A Dual Role Reductase from Phytosterols Catabolism Enables the Efficient Production of Valuable Steroid Precursors

Article abstract of DOI:10.1002/anie.202015462

4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.

Full text of DOI:10.1002/anie.202015462

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: A Dual Role Reductase from Phytosterols Catabolism Enables
the Efficient Production of Valuable Steroid Precursors
Authors: Haidong Peng, Yaya Wang, Kai Jiang, Xinru Chen, Wenlu
Zhang, Yanan Zhang, Zixin Deng, and Xudong Qu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202015462
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10.1002/anie.202015462
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Dual Role Reductase from Phytosterols Catabolism Enables the
Efficient Production of Valuable Steroid Precursors
Haidong Peng,[a][b] Yaya Wang,[a] Kai Jiang,[a] Xinru Chen,[a] Wenlu Zhang,[a] Yanan Zhang,[a] Zixin
Deng,[a][b] and Xudong Qu*[a][b]
[a]
Dr. H. Peng, Dr. Y. Wang, K. Jiang, X. Chen, W. Zhang, Dr. Y. Zhang, Prof. Dr. Z. Deng, Prof. Dr. X. Qu
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education, School of Pharmaceutical Sciences
Wuhan University
1 Luojiashan Rd., Wuhan 430071, China
E-mail: quxd19@sjtu.edu.cn
[b]
Dr. H. Peng, Prof. Dr. Z. Deng, Prof. Dr. X. Qu
State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology
Shanghai Jiao Tong University,
800 Dongchuan Rd. Shanghai 200240, China
Supporting information for this article is given via a link at the end of the document.
Abstract: 4-androstenedione (4-AD) and progesterone (PG) are two
of the most important precursors for synthesis of steroid drugs,
however their current manufacturing processes suffer from low
efficiency and severe environmental issues. In this study, we decipher
a dual-role reductase (mnOpccR) in the phytosterols catabolism,
which engages in two different metabolic branches to produce the key
intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a
4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA)
and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-
OPA), respectively. Inactivation or overexpression of mnOpccR in the
Mycobacterium neoaurum can achieve exclusive production of either
4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-
HBC can be efficiently converted to PG in a scalable manner (100
gram scale). This study deciphers a pivotal biosynthetic mechanism
of phytosterol catabolism and provides very efficient production routes
of 4-AD and PG.
To replace the polluting diosgenin degradation process, several
approaches using semi-synthesis and synthetic biology
techniques were developed.[8-11] One particularly relevant method
involves the semi-synthesis of PG from 4-HBC. This process uses
only 2 synthetic steps, with less hazardous chemical reagents
(Figure S1), and is more efficient than the diosgenin degradation
process. Additionally, 4-HBC can be produced from the
biodegradation of phytosterols; an abundant side-product from
the industrial refinement of vegetable oil, whereas diosgenin
needs to be isolated from commercial plants. For this reason,
production of PG using the 4-HBC route is both more economical
and environmentally friendly. The major obstacle for the
application of this strategy is the low production yield of 4-HBC
from phytosterols biodegradation. Interestingly, the solution to
increase 4-HBC production yield is essentially opposite to the
other aim to abolish 4-HBC production in 4-AD production process
from phytosterol biodegradation. Thus, the key for addressing
these two long-term issues within the steroid industry requires the
investigation into an improved method to regulate the production
of 4-HBC.
