Oxidative Carbon Backbone Rearrangement in Rishirilide Biosynthesis

Article abstract of DOI:10.1021/jacs.9b12736

The structural diversity of type II polyketides is largely generated by tailoring enzymes. In rishirilide biosynthesis by Streptomyces bottropensis, ¹³C-labeling studies previously implied extraordinary carbon backbone and side-chain rearrangements. In this work, we employ gene deletion experiments and in vitro enzyme studies to identify key biosynthetic intermediates and expose intricate redox tailoring steps for the formation of rishirilides A, B, and D and lupinacidin A. First, the flavin-dependent RslO5 reductively ring-opens the epoxide moiety of an advanced polycyclic intermediate to form an alcohol. Flavin monooxygenase RslO9 then oxidatively rearranges the carbon backbone, presumably via lactone-forming Baeyer-Villiger oxidation and subsequent intramolecular aldol condensation. While RslO9 can further convert the rearranged intermediate to rishirilide D and lupinacidin A, an additional ketoreductase RslO8 is required for formation of the main products rishirilide A and rishirilide B. This work provides insight into the structural diversification of aromatic polyketide natural products via unusual redox tailoring reactions that appear to defy biosynthetic logic.

Full text of DOI:10.1021/jacs.9b12736

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Communication
Oxidative carbon backbone rearrangement in rishirilide biosynthesis
Olga Tsypik, Roman Makitrynskyy, Britta Frensch, David L.
Zechel, Thomas Paululat, Robin Teufel, and Andreas Bechthold
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12736 • Publication Date (Web): 17 Mar 2020
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Journal of the American Chemical Society
Oxidative carbon backbone rearrangement in rishirilide biosynthesis
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Olga Tsypik†, Roman Makitrynskyy†, Britta Frensch‡, David L. Zechel§, Thomas Paululat⁑, Robin
Teufel‡,*, and Andreas Bechthold†,*
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† Department of Pharmaceutical Biology and Biotechnology, Institute of Pharmaceutical Sciences, Albert-Ludwigs-
Universität Freiburg, Stefan-Meier-Strasse 19, 79104, Freiburg, Germany.
‡ Faculty of Biology, Schänzlestr. 1, 79104, Freiburg, Germany.
§ Department of Chemistry, Queen's University, 90 Bader Lane, Kingston, K7L 3N6 Ontario, Canada.
⁑ Organic Chemistry, University of Siegen, Adolf-Reichwein-Str. 2, 57068 Siegen, Germany.
Supporting Information Placeholder
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biosynthetic intermediates or shunt products (e.g., 4) not observed
in the native strain (Fig. 1B). To identify the substrate of RslO5,
the enzyme was heterologously produced with an N-terminal His6
tag and isolated with bound flavin mononucleotide (FMN) from
Escherichia coli BL21 (DE3) StarTM (Figs. S1A and S2). Addition
of RslO5 to a crude extract from the S. bottropensis ∆rslO5 mutant
in the presence of additional FMN and NAD(P)H led to the
conversion of 4 (m/z = 367.117, [M-H]–) into 5 (m/z = 369.133, [M-
H]–) (Fig. 1C, S3 and S4). Moreover, when RslO5 was incubated
with purified 4 as a substrate, a clean reduction to 5 was observed
in the presence of NADPH or NADH (Figs. 2A and B, S5, S6, &
S7). In contrast, 4 was neither converted in assays with boiled
RslO5, nor in assays containing only FMN and NADPH (Fig. S7).
Next, 4 and 5 were purified from the respective mutants and
analyzed by NMR spectroscopy (see Supporting Information). 4
proved to be an advanced intermediate with fully cyclized but non-
rearranged carbon backbone and a surprising C3-C16 epoxide
functionality. By contrast, 5 contained a C16-hydroxyl group most
likely arising from the reductive epoxide ring opening of 4
(Scheme 1).
ABSTRACT: The structural diversity of type II polyketides is
largely generated by tailoring enzymes. In rishirilide biosynthesis
by Streptomyces bottropensis, 13C-labelling studies previously
implied extraordinary carbon backbone and sidechain
rearrangements. In this work, we employ gene deletion
experiments and in vitro enzyme studies to identify key
biosynthetic intermediates and expose intricate redox tailoring
steps for the formation of rishirilides A, B, D and lupinacidin A.
