Engineering the "missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase

Article abstract of DOI:10.1021/acscatal.7b04115

The realization of a synthetic biology approach to microbial (1R,2S,5R)-(-)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

Full text of DOI:10.1021/acscatal.7b04115

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Article
Engineering the ‘Missing Link’ in Biosynthetic (–)-
Menthol Production: Bacterial Isopulegone Isomerase
Andrew Currin, Mark S. Dunstan, Linus O. Johannissen, Katherine Anne
Hollywood, Maria Vinaixa, Adrian Jervis, Neil Swainston, Nicholas Rattray, John
Gardiner, Douglas B Kell, Eriko Takano, Helen S. Toogood, and Nigel S. Scrutton
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b04115 • Publication Date (Web): 24 Jan 2018
Downloaded from http://pubs.acs.org on January 24, 2018
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Engineering the ‘Missing Link’ in Biosynthetic (–)ꢀMenthol
Production: Bacterial Isopulegone Isomerase
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Andrew Currin,† Mark S. Dunstan,† Linus O. Johannissen,†Katherine A. Hollywood,†
Maria Vinaixa,† Adrian J. Jervis,† Neil Swainston,† Nicholas J. W. Rattray,† John M.
Gardiner, † Douglas B. Kell,† Eriko Takano,†‡ Helen S. Toogood,‡and Nigel S.
Scrutton†‡*
†Manchester Centre for Fine and Speciality Chemicals (SYNBIOCHEM) and ‡School of
Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN,
United Kingdom
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CORRESPONDING AUTHOR
*Professor Nigel S. Scrutton, Phone: +44 (0)1613065152. Fax: +44 (0)1613068918. Eꢀmail:
nigel.scrutton@manchester.ac.uk
ABSTRACT
The realisation of a synthetic biology approach to microbial (1R, 2S, 5R)ꢀ(–)ꢀmenthol (1)
production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only
enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that ꢁ5ꢀ3ꢀ
ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)ꢀ(+)ꢀ
pulegone (R)ꢀ2 from (+)ꢀcisꢀisopulegone (3). Using a roboticsꢀdriven semiꢀrational design strategy,
we identified a key KSI variant encoding four active site mutations, which confer a 4.3ꢀfold
increase in activity over the wildꢀtype enzyme. This was assisted by the generation of crystal
structures of four KSI variants, combined with molecular modelling of 3 binding to identify key
active site residue targets. The KSI variant was demonstrated to function efficiently within
cascading biotransformations with downstream Mentha enzymes pulegone reductase and (–)ꢀ
menthone:(–)ꢀmenthol reductase to generate 1 from 3. This study introduces the use of a
recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the
production of 1 in microorganisms.
KEYWORDS
Biosynthetic (–)ꢀmenthol production; ketosteroid isomerase; isopulegone isomerase; enzyme
engineering; robotics.
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INTRODUCTION
Limonene and its related derivatives are the most abundant, naturally sourced monoterpenoids
known.1 These natural products are commonly used in the perfume, fragrance and flavour
industries.2ꢀ3 For example, (R)ꢀlimonene ((R)ꢀ4) is the major essential oil constituent of orange
peels, and is often found in cleaning products.4 In contrast Mentha x piperita (peppermint)
generates (S)ꢀlimonene ((S)ꢀ4), which is subsequently converted into menthol isomers (Scheme
1A).2, 5 The most commercially useful isomer is 1, known by its characteristic cooling anaesthetic
effects and aroma.6 It also has antibacterial, anticancer and antiꢀinflammatory activities, making it a
valuable natural product.7ꢀ10
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Commercially, 1 is utilised both as a pure compound and as a component of the essential oil of
peppermint, with an estimated 30,000 tonnes consumed annually.11 Whilst the majority of 1 is
extracted from Mentha canadensis, a large proportion is generated synthetically (US$ 300 million
annually).12 The two major synthetic routes to 1 are the HaarmannꢀReimer route from mꢀcresol and
the Takasago synthesis utilising βꢀpinene.13 However, the flavour and fragrance industries and/or
consumers may demand that these compounds are supplied from natural sources, particularly when
used as food additives.