Introduction
Recently,
the
17-hydroxysteroid/
22-OH-BNC-CoA
Steroid drugs are the second largest catalogue of
pharmaceuticals, containing more than 400 drug entities (~17%
of FDA approved drugs), used in the clinics for a wide range of
diseases.[1,2] To meet the large demand, over 1,000,000 tons of
steroid drugs with a sale value of ~100 billion USD are produced
annually.[3,4] Due to their structural complexity production of
steroid drugs primarily relies on semi-synthesis, with
progesterone (PG, 1) and 4-androstenedione (4-AD, 2) (Figure 1)
being two of the most important precursors for manufacturing.[4]
In industrial settings, 4-AD and PG are mainly produced by
biodegradation of phytosterols and chemical degradation of
diosgenin respectively.[2,4-6] Despite being used for many years
these processes still have problems that require solutions,
including: i) the co-production of side product 20-hydroxymethyl
dehydrogenase; Hsd4A, was found to be relevant to 4-HBC
formation.[12] Inactivation of hsd4A and 3-ketosteroid Δ1-
dehydrogenases (kstDs) in the Mycobacteria neoaurum ATCC
25795 enabled the production of 4-HBC from phytosterols and
resulted in a conversion efficiency of 47-49% (Molar yield).[12]
Although manipulation of nutrient and physiological factors can
further improve the conversion of 4-HBC,[1,13,14] the lack of a
detailed biosynthetic mechanism is a major bottleneck for
metabolic engineering that aims to either eliminate (for 4-AD
production) or enhance the production of 4-HBC (for PG
synthesis). To address this, we performed an investigation on the
biosynthesis of 4-HBC. Our study identified an unusual reductase
(mnOpccR) engaging in two independent branches of the
phytosterols catabolism pathway to produce 4-HBC. Manipulation
of this gene in the Mycobacterium neoaurum CCTCC AB2019054
can shift the metabolic flux between 4-AD and 4-HBC, leading to
the exclusive production of 4-AD or 4-HBC from phytosterols. 4-
HBC is then transformed into PG through an optimized two-step
synthetic procedure. Therefore, we provide a practical solution to
both PG and 4-AD manufacturing by addressing the
aforementioned long-standing issues.
pregn-4-ene-3-one (4-HBC, also named HMP and 4-BNA, 3 in
[7]
Figure 1) during 4-AD fermentation,
which reduces the
conversion of phytosterols and complicates 4-AD purification due
to their identical chemical properties; and ii) poor overall
production yield of PG (50%) and serious environmental issues of
the diosgenin chemical degradation process, which requires 8
synthetic-steps and the use of strong acids and oxidants (Figure
S1).[5-6]
1
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10.1002/anie.202015462
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RESEARCH ARTICLE
Figure 1. The Physterol Biodegradation Pathway in Mycobacterium neoaurum. Physterols (green) which are a mixture of β-sitosterol, stigmasterol, campersterol
and brassicasterol (contents see supplymentary inforamtion), 3-OPC-CoA (purple), steroid compounds produced by physterols degradation (light blue) and
semi-synthesis (mageta) are shaded in colors. Enzymes with question marks and intermediates with brackets are putative and haven’t been verified in
Mycobacterium. ADH, alchohol dehydrogenase; Sal, steroid aldolase; MT, methyltransferase; TE, thioesterase.
gene (97% identity to KstD1 of ATCC 25795). Since ADD is not a
desired component, we deleted the kstD gene. As expected, the
resulting strain M. neoaurum mJTU1 only produces 4-AD and 4-
HBC as the two major degradation products (Figure 2, trace II),
Results and Discussions
thus it was used as the starting strain for further study.
Analysis of the Genome of M. neoaurum CCTCC AB2019054
and Elimination of ADD by Deleting kstD.
Industrial phytosterol biodegradation is mainly achieved using
the fast growing Mycobacterium strain M. neoaurum. This
process requires a set of enzymes (Figure 1)[15] in which
cholesterol oxidase (ChoM1, ChoM2) and 3β-hydroxysteroid
dehydrogenase/ isomerase (Hsds) first introduce a 3-ketone
group and a Δ5→4 isomerization in the A-ring of phytosterols.[16-
20]
The intermediates are then oxygenated at C27 by P450
enzymes (CYP125)[21-23] and converted into their CoA form by a
ligase (FaD19).[24] These activated intermediates enter into a β-
oxidation-like route including dehydrogenases (ChsE4-E5, ChsE3,
[27]
ChsE1-E2, Hsd4A),[12,25-26] hydratase (ChsH1-ChsH2),
thiolase (FadA5),[28] and an aldolase complex (Ltp2-ChsH2
)
[29-30] that sequentially remove the side chain to generate 4-
DUF35
AD. Further processing by KstD[31-32] and 9α-hydroxylase
KshAB[33-34], in an individual or collaborative manner, results in 4-
AD conversion into 1, 4-androstadienedione (ADD, 4), 9α-
hydroxyandrostenedione
(9-OH-AD,
5)
and
9α-
hydroxyandrostadienedione (9-OH-ADD, 6). Finally, 9-OH-ADD is
unstable and readily undergoes ring-opening, further degrading
into small carboxylic acids.[4]
M. neoaurum CCTCC AB2019054 is a laboratory strain able to
transform phytosterols into 4-AD (68%), ADD (15%) and 4-HBC
(17%) (Figure 2, trace I). To access the genetic information of
phytosterol catabolism, its genome was sequenced. CTCC AB
2019054 shows an identical sequence pattern to the another M.