First, the flavin-dependent RslO5 reductively ring opens the
epoxide-moiety of an advanced polycyclic intermediate to form an
alcohol. Then, flavin monooxygenase RslO9 oxidatively
rearranges the carbon backbone, presumably via lactone-forming
Baeyer-Villiger oxidation and subsequent intramolecular aldol
condensation. While RslO9 can further convert the rearranged
intermediate to rishirilide D and lupinacidin A, an additional
ketoreductase RslO8 is required for formation of the main products
rishirilide A and rishirilide B. This work provides insight into the
structural diversification of aromatic polyketide natural products
via unusual redox tailoring reactions that appear to defy
biosynthetic logic.
RslO5 is homologous to reductases of the Old Yellow Enzyme
(OYE) family19. These enzymes typically catalyze two electron
reductions of double bonds via hydride transfer, including those of
α-β-unsaturated carbonyls and nitro-olefins. An amino acid
sequence alignment of RslO5 with members of the OYE family
(Fig. S8) revealed the conservation of Y177, H172, and N175. The
corresponding amino acid residues H191 and N194 in OYE
stabilize the transient enolate anion arising from initial hydride
transfer, followed by Y196-dependent protonation to afford the
reduced product.19–21 For the conversion of 4 into 5, we thus
envisage a mechanism analogous to OYE for RslO5 (Scheme 1),
possibly via a transient C17 oxyanion intermediate stabilized by
H172 and N175. To further examine RslO5, enzyme variants were
generated by site-directed mutagenesis. Unlike wild type RslO5,
solutions of purified RslO5-H172N were clear, indicating a
deficiency in FMN binding. Moreover, the RslO5-H172N variant
was inactive when incubated with an extract containing 4 in the
presence of FMN and NADPH (Fig. 2C). In contrast, the N175H
and Y177F enzyme variants maintained the ability to bind FMN
but showed significantly reduced activity (30 % and 11 % relative
to wild type RslO5, respectively) (Figs. 2D and E, S5 and S6),
consistent with the proposed catalytic roles of the side chain
residues.
Streptomycetes generate structurally diverse natural products
with a wide spectrum of bioactivities, such as the type II
polyketides1 rishirilides A (1), B (2) and D (3) from S. bottropensis.
These compounds act as inhibitors of α2-macroglobulin (1 and 2)2
and S-glutathione reductase (2)3 (Scheme 1). While four total
syntheses of 2 have been reported,4–7 little is known about
rishirilide biosynthesis. Previous 13C-labelling and NMR
spectroscopic studies revealed that 2 is derived from an isobutyrate
starter unit (generated from valine and malonyl-CoA) and eight
malonyl-CoA extender units, the last of which undergoes an
additional decarboxylation (Scheme 1).8 The 13C-labelling pattern
furthermore implied an unusual oxidative rearrangement of the
carbon backbone2 (Scheme 1). In this study, the redox tailoring
enzymes RslO5, RslO8, and RslO9, encoded by the rishirilide (rsl)
biosynthetic gene cluster,9 are shown to facilitate the key tailoring
steps in rishirilide biosynthesis.
Initially, the roles of the predicted flavin-dependent enzymes
RslO5 and RslO9 were examined, as flavoenzymes are
mechanistically versatile10–15 and often impart structural
complexity to polyketide natural products.11,13,16–18 The individual
genes in S. bottropensis were replaced via double cross-overs with
spectinomycin (rslO5 mutant) or apramycin resistance genes
(rslO9 mutant) and the resulting mutants assayed for production of
rishirilides by high resolution liquid chromatography mass
spectrometry (HR-LCMS).
Relative to S. bottropensis (Fig. 1A), the ∆rslO5 strain was
deficient in the production of 1, 2, and 3, but accumulated putative
Scheme 1. Proposed biosynthesis of rishirilides and side products by S. bottropensis. Compounds in boxes were characterized by NMR
spectroscopy. The oxygen atom derived from O2 is colored in red. Black and grey arrows indicate main enzyme reactions and (enzymatic
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and non-enzymatic) side reactions, respectively. The pathway likely involves reduced hydroquinonic intermediates, which promote
subsequent redox transformations (such as the epoxidation to 4)22–25
1
.