Scheme 1. A) The biosynthesis of
1 and (+)ꢀneoisomenthol (5) from (S)ꢀ4. Abbreviations: L3H = (–)ꢀ
limoneneꢀ3ꢀhydroxylase; IPDH = (–)ꢀtransꢀisopiperitenol dehydrogenase; IPR = (–)ꢀisopiperitenone
reductase; IPGI = (+)ꢀcisꢀisopulegone isomerase; PGR = (+)ꢀpulegone reductase; MMR = (–)ꢀmenthone: (–)ꢀ
menthol reductase and MNMR = (–)ꢀmenthone: (–)ꢀneomenthol reductase.2 B) Proposed KSI catalytic
mechanism with 3, based on the known mechanism of the enzyme towards ꢁ5ꢀ3ꢀketosteroids.14
Naturally sourced peppermint oil can vary in both relative composition of its monoterpenoid
constituents and overall yields due to fluctuating environmental conditions. This leads to price
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volatility, and increased arable land competition for more profitable crops, such as biofuel biomass
production. Optimisation of peppermint oil production through conventional breeding methods is
difficult as it is a sterile hybrid plant.2, 15 Additionally purification of 1 from essential oil requires
expensive and lowꢀyielding steam distillation and filtration processes to generate a usable
product.16ꢀ17 Therefore, alternative clean biosynthetic routes to these compounds are a commercially
attractive prospect to generate 1 with a ‘natural’ label.
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One alternative ‘natural’ route is to introduce the biosynthetic pathway of 1 into cost effective and
even foodꢀcompatible microorganisms.18ꢀ19 This general synthetic biology approach generates
biological factories, where rapid fine chemicals production can be achieved sustainably using nonꢀ
petroleum renewable feedstocks, with a reduced land burden and higher product consistency and
purity.20ꢀ22 Such an approach has proved commercially successful in the semisynthetic industrialꢀ
scale production (ca. 35 tonnes per annum) by Sanofi of artemisinin, a major active ingredient in
modern malarial treatments. Similarly, the semisynthetic production of Taxol has been
demonstrated, where microorganism biofactories generated the precursor taxadiene using
introduced pathways.20
We recently described the incorporation of the M. piperitaꢀbased biosynthetic pathway from (R)ꢀ2
to 1 into Escherichia coli.23 An in vitro protocol was established where 1 was successfully
generated, utilising E. coli cell extracts as the biocatalyst source. However, a more costꢀeffective
route would be an in vivo approach, where the full pathways from simple carbon sources to 1 would
be incorporated, and the microorganisms would be grown on waste feedstock. The six enzymatic
steps from (S)ꢀ4 to 1 in Mentha sp. have been well characterised,5, 24ꢀ30 and the native enzymes for
five of these reactions have been sequenced and functionally expressed in E. coli (Scheme 1A).4ꢀ5,
23, 28, 30ꢀ31
The remaining enzyme is isopulegone isomerase (IPGI), which catalyses the double bond
migrating isomerisation (C3ꢀC4 to C2ꢀC3) of 3 to (R)ꢀ2 (Scheme 1AꢀC).26 Early studies with
impure native Mentha IPGI identified a 54 kDa protein complex2, 24 exhibiting cofactorꢀfree
isomerisation, utilising a mechanism similar to bacterial ꢁ5ꢀ3ꢀketosteroid isomerases (KSI).26
Therefore, the IPGI mechanism likely involves deprotonation of the C2α hydrogen of 3 by an
enzymatic proton acceptor (D40 in KSI), to form an enolate intermediate (Scheme 1BꢀC), stabilised
by hydrogen bonding residues (Y16 and D103 in KSI). A protonꢀdonating residue then transfers its
proton to C4, leading to double bond migration to C2ꢀC3 and reformation of the carbonyl group. As
no significant ketosteroid isomeraseꢀlike proteins have been annotated in plant genomes, the protein
sequence of native IPGI is likely to remain elusive until the completion of the full annotation of the
Mentha longifolia genome sequence.32
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To complete the construction of a pathway from (S)ꢀ4 to 1 in E. coli, we investigated a wellꢀstudied
bacterial ꢁ5ꢀ3ꢀketosteroid isomerase from Pseudomonas putida,14, 33ꢀ34 and found it exhibited low
activity towards 3. To improve the IPGI activity, we performed three rounds of automated
mutagenesis to improve yields of 2. The crystal structures of four KSI variants were generated, to
assist the structureꢀguided mutagenesis approach to improve the binding of 3 to the enzyme.
