces marxianus
(CECT 1018), Pichia glucozyma (CECT 11449), Meyerozyma
guilliermondii (CECT 11379), Schwanniomyces polymorphus var.
polymorphus (CECT 10060), Torulospora delbrueckii (CECT
1015) and Pichia fermentans (CECT 1455). S. cerevisiae
FY86[37] proceeds from our laboratory stock of yeast strains.
Debaryomyces hansenii var hansenii CBS767, commonly used
as the D. hansenii-type strain, was provided by Dr. José Ramos
(Universidad de Córdoba).
Cells were grown overnight to an OD600 ranging from 15 to 50
(depending on the strain) by incubation in an orbital shaker
(shaking diameter 25 mm, 150 rpm) at 30°C in a 50 mL
Erlenmeyer flask containing 10-20 mL of YPD medium (1% w/v
yeast extract, 2% w/v peptone, 2% w/v glucose) or SW-YPD
medium (with the same YPD composition but prepared with
seawater). The latter medium was filtered, sterilized and
supplemented with chloramphenicol to a final concentration of
100 g mL-1. To avoid any differential effect of this antibiotic on
yeast growth, it was also added to the YPD medium prior to the
inoculation of cells.
Conclusion
The screening of several unconventional yeasts in saline-rich
media allowed the isolation of halotolerant strains to be used as
biocatalysts in seawater. Their regio- and stereoselectivity at
different temperatures in the reduction reaction of 1 were
checked to obtain acyloins 2 and 3. The results were dependent
on the strain, temperature and also on the reaction medium
nature. Although many of the tested strains provided interesting
results to obtain 2, we found only two (S. cerevisiae FY86 and K.
marxianus) with which acyloins 2-S, and 3-R or 3-S were
obtained with good to excellent enantiomeric excess, which
occurred in seawater. Reuse cells for several rounds, and
maintaining stereoselectivity, were also possible. In conclusion,
we have demonstrated the possibility of using halotolerant
strains as biocatalysts in organic synthesis processes in
seawater. This possibility looks promising from a bio-sustainable
point of view when large quantities of water are required. Further
molecular analyses are required to understand the reasons for
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10.1002/cbic.202000023
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FULL PAPER
To follow the growth of yeast strains in different liquid media,
overnight cultures were diluted to an OD600 of 0.15 and
incubation was continued for 48 h at 30°C. The optical density
was measured over time.
oil
1H-RMN (300 MHz, CDCl3): δ= 2.1, (s, 3H; CH3), 4.3 (d,3J(H,H)=
4.3 Hz, 1H; OH), 5.1 (d,3J(H,H)= 4.2 Hz, 1H; CH), 7.3-7.4 (m,
5H; CH Ar) ppm; 13C-RMN (75 MHz, CDCl3): δ= 25.2, 80.0,
127.2, 128.0, 128.9, 137.8, 207.5 ppm.
Chiralcel ODH column (Hex/iprOH 94:6, flow= 0.4 mL min-1,
wavelength= 214 nm) tR= 24.8 min ((S)-isomer), tR= 28.7 min
((R)-isomer); []D20= - 131 (c 0.55 in EtOH, 91% ee, R), (lit -
133.1 c= 1.1 in EtOH)[35]
Growth experiments were also carried out in culture plates of
YPD-based medium that contained 2% (w/v) agar,
supplemented with NaCl to a final concentration within the 3.5-
7.5% (w/v) range.
Procedure for the biocatalyzed reduction of 1 by whole cells
Viability determination by the blue trypan assay
The volume that corresponded to 100 OD600 units from the
above-described overnight cultures was centrifuged, and cells
were washed and resuspended in the reaction medium, FWD
(2% (w/v) glucose in freshwater) or SWD (2% (w/v) glucose in
seawater). The cells were suspended in a final 10 mL volume in
50-mL Erlenmeyer flasks. The mixture was incubated at 30°C,
45°C or 4°C for 30 min, with orbital shaking before adding the
substrate (10 mg). The mixture was maintained with orbital
shaking for 2 h at 30°C and 45°C or for 22 h in the case of
The viability of yeast cells after each reuse was determined by
the trypan blue exclusion assay, which allows the direct
identification and enumeration of live (unstained) and dead
(blue) cells in a given population.[38] Experiments were carried
out in triplicate.
reactions carried out at 4°C. Then it was centrifuged (3 min at Acknowledgements
This work was supported by the Universitat de València (UV-
3000 rpm) and the aqueous supernatant was extracted with
methylene chloride (2x8 mL). The organic phases were
combined and dried over sodium sulfate. After solvent
evaporation, the crude material was analyzed by 1HNMR and
the percentage of each product was determined by integrating
the signals corresponding to each one. The enantiomeric excess
of the obtained acyloins was determined by chiral HPLC.
Cells’ ability to be recycled was also analyzed. Reactions,
performed under the conditions described above, were carried
out in parallel using FWD or SWD at 30°C and at 4°C. After 2 h
in the first case and 24 h in the second, each one was stopped
by centrifuging. The supernatant was removed, the pellet was
washed with the same medium (2 mL) and the reactions were
set up again under the same conditions using fresh solutions.
The process was repeated for several rounds. In each one, the
crude material was analyzed to determine the conversion and
the enantiomeric excess of each isomer as explained above.
In order to isolate both acyloins and determine its optical rotation,
equivalent reactions to those described above were performed
with S. cerevisiae FY86 as biocatalyst at 4°C, but using 1000
OD units of whole cells and 100 mg of 1 in 50 mL of seawater.
After 2 h, the work up described above was carried out and the
crude material was purified by flash chromatography using a
mixture of hexane and ethyl acetate (9:1). A first fraction of pure
acyloin 2 was obtained, and a second one mainly composed by
3.
INV-AE15-323062). We gratefully acknowledge Dr J. Ramos for
providing us with the Debaryomyces hansenii strain CBS767
and the SCSIE (Universitat de València) for access to its
instrumental facilities.
Conflict of Interest
The authors declare no conflict of interest
Keywords: Biocatalysis, Chiral acyloins, Green Chemistry,
Halotolerant Yeasts, Seawater.
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Entry for the Table of Contents
Halotolerant yeasts were used to reduce 1 in sea/freshwater.
The regio- and stereoselectivity to obtain acyloins 2 and 3
depended on the strain, temperature and reaction medium.
Interesting results to obtain 2-S were mostly found, but only with
FY86 and K. marxianus in seawater, 3-R or 3-S were obtained
with good to excellent ee. This possibility looks promising from a
bio-sustainable point of view when large quantities of water are
required.
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000023
ChemBioChem
FULL PAPER
This article is protected by copyright. All rights reserved.