2
3
4
5
6
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9
1 x
O
O
3
1 OH
OH
O
O
2
O
O
2
SCoA
SCoA
2
3
PKS II
[R]
[R]
O
O
[O]
8 x
OH OH OH
O
2
4 H2O
CO2
OH OH
O
OH OH
O
HO
[O]
O
O
3
H
3
Baeyer-Villiger
oxidation
6
8
O
O
H+
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O
OH
17
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17
RslO9
RslO9
RslO5
16
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O
O
14
11
13
-H+
14
14
OH
OH
15
FADH2
O2
FAD
H2O
FMNH2
FMN
OH OH
O
O
OH OH
O
O
O
O
O
O
O
OH OH
OH
O
5
4
+H+
RslO9
H
H
H
O
O
O
a)
RslO8
16 OH
17
15
RslO9
OH
OH
15
16
17
13
16
14
OH
15
14
keto
reduction
NADPH
+ H+
17
O
lactone
opening
O
COOH
O
HO
OH
13
COOH
HO
O
H2O
OH OH
HO
OH
H
O
HO
NADP+
HO
OH
H+
H+
H+
FADH2
FAD
6
1
2
(rishirilide B)
(rishirilide A)
b)
FADH2
RslO9
lactone opening
FAD
pH  
O
OH
OH
O
O
H
O
H
[O]
14
16
17
16 OH
14
16 OH
17
17
OH
OH
OH
13
COOH
13
OH
O
COOH
15
OH OH
15
OH OH
OH OH
HO
OH
O
HO
CO2
H2O
3
7
(lupinacidin A)
(rishirilide D)
Figure 1. Gene inactivation studies of rslO5 and rslO9. (A) TIC
analysis of a culture extract from S. bottropensis. (B) Extract from
S. bottropensis ∆rslO5. (C) Reaction of RslO5 with a crude extract
from the ∆rslO5 mutant with FMN and NADPH. (D) Extract from
S. bottropensis ∆rslO9. The compound marked with asterisks (*) is
a degradation product of 5 (structure not elucidated). The peak
numbering corresponds to compounds in Scheme 1.
Figure 2. LC-MS (TIC) analysis of the conversion of 4 by RslO5
and active site variants. (A) Purified 4; (B) 4 + RslO5 + FMN +
NADPH; (C) 4 + RslO5-H172N + FMN + NADPH; (D) 4 + RslO5-
N175H + FMN + NADPH; (E) 4 + RslO5-Y177F + FMN +
NADPH. See Figs. S5 and S6 for uncropped traces and EIC’s.
As 5 also accumulated in the ∆rslO9 mutant (but was prone to
degradation) (Fig. 1D), it may represent RslO9’s substrate. RslO9
is a prime candidate for catalyzing the central oxidative
rearrangement steps in rishirilide biosynthesis because of
homology to FAD-dependent Baeyer-Villiger monooxygenases
such as MrqO6 (52% sequence identity / 95% sequence coverage),
BexE (31% / 92%) and MtmOIV (36% / 46 %). Both MrqO6 and
BexE perform key oxidative rearrangements during the
biosynthesis of the angular aromatic polyketides murayaquinone
and BE-7585A, respectively26,27
, while MtmOIV catalyzes
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oxidative C–C bond cleavage in mithramycin biosynthesis.28
RslO9 was heterologously produced with an N-terminal His6 tag
1
and contained a bound FAD cofactor after purification (Figs. S1B
and S2). Indeed, RslO9 converted 5 to 3 (m/z = 387.144, [M-H]–)
in the presence of NADPH or NADH. In contrast, 5 was not
converted in assays containing heat-denatured RslO9 or in assays
lacking NAD(P)H (Fig. 3 and Figs. S9, S10, & S11).
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Figure 4. LC-MS (TIC) analysis of 6 formation during time-course
reactions of RslO9 ± RslO8. (A) Chromatogram for purified 5. (B)
Incubation of 5 with RslO9 for 0.5 min, (C) 1 min, (D) 1.5 min and
(E) 5 min. (F) Reaction of 5 with RslO9 and RslO8 incubated for
0.5 min, (G) 1 min, (H) 1.5 min and (I) 5 min. See Figs. S13 and
S14 for uncropped traces and EIC’s. All reactions included
NADPH.