Molecular dynamics simulations and DFT modeling were applied to the most successful variant, to
investigate the mode of increase in activity. The performance of the optimised KSI variant within
both in vitro and in vivo biocatalysis cascades from 3 to 1 was demonstrated. Therefore, the
identification and optimisation of a substitute IPGI is a critical step in the future generation of a
potentially industrially E. coli strain capable of producing ‘natural’ 1.
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EXPERIMENTAL SECTION
Design and synthesis of KSI variants. The wild type gene sequence encoding the ꢁ5ꢀ3ꢀ
ketosteroid isomerase from P. putida (UniProt ID: P07445) was designed using the GeneGenie
online tool, containing a Cꢀterminal His6ꢀtag.35 Mutagenic oligonucleotides for the firstꢀgeneration
libraries encoding ambiguous codons for 16 residues were designed optimised for E. coli expression
using CodonGenie (Tables S1ꢀS2).36 Eight overlapping DNA oligonucleotides (up to 80 nt in
length) were used to assemble the full length genes using the SpeedyGenes gene synthesis method
(Supporting Information Section S2.5).37ꢀ38 Purified genes were ligated into linearised pET21b
(Novagen) to generate a Cꢀterminally His6ꢀtagged enzyme by InꢀFusion cloning (Clontech),
according to the manufacturers protocol. Cloning products were transformed into T7 Express
competent E. coli cells and grown on LB agar plates containing 100 ꢂg/ml ampicillin, followed by
incubation overnight at 37 °C.
The secondꢀ and thirdꢀgeneration variants were constructed using asymmetric PCR mutagenesis,39ꢀ
40
followed by ligation into linearised pET21b by InꢀFusion cloning. These mutations were
designed as double or triple variants of the first generation residues identified as providing hits
(V88I, L99X and D103S). The final variant was a combination of 4 individual positive mutations
(V88I/L99V/V101A/D103S).
Gene synthesis of Mentha biocatalysts. The protein sequences for the following enzymes from M.
piperita were obtained from UniProt (http://www.uniprot.org): pulegone reductase (MpPGR;
Q6WAU0) and (–)ꢀmenthone:(–)ꢀmenthol reductase (MMR; Q5CAF4). The respective gene
sequences were designed and synthesised and subcloned into pET21b by GeneArt (ThermoFisher),
incorporating a Cꢀterminal His6ꢀtag and applying codon optimisation techniques of rare codon
removal for optimal expression in E. coli. Each construct was transformed into E. coli T7 Express,
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and grown on LB agar plates containing 100 ꢂg/ml ampicillin, followed by incubation overnight at
37 °C.
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Roboticsꢀdriven production and purification of variant libraries. Robotic colony picking and
auto induction media inoculation (Formedium, 100 ꢂg/mL ampicillin) of first generation libraries
was performed into deep well plates (Hamilton Robotics), including replica plating into LB
(Formedium, 100 ꢂg/mL ampicillin) for glycerol stock generation. Plates were covered with a
Breathseal sealer (Greiner BioꢀOne) and incubated at 30 °C, 1000 rpm for 24 h. Each variant
underwent robotic cell lysis, extraction and purification using NiꢀNTA resin (Qiagen; 50 ꢂL per
variant) prior to enzymatic assays (see Supporting Information Section S3.1 for details). Enzyme
concentrations were determined using the Bradford assay (BioꢀRad) following the manufacturers
protocol.