neoaurum strain VKM Ac-1815D.[7] Unlike the M. neoaurum
ATCC 25795, which has three KstD genes (kstD1-3),[35] both
CCTCC AB2019054 and VKM Ac-1815D only bear a single KstD
Figure 2. Production of Steroid Precursors in the M. neoaurum Wild Type
and Mutant Strains. (I) CCTCC AB2019054; (II) mJTU1 (ΔkstD), x is a
marginal steroid product different from ADD; (III) mJTU2 (ΔkstD-Δhsd4A);
(IV) mJTU3 (ΔkstD-ΔchsE1-E2-ΔchsH1-H2); (V) mJTU4 (ΔkstD-
ΔmnOpccR).
2
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10.1002/anie.202015462
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RESEARCH ARTICLE
Unveiling 3-OPC-CoA Reduction Involves the Formation of 4-
HBC and Inactivation of ChsE1-E2 and ChsH1-H2 to Produce
3-OPC and 3-OPDC.
Surprisingly, the resulting mutant mJTU3 not only increased 4-
HBC by 12%, but also produced two new major steroidal products
(Figure 2, trace IV). Structural characterization revealed that
these products are 3-OPC methyl ester (3-OPCM, 11)[36] and 3-
oxo-4,17-pregnadiene-20-carboxylic methyl ester (3-OPDCM,
12). These are possibly generated through successive thioester
hydrolysis and carboxyl methylation from their CoA intermediates
(Figure 1). Although (11) and (12) are not expected products, they
are very useful for the synthesis of many steroidal 17-
carboxylates.[37] These products, or their free acids, are not able
to be detected in the mJTU1 suggesting the branched pathways
to their biosynthesis are silenced in the ordinary phytosterol
degradation condition. The accumulation of 3-OPC-CoA (10) in
high concentrations could activate these pathways to produce
(11) and (12), which appears to be heavily favored over the 4-
HBC pathway. The low conversion of 3-OPC-CoA (10) to 4-HBC
suggests that identifying the putative 3-OPC-CoA reductase is
necessary to improve the conversion and subsequent yield of 4-
HBC.
With the objective of improving the production of 4-HBC, we first
replicated the reported strategy in ATCC 25795 to delete hsd4A
in mJTU1, generating the strain mJTU2.[12] Blocking the
conversion of 22-OH-BNC-CoA (7) to 22-oxo-BNC-CoA (8) was
assumed to direct 7 into entering the retro-aldol elimination route.
Subsequently, this would yield 3-oxo-4-pregnene-20-aldehyde (3-
OPA, 9), leading to the conversion of 4-HBC by aldehyde
reductase (Figure 1). However, our experimental results of hsd4A
deletion (97.4% identity to the Hsd4A in ATCC 25795), show no
effect on the profile of products (Figure 2, trace II-III). These
results suggest that CCTCC AB2019054 may contain other
enzymes which can compensate for the function of Hsd4A.
The failure to boost 4-HBC production through inactivation of
hsd4A motivated us to focus on the 4-HBC biosynthesis itself.
Except for the retro-aldol elimination route,[12] we hypothesize it is
also possible to synthesize 4-HBC via reduction of 3-oxo-4-
pregnene-20-carboxyl-CoA (3-OPC-CoA, 10), although the
presently known CoA reductases only accept alkyl acyl-CoAs. To
verify this assumption, we performed an in vitro reduction assay
of 3-OPC-CoA (10) using the crude lysate of mJTU1 cells. HPLC
analysis reveals that both 4-AD and 4-HBC can be produced
when synthetic 3-OPC-CoA (10) and reducing equivalents of
NADPH were both added into the cell lysate (Figure 3, trace V).
The ratio of 4-HBC to 4-AD (12%: 88%) is lower than that
observed in the phytosterols fermentation broth (17%: 83%)
indicating that 4-HBC is indeed partially produced via the
reduction of 3-OPC-CoA.
Identification of mnOpccR and Elimination of 4-HBC.