Figure 3. LC-MS (TIC) analysis of the reactions of RslO8 and
RslO9 in vitro. (A) Chromatogram for purified 5. (B) Incubation of
5 with heat inactivated RslO9. (C) Reaction of 5 with RslO9. (D)
Reaction of 5 with RslO9 and RslO8. Reactions B, C, and D all
included FAD and NADPH. See Figs. S9 and S10 for uncropped
traces and EIC’s.
The proposed structure of 6 implies that reduction of the C13-
ketone to the respective hydroxyl group could afford 1. We
hypothesized that this step could be catalyzed by RslO8, which is
50% identical to the proposed enoyl-reductase ActVI-ORF2 from
actinorhodin biosynthesis33 and resembles medium chain
dehydrogenases/reductases. These enzymes are typically
composed of two domains: an N-terminal catalytic domain with
homology to the folding chaperones GroEL and GroES; and a C-
terminal Rossmann fold that binds NAD(P)H. To gain more
insights into the function of RslO8, the S. bottropensis ΔrslO8
mutant was generated. Compared to the wild type, inactivation of
rslO8 eliminated production of 1 and 2 in S. bottropensis, but
increased the formation of 3 (Figs. S18 and S19), thus supporting
a role for RslO8 in late stage rishirilide tailoring.
To further investigate the anticipated oxygenase functionality of
RslO9, we conducted isotope-labelling experiments with 18O2 (Fig.
S12). Indeed, when 5 was converted by RslO9 in presence of 18O2,
the incorporation of one 18O-atom into 3 was observed by HR-
LCMS. Hence, we propose that RslO9 first inserts an oxygen atom
between C14 and C15 via Baeyer-Villiger oxidation, before a C14-
carbanion attacks the C17-ketone of the side chain to produce the
postulated intermediate 6 (Scheme 1). Consistent with this
mechanism, no incorporation of 18O was observed when 5 was
converted to 3 in the presence of H218O (Fig. S12). RslO9 may then
further transform 6 into 3 via reduction of the central ring under
concomitant lactone opening and following tautomerization
(Scheme 1).
To further investigate this, N-terminal His6 tagged RslO8 was
produced and isolated from E. coli (Fig. S1C). As anticipated, in
vitro assays of RslO8 and RslO9 with 5 as substrate afforded 1 and
only minor amounts of 3 (Fig. 3D), whereas RslO8 alone did not
convert 5. RslO8 thus likely regio- and stereoselectively reduces
the C13-ketone of 6 and thereby redirects the pathway toward 1
production. Consistent with this proposal, 3 was not accepted as a
substrate by RslO8 in in vitro assays (Fig. S17). Crucially, isolated
6 could be reduced to 1 by RslO8 in presence of either NADH or
NADPH, thus corroborating our biosynthetic proposal (Figs. S15
and S16). The structure of 1 suggests a possible lactone opening
and conversion to 3 at higher pH values triggered by the
deprotonation of C13. In fact, incubation of 1 at pH values ≥ 7.5
resulted in the spontaneous formation of 3 (Scheme 1, Fig. S20). 3
may thus represent both a shunt product arising from spontaneous
degradation of 1 as well as an enzymatic side product from the
competing activity of RslO9 and RslO8 for 6. Finally, the
generation of 2 from main product 1 could be rationalized by
RslO9-mediated reduction of the central ring resulting in lactone
opening, followed by water elimination (Scheme 1). Indeed, 1 was
slowly converted to 2 in in vitro assays containing RslO9 and
NADPH, in contrast to assays with boiled enzyme (Figs. S21 and
S22). Taken together, 1 and 2 likely represent the main pathway
products; yet the instability of 1 at higher pH values also promotes
formation of the side products 3 and 7.