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KSI variant biotransformation screen. Substrate 3 (85% purity) was synthesised by Dr Aisling
Miller (University of Manchester), using a combination of chemical and enzymatic
transformations.41 Reactions (200 ꢂL) were performed in 50 mM Tris pH 7.0 containing 3 (1 mM)
and KSI extract (189 ꢀL) and incubated at 30 °C for 24 h (180 rpm). Samples were prepared for
GCMS analysis by solvent extraction with 180 ꢂL ethyl acetate containing 0.01% secꢀbutylbenzene
as the internal standard, and dried with anhydrous MgSO4. GCꢀMS achiral quantitative analysis was
conducted on a 7890B GC coupled to a 5975 series MSD quadrupole mass spectrometer and
equipped with a 7693 autosampler (Agilent Technologies). Chiral product analysis was performed
by analysing reactions by GC using an Agilent Technologies 7890A GC system with an FID
detector and a ChirasilꢀDEXꢀCB column (Agilent; 25 m, 0.32 mm, 0.25 ꢂm). Further
chromatography details can be found in Supporting Information Section S3.2.
Yields of (R)ꢀ2 were corrected for the initial concentration present in substrate 3 (15%). Due to
limitations in the quantity of inꢀhouse synthesised 3, three colonies per plate were pooled together
to test for an increase in yield of (R)ꢀ2. Pooled samples exhibiting an increased (R)ꢀ2 yield
compared to wildꢀtype KSI were rescreened individually, as above, to identify the well(s)
responsible for the improved yield. Quantification was performed by comparing the peak areas to
the calibration curves of authentic standards. Vendor binary files were converted to open mzXML
data format42 using ProteoWizard msConvert.43 Automated peak profiling and quantification was
conducted using inꢀhouse scripts written in R.
Comparative activity of the variants. First generation KSI variant hits and later mutants generated
by siteꢀspecific mutagenesis were cultured from glycerol stocks in LB medium containing 100
ꢂg/mL ampicillin as above. The KSIꢀencoding plasmids were extracted and purified (Machereyꢀ
Nagel), and underwent Sanger sequencing to identify the mutation present. Larger scale cultures
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(500 mL) were generated for each variant in the same antibioticꢀselective LB medium, and
incubated at 37 °C (180 rpm) to an OD600nm of 0.6ꢀ0.8, followed by induction with 0.1 mM
isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG). Cultures were incubated overnight at 25 °C (180 rpm)
and the cells harvested by centrifugation (3000 x g for 5 min). Each variant underwent extraction (4
mL lysis buffer) and purification (0.5 mL NiꢀNTA resin) using the same protocol and buffers as in
the initial screen (Supporting Information Section S3.1). Biotransformations were performed with 1
mM 3 as above (10 ꢀM KSI), and the variants that did not display improved yield of (R)ꢀ2 above
wild type (false positives) were discarded.
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Steadyꢀstate reactions with (+)ꢀcisꢀisopulegone. Continuous steady stateꢀreactions with 3 were
performed with selected purified variants to investigate the effect of the mutation(s) on the reaction
rate. Reactions (100 ꢂL) were composed of 50 mM Tris pH 7.0 containing 1 mM 3 within UVꢀStar
microplates (Greiner BioꢀOne) and covered with a ClearVue sealing sheet. Product formation was
detected by monitoring the absorbance at 260 nm for 1 h at 20 °C using a CLARIOstar microplate
reader (BMG Labtech). Concentrations were determined by comparison to a standard curve (Figure
S1), and data were corrected for both 3 and (R)ꢀ2 loss over 1 h (control substrate and product data).