Synthesis of alcohol from acyl-CoA normally requires two
consecutive two-electron (2-e) reduction steps: reduction of acyl-
CoA to aldehyde and reduction of aldehyde to alcohol.[38] In a few
cases, this conversion can be fulfilled through a single four-
electron (4-e) reduction.[38,39] As no aldehyde intermediate was
detected in the crude lysate assay system, we assume that 4-
HBC is formed through the 4-e reduction route from a acyl-CoA
precursor by a 4e reductase. So far, only two types of four-
electron fatty acyl-CoA reductase have been identified: I) the first,
existing in prokaryotes and eukaryotes, can catalyze the
conversion of both fatty acyl-CoA and fatty acyl-ACP (acyl carrier
protein) to fatty acyl alcohols;[40-44] II) the second type contains an
N- and C- terminal didomain, observed in a few bacterial
species,[39,45] e.g. maFACoAR (Maqu_2507) in Marinobacter
Next, considering that the metabolic branch of 4-HBC resides
in front of the dehydrogenation of 3-OPC-CoA (10), we envision
that abolishing the late stage steps in the β-oxidation route will
divert the production from 4-AD to 4-HBC. Therefore, both the 3-
OPC-CoA dehydrogenase (ChsE1-E2) and 3-OPDC-CoA
hydratase (ChsH1-H2) genes were deleted from the strain mJTU1. aquaeolei VT8 which only accepts fatty acyl-CoAs.[39]
Through blast analysis, we found a maFACoAR homologous
gene (mnOpccR, accession no. MT747422, 43% identity to
maFACoAR, Figure S2) in CCTCC AB2019054. To test its
function, mnOpccR was overexpressed in the model strain
Mycobaterium smegmatis MC2 155. The purified protein was able
to efficiently convert 3-OPC-CoA (10) into 4-HBC using NADPH
as an exclusive reductant (Figure 4, trace IV) (NADH is not active,
Figure S3). Considering that maFACoAR-type enzymes are also
able to reduce aldehydes, we further assayed its activity on 3-
OPA (9). To our delight, mnOpccR is even more efficient in the
reduction of 3-OPA (9) (Km = 0.2736 ± 0.06 mM, kcat = 18. 13 ±
1.36 min-1, kcat / Km = 66.25 ± 13.84 mM-1 min-1) than 3-OPC-CoA
(10) (Km=0.08 ± 0.006 mM, kcat = 2. 74 ± 0.08 min-1, kcat/ Km =
34.33 ± 3.6 mM-1 min-1) (Figure 4, trace V and Figure S4). This
activity suggests that the mnOpccR also engages in the retro-
aldol elimination route to produce 4-HBC by reduction of 3-OPA
(9). As expected, deletion of this dual role gene in mJTU1 can
completely abolish the production of 4-HBC (Figure 2, trace V).
Additionally, the blockage of the metabolic flux to 4-HBC
increases the yield of 4-AD by 16% compared to wild-type.
Furthermore, the elimination of 4-HBC in the degradation
Figure 3. Biochemical Assay Investigating the Conversion of 3-OPC-CoA
products also drastically facilitates the purification of 4-AD.
Approximately 95% chemical purity of 4-AD can be achieved by a
simple ethyl acetate extraction, creating an applicable solution for
industrial 4-AD manufacturing.
Using Crude Cell Lysate of mJTU1. (I) standard 4-HBC; (II) standard 4-
AD; (III) standard 3-OPC-CoA; (IV) control reaction, omitting the 3-OPC-
CoA; (V) standard reaction system, converting the 3-OPC-CoA into 4-
HBC and 4-AD in 2 hours.
3
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10.1002/anie.202015462
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RESEARCH ARTICLE
(S375A-G377A-R398A) of the full-length enzyme was successful.
Activity assays reveal that these two mutants lose activity on 3-
OPC-CoA (10), but retain activity to efficiently reduce the
aldehyde 3-OPA (9) (Figure 4, trace VI to IX). This confirms that
the NtD is responsible for the conversion of aldehyde to alcohol
and the CtD appears to catalyze the reduction of 3-OPC-CoA (10)
to aldehyde. In addition, the 3-OPA (9) was not able to be
detected during the reduction of 3-OPC-CoA (10), suggesting that
this aldehyde intermediate was shuttled within an internal tunnel
between the N- and C- terminal domains.