In order to corroborate the proposed pathway, we examined the
RslO9-catalyzed reaction in more detail via discontinuous enzyme
assays. In fact, the transient formation of an unstable compound
with the expected mass of 6 (m/z = 385.129 [M-H]–) was observed
during the conversion of 5 into 3 (Fig. 4, S13 and S14). Moreover,
following the isolation of 6 and subsequent incubation with RslO9
in in vitro assays, 3 was formed by RslO9 in addition to degradation
products such as the predominant 7, thus unambiguously
identifying 6 as an intermediate in rishirilide biosynthesis (Scheme
1, Figs. S15 and S16). Although substrate reduction by a flavin
monooxygenase as suggested for RslO9 appears perplexing, it has
been reported for flavin monooxygenase ActVA involved in the
biosynthesis of the type II polyketide actinorhodin29. It is
noteworthy that 3 proved unstable and decomposed spontaneously
to 7 (m/z = 339.123, [M-H]–) and other minor compounds (Figs. 1,
3 & 4 and Fig. S17). Based on MS and UV-Vis data30, 7 is most
likely the anti-tumorigenic lupinacidin A31 that was recently
reported to be produced by the rishirilide gene cluster32. 7
formation can be envisaged by decarboxylation of C15 of 3 and
concurrent water elimination at C17, followed by tautomerization
and autooxidation, as supported by 18O-isotope labelling
experiments (Scheme 1, Fig. S12).
In summary, this study establishes the key enzymatic tailoring
reactions steps in rishirilide biosynthesis (for an overview of the
gene cluster and the predicted functions of other encoded proteins,
3
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(7) Odagi, M.; Furukori, K.; Takayama, K.; Noguchi, K.; Nagasawa, K.
see Fig. S23). The interplay between epoxide reductase RslO5,
flavin monooxygenase RslO9, and ketoreductase RslO8 enables
the intricate oxidative rearrangement of the polyketide carbon
backbone and ultimately gives rise to structurally distinct
rishirilides and lupinacidin A.
Total Synthesis of Rishirilide B by Organocatalytic Oxidative Kinetic
Resolution: Revision of Absolute Configuration of (+)-Rishirilide B.
Angew. Chem. Int. Ed. Engl. 2017, 56, 6609–6612.
1
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(8) Schwarzer, P.; Wunsch-Palasis, J.; Bechthold, A.; Paululat, T.
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Biosynthesis of Rishirilide B. Antibiotics (Basel) 2018, 7.
6
7
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ASSOCIATED CONTENT
Supporting Information
(9) Yan, X.; Probst, K.; Linnenbrink, A.; Arnold, M.; Paululat, T.; Zeeck,
A.; Bechthold, A. Cloning and heterologous expression of three type II
PKS gene clusters from Streptomyces bottropensis. Chembiochem 2012,
13, 224–230.
The Supporting Information is available on the ACS Publications
website at DOI:
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(10) Matthews, A.; Saleem-Batcha, R.; Sanders, J. N.; Stull, F.; Houk, K.
N.; Teufel, R. Aminoperoxide adducts expand the catalytic repertoire of
flavin monooxygenases. Nat. Chem. Biol. [Online early access]. DOI:
10.1038/s41589-020-0476-2.
Experimental procedures and compounds characterization data.
AUTHOR INFORMATION
Corresponding Authors
(11) Walsh, C. T.; Wencewicz, T. A. Flavoenzymes: versatile catalysts in
biosynthetic pathways. Nat. Prod. Rep. 2013, 30, 175–200.
Prof. Dr. Andreas Bechthold, Department of Pharmaceutical
Biology and Biotechnology, Institute of Pharmaceutical Sciences,
Albert-Ludwigs-Universität Freiburg, Stefan-Meier-Strasse 19,
(12) Teufel, R.; Miyanaga, A.; Michaudel, Q.; Stull, F.; Louie, G.; Noel,
J. P.; Baran, P. S.; Palfey, B.; Moore, B. S. Flavin-mediated dual oxidation
controls an enzymatic Favorskii-type rearrangement. Nature 2013, 503,
552–556.
79104,
Freiburg,
Germany.
E-mail:
andreas.bechthold@pharmazie.uni-freiburg.de
(13) Teufel, R.; Agarwal, V.; Moore, B. S. Unusual flavoenzyme
catalysis in marine bacteria. Curr. Op. Chem. Biol. 2016, 31, 31–39.
Dr. Robin Teufel, Faculty of Biology, Schänzlestr. 1, 79104,
Freiburg, Germany. E-mail: robin.teufel@zbsa.uni-freiburg.de.
(14) Fraaije, M. W.; Mattevi, A. Flavoenzymes: diverse catalysts with
recurrent features. Trends Biochem. Sci. 2000, 25, 126–132.
Notes
The authors declare no competing financial interests.