The previously known maFACoAR-type enzymes are all
specific to the medium-to-long chain fatty acyl-CoAs for wax
biosynthesis.[39,45] To verify whether mnOpccR is also active
towards fatty acyl-CoA or other type of CoAs, including lauryl-CoA
(C12 fatty acyl-CoA), butyryl-CoA, crotonyl-CoA and cinnamic-
CoA were employed for the activity assay. LC-MS and GC-MS
analysis shows that mnOpccR is not able to convert these
substrates at all (Figure S6), suggesting it is exclusively involved
in steroid catabolism. Exploration of its distribution in other
organisms revealed that the mnOpccR homologues widely exist
in many other Mycolicibacteria (Figure S7), including both the
slow growth pathogenic and fast growth non-pathogenic strains
such as M. smegmatis. To verify this discovery, the homologous
gene msOpccR (identity 78%, accession number: AIU06854) of
M. smegmatis was overexpressed and purified. Interestingly, this
enzyme shows no reductive activity of 3-OPC-CoA (10) (Figure 4,
trace X) as well as above the aforementioned fatty acyl-CoA
substrates (data not shown). However, it does appear capable of
reducing 3-OPA (9) to 4-HBC (Figure 4, trace XI). By replacing
the CtD (365-681aa) of msOpccR with the mnOpccR CtD (362-
667aa), the reducing activity of 3-OPC-CoA (10) into 4-HBC can
be restored (Figure 4, trace XII). This confirms that the
msOpccR’s CtD is naturally inactive. It suggests that M.
smegmatis may only have the retro-aldol elimination route to
produce 4-HBC and msOpccR catalyzes the conversion of 3-OPA
to 4-HBC. In addition, the interchangeable CtDs between
mnOpccR and msOpccR suggest that the two half-reactions are
performed independently in the two domains. Based on these
results, two possible modes for maFACoAR-type enzyme
reactions exist, these are: (I) the 4-e reduction mode, where 3-
OPC-CoA (10) first enters into the cavity of the CtD. After the first
2-e reduction, the yielding aldehyde intermediates 3-OPA (9) are
shuttled from the C-terminal cavity to the N-terminal cavity
through an internal tunnel, where they are further reduced into
alcohol through the second 2-e reduction; (II) The 2-e reduction
mode, 3-OPA (9) may directly enter into the N-terminal cavity for
reduction or transport through the C-terminal cavity and the
internal tunnel. In the phytosterols catabolism of CCTCC
AB2019054 both modes co-exists and mnOpccR plays dual roles
for the generation of 4-HBC.
Figure 4. Biochemical Assay Investigating the Conversion of 3-OPC-CoA
by mnMnOpccR. (I) standard 4-HBC; (II) standard 3-OPA; (III) standard 3-
OPC-CoA; (IV) mnOpccR reaction, converting 3-OPC-CoA into 4-HBC; (V)
mnOpccR reaction, converting 3-OPA into 4-HBC; (VI) mnOpccR-NtD
reaction, showing no activity on 3-OPA-CoA; (VII) mnOpccR-NtD reaction,
converting 3-OPA into 4-HBC; (VIII) mnOpccR-S375A-G377A-R398A
reaction, showing no activity on 3-OPC-CoA; (IX) mnOpccR-S375A-
G377A-R398A reaction, converting 3-OPA into 4-HBC; (X) msOpccR
reaction, showing no activity on 3-OPC-CoA; (XI) msOpccR reaction,
converting 3-OPA into 4-HBC; (XII) msOpccR-NtD-mnOpccR-CtD chimeric
protein’s reaction, converting 3-OPC-CoA to 4-HBC.
Deciphering the Catalytic Mechanism of mnOpccR.
Currently, the reaction mechanisms of the maFACoAR-type
enzyme remain poorly understood, particularly the catalytic roles
of the N- and C- terminal domains (NtD and CtD).[39,45] Sequence
analysis revealed that both the NtD and CtD of mnOpccR have a
conserved pyridine nucleotide binding region (Figure S5) and
resemble the Re domain of the non-ribosomal peptide synthetase
(NRPS) MxaA[46] (30% identity) and the acyl-CoA reductase
Acr1[47] (42% identity) respectively (Figure S5). As both MxaA-Re
and Acr1 are able to catalyze the reduction of thioesters, the
function of the NtD and CtD of mnOpccR cannot be readily
distinguished. To verify their exact roles, we dissected the
mnOpccR by individually expressing the NtD (1-361aa) and the
CtD (362-667aa) (Figure S5). Efforts to solubilize the CtD or
introduce inactive mutations (G13A) within the N-terminal
NADPH-binding motif were unsuccessful; both for the full length
mnOpccR and the NtD (Table S1). Fortunately, expression of NtD
alone and inactivation of the NADPH binding motif in the CtD
Efficient Production of 4-HBC and the Dual Role of mnOpccR
in the Phytosterol Catabolism.
After elucidating mnOpccR, efforts were directed towards
enhancing its activity to overproduce 4-HBC. The Mycobacterium
system has a unique gene organization, with around half of its
genes containing no 5’-UTR, e.g. the Shine-Dalgarno (SD)
sequence.[48,49] From sequence analysis, mnOpccR seems to
have no 5’-UTR (Figure S8). To ensure the success of
overexpression of mnOpccR, we designed three types of
4
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10.1002/anie.202015462
Angewandte Chemie International Edition
RESEARCH ARTICLE
constructs: (I) with the native 100 bp sequence upstream of the
start codon; (II) with a standard mycobacterial SD sequence
(-
[48,49]
16)GAGAAAGGG(-8)
and (III) without 5’-UTR. Those three
cassettes were placed under the strong constitutive promoter
pG13 and transferred into the mJTU1 for integration into its
genome. Surprisingly, none of these strains show significant
improvement in 4-HBC production. The highest increase in 4-
HBC conversion was only by about 10%, using the strain with
native 100 bp upstream sequence (mJTU5) compared with
mJTU1 (Figure 5, trace II). This suggests the 4-AD pathway is still
stronger than the accumulation of the enhanced 4-HBC and the
retro-aldol elimination branch.
Considering the inactivation of hsd4A can weaken the
mainstream pathway to 4-AD by diverting part of metabolic flux
into the retro-aldol elimination route, the constructs with native
100 bp sequence pG13-mnOpccR were further introduced into
the strain mJTU2 (ΔkstD1-hsd4A) to generate mJTU8. The
fermentation of this recombinant strain revealed a dramatic
increase in the yield of 4-HBC, almost completely diverting the
production of 4-AD to 4-HBC (93% conversion from phytosterols
in molar ratio, Figure 5, trace III). These results demonstrate that
the 2-e reduction by mnOpccR is indeed more crucial for the
production of 4-HBC than the 4-e reduction route, and the
cumulative effect of both routes is able to surpass the 4-AD
pathway when hsd4A was inactivated.
The in vitro and in vivo results provide a comprehensive
explanation of the catalytic role mnOpccR plays in phytosterols
catabolism. Evidently, mnOpccR plays a dual role in the
conversion of 3-OPC-CoA and 3-OPA by performing both 4-e and
2-e reduction respectively. Within the wild type strain, a
preference for the β-oxidation pathway is favored, with only 17%
of metabolic products being converted into 4-HBC through the 3-
OPC-CoA reduction and retro-aldol elimination branches. From
the ratio of 4-AD and 4-HBC in the crude lysate reaction, it is
estimated about 12% of 4-HBC was generated through the 3-
OPC-CoA reduction and the remaining 5% of 4-HBC being
produced through retro-aldol elimination. Due to the preferential
direction towards the 4-AD pathway, it is necessary to weaken it
by inactivation of hsd4A, however, the sole inactivation of hsd4A
is not able to direct the metabolic flux towards the formation of 4-
Scheme 1. Production of PG from phytosterols.
HBC. Analysis of the CCTCC AB2019054 genome revealed that
there are 6 proteins with >38% identity and 10 proteins with 31%-
38% identity to the Hsd4A (accession no. MW228416-
MW228431) which may compensate its function. To confirm this
assumption, we deleted the most identical gene hsd4A2 (45%
identity to Hsd4A) on the basis of strain mJTU2 (ΔkstD-Δhsd4A).
Fermentation analysis of the resulting mutant strain mJTU9
(ΔkstD-Δhsd4A-Δhsd4A2) revealed that it indeed can produce 4-
HBC with a significant increased ratio (85% in the products profile)
comparing to the stain of mJTU2 (with 17% 4-HBC in the products
profile) (Figure 5, trace IV). This result indicated that Hsd4A2
plays a more important role than Hsd4A in conversion of 7 to 8
(Figure 1). Furthermore, as mJTU9 still produces 4-AD (15% in
the products profile), other isoenzymes should still exist in the
strain, waiting for further study to verify their function.