(15) Huijbers, M. M. E.; Montersino, S.; Westphal, A. H.; Tischler, D.;
van Berkel, W. J. H. Flavin dependent monooxygenases. Arch. Biochem.
Biophys. 2014, 544, 2–17.
ACKNOWLEDGMENTS
We thank Prof. Dr. J. Rohr, University of Kentucky, for fruitful
discussion of our results and Dr. M. Myronovskyi, University of
Saarbrucken, for kind assistance in HRMS analysis. The project
was funded by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) 235777276/GRK1976 (awarded to
AB) and TE 931/2-1 (awarded to R.T.).
(16) Teufel, R.; Stull, F.; Meehan, M. J.; Michaudel, Q.; Dorrestein, P.
C.; Palfey, B.; Moore, B. S. Biochemical Establishment and
Characterization of EncM’s Flavin-N5-oxide Cofactor. J. Am. Chem. Soc.
2015, 137, 8078–8085.
(17) Yunt, Z.; Reinhardt, K.; Li, A.; Engeser, M.; Dahse, H.-M.;
Gütschow, M.; Bruhn, T.; Bringmann, G.; Piel, J. Cleavage of four
carbon-carbon bonds during biosynthesis of the griseorhodin a spiroketal
pharmacophore. J. Am. Chem. Soc. 2009, 131, 2297–2305.
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New Insights into the Conversion of Versicolorin A in the Biosynthesis of
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the total synthesis and determination of the absolute configuration of
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O
1
OH
OH
O
O
1 x
8 x
O
O
1
2
3
4
5
6
O
O
2
2
3
SCoA
SCoA
3
PKS II
[R]
[R]
O
[O]
OH OH OH
O
4 H2O
CO2
OH OH
O
OH OH
O
HO
[O]
7
8
9
O
O
H
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Baeyer-Villiger
oxidation
2
6
8
2
O
O
3
H+
3
O
OH
17
17
RslO9
RslO9
RslO5
16
15
16
17
17
16
15
O
O
14
11
13
-H+
14
14
OH
OH
15
FADH2
O2
FAD
H2O
FMNH2
FMN
OH OH
O
O
OH OH
O
O
O
O
O
O
O
OH OH
OH
O
5
4
+H+
RslO9
H
H
H
O
O
O
a)
16 OH
15
RslO8
RslO9
lactone
OH
OH
17
16
13
16
14
OH
15
14
17
keto
17
15
O
HO
O
COOH
O
reduction
HO
OH
13
COOH
O
H2O
OH OH
HO
OH
H
O
opening
HO
NADP+
HO
OH
H+
NADPH
+ H+
H+
H+
FADH2
FAD
6
1 (rishirilide A)
2 (rishirilide B)
b)
FADH2
RslO9
lactone opening
FAD
pH
O
OH
OH
O
O
H
O
H
[O]
14
16
17
16
OH
COOH
14
16
OH
17
OH
17
OH
OH
13
13
OH
O
COOH
OH OH
15
OH OH
OH OH
15
HO
OH
O
HO
CO2
H2O
3 (rishirilide D)
7 (lupinacidin A)
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A) wt
B) ∆rslO5
C) ∆rslO5 + RslO5 + FMN + NADPH
D) ∆rslO9
*
2.0
3.0
4.0
5.0
6.0
Time (min)
7.0
8.0
9.0
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5
4
A) 4
B) 4 + FMN + NADPH + RslO5
C) 4 + FMN + NADPH + RslO5-H172N
D) 4 + FMN + NADPH + RslO5-N175H
E) 4 + FMN + NADPH + RslO5-Y177F
2.0
3.0
4.0
5.0
6.0
7.0
8.0
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3
5
7
A) 5
B) 5 + FAD + NADPH + boiled RslO9
C) 5 + FAD + NADPH + RslO9
D) 5 + FAD + NADPH + RslO9 + RslO8
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
Time (min)
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A) 5
B) 5 + RslO9, 0.5 min
C) 5 + RslO9, 1 min
D) 5 + RslO9, 1.5 min
E) 5 + RslO9, 5 min
F) 5+ RslO9 + RslO8, 0.5 min
G) 5+ RslO9 + RslO8, 1 min
H) 5 + RslO9 + RslO8, 1.5 min
I) 5 + RslO9 + RslO8, 5 min
2.0
3.0
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5.0
6.0
7.0
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