Although the strain mJTU9 can significantly improve the ratio
of 4-HBC in the products profile, its conversion (68% from
phytosterols in molar ratio) is still much lower than the strain
mJTU8 (ΔkstD-Δhsd4A and pG13-mnOpccR, 93% from
phytosterols in molar ratio). This is likely due to the toxicity of the
aldehyde 3-OPA (9) and the reversibility of retro-aldol elimination
reaction, restricting the flow of metabolic flux. The overexpression
of mnOpccR rapidly removes the accumulation of 3-OPA (9), and
the metabolic flux can be driven towards retro-aldol elimination,
resulting in a dramatic increase in 4-HBC production. These
results confirm that the mnOpccR plays a determinant role in the
retro-aldol elimination branch.
Preparation of PG from 4-HBC and Phytosterols.
To demonstrate the practical utility of the 4-HBC route, we
performed a preparative scale biotransformation of phytosterols
using flask fermentation. 10 g L-1 of phytosterols were fed to the
culture uniformly at an interval of 24 h between the 1rd - 3th day of
the fermentation period, resulting in a total of 7.41 g L-1 of pure 4-
HBC (93% molar ratio) obtained by day 6 (Scheme 1). Using a
simple ethyl acetate method of extraction from the fermentation
broth 4-HBC was obtained > 95% purity, substantially improving
the existing purification process. To the best of our knowledge,
this is the highest yield of 4-HBC ever reported. It almost doubles
the observed conversion (47-49%) previously achieved using
ATCC 25795-Δhsd4A-ΔkstD1-3,[12] addressing
bottleneck for the 4-HBC route in the production of PG.
a
major
Synthesis of PG from 4-HBC has been previously
documented.[9-11] The synthetic procedure contains two separate
steps; the conversion of 4-HBC to 4-HBC aldehyde, and the
conversion of the aldehyde to PG. Although pyridinium
dichromate (PDC) has proven to be efficient in oxidizing the
Figure 5. Production of 4-HBC in the M. neoaurum mutant strains. (I) mJTU1
(ΔkstD); (II) mJTU5 (ΔkstD and pG13-mnOpccR); (III) mJTU8 (ΔkstD-Δhsd4A
and pG13-mnOpccR); (IV) mJTU9 (ΔkstD-Δhsd4A-Δhsd4A2)
5
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10.1002/anie.202015462
Angewandte Chemie International Edition
RESEARCH ARTICLE
alcohol of 4-HBC (73% yield),[11] this reagent is highly toxic and
highly restricted by EHS in China. By exploring a few reagents,
we found that TEMPO oxidation can provide excellent conversion
to produce 4-HBC aldehyde with 95% yield (Scheme 1).
Furthermore, through minor optimization of the second reaction
condition,[10] PG can be efficiently produced (90% yield) through
the copper-mediated catalytic radical oxygenation of 4-HBC
aldehyde (Scheme 1). Finally, to demonstrate its practical utility,
we scaled up the reaction to a preparative level. Based on these
conditions, 80.5 g PG (84.6% yield) was obtained from 100 g 4-
HBC. By accounting for the yield of phytosterols degradation, the
total yield of phytosterols-based PG can be up to 78.7%, which is
much more efficient than the diosgenin degradation route (50%
yield over 8 steps,[5,6] Figure S1). Therefore, this process is both
more economical and sustainable than the diosgenin degradation
route for industrial production of PG.
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In summary, we have deciphered the biosynthetic mechanisms
of 4-HBC in the phytosterols biodegradation pathway of M.
neoaurum CCTCC AB2019054. 4-HBC is formed by two
independent pathways, in which mnOpccR catalyzes the pivot
reactions between 4-e reduction of 3-OPC-CoA (10) and 2-e
reduction of 3-OPA for its generation. The 4-e reduction uses the
C-terminal domain of MnOpccR to convert 3-OPC-CoA into 3-
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4-HBC can be efficiently converted into PG in a scalable way.
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RESEARCH ARTICLE
Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
An unusual reductase (mnOpccR) in phytosterol catabolism is found to catalyze the formation of 4-HBC through two different routes.
Inactivation or overexpression of mnOpccR leads to exclusive production of 4-androstenedione (4-AD) and 4-HBC from phytosterols.
By a two-step synthesis, 4-HBC can be further efficiently converted into progesterone (PG). This work provides two green and
economical methods for 4-AD and PG manufacturing.
Table of Contents
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