Cascade biocatalysis. The Mentha enzymes MpPGR and MMR were produced (500 mL culture;
20 °C) and purified according to the method for the KSI variants (Supporting Information Section
S3.1). Reactions (200 ꢂL) were performed in 50 mM Tris pH 7.0 containing 10 ꢂM KSI, 1 ꢂM
MpPGR, 0.3 ꢂM MMR, 1 mM 3 and a cofactor recycling system (10 U Sigma glucose
dehydrogenase, 10 ꢂM NADP+ and 15 mM Dꢀglucose) to supply MpPGR and MMR with
NADPH.23 Reactions were incubated at 30 °C for 24 h (180 rpm), and the extracts were analysed by
GCꢀMS as before.
Pathway assembly and in vitro activity. The genes encoding KSI variant V88I/L99V/V101A/
D103S, MpPGR and MMR and their respective ribosomal binding sequences (RBS) were amplified
by PCR and ligated into pET21b using InꢀFusion cloning to create an expression construct under
the control of one T7 promoter. The RBS of MMR was modified by PCR mutagenesis to generate
three constructs with lower predicted translation initiation rates (Table S3). Each construct was
transformed into competent E. coli NEB5α cells, and underwent culture growth, plasmid
purification and DNA sequencing as before.
The plasmids were transformed into competent E. coli NiCo21 (DE3) expression cells and cultured
on LB agar containing 100 ꢂg/mL ampicillin. Single colonies were selected and grown in 200 mL
phosphate buffered Terrific Broth pH 7.0 containing 0.4% glycerol and 100 ꢂg/mL ampicillin,
followed by induction with 0.1 mM IPTG. The cultures were incubated overnight at 20 °C, and
harvested by centrifugation (3000 x g). Soluble cell extracts were generated using lysis buffer (3
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mL pH 7.0), sonication and centrifugation as above. Cascade biocatalysis reactions were set up as
before, except the purified enzymes were replaced with 50 ꢂL of cell extract.
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Protein crystallization and structure solution. Purified KSI variants D103S, V88I/L99V,
V88I/L99V/D103S and V88I/L99V/V101A/D103S (10 mg/mL) were crystallised using a Mosquito
robot (TTP Labtech) and the following crystallisation solutions: i) 10ꢀ25% PEG3350 with 0.2 M
MgCl2; ii) 10ꢀ25% PEG3350 with 0.2 M ammonium acetate (pH 4.6) and iii) 10ꢀ25% PEG3350
with 0.2 M ammonium acetate (pH 5.5). Crystals were flash frozen in 30 % glycerol and data
collected at the Diamond light source (beamlines IO4 and IO3). Reflections were merged and
scaled with Xia2.44 All mutant structures were solved by molecular replacement using PhaserꢀMR
(CCP4)45 and wild type KSI (PDB code: 1OPY) as a search model. Initial model building was done
with AutoBuild (PHENIX)46 followed by iterative cycles of manual model building and refinement
in COOT47 and Phenix.refine48 respectively. Mutations could be clearly identified in Fo-Fc
difference maps and were modeled accordingly. The final data collection and refinement statistics
are found in Table S4. The atomic coordinates and structure factors (6F4Y, 6F50, 6F53 and 6F54)
have been deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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Molecular modelling. DFT modelling was performed using Gaussian09 revision D.01.49 An active
site model was created from the crystal structure of KSI with bound androstenedione (6; PDB ID
1OHS) including the sideꢀchains and αꢀcarbons of the 15 firstꢀshell residues shown in Figure S4,
with substrate 3 built in using the coordinates of the corresponding ligand 6 atoms as the starting
point. Energy minimisation was carried out using the B3LYP hybrid functional and 6ꢀ31G(d) basis
sets50 with the D3 version of Grimme’s dispersion.51 Since the active site is solvent exposed, a
continuum solvent model was used to mimic the shielding effect of water. Molecular dynamics
simulations were carried out using Gromacs 4.6.1 with the Gromos 53A6 force field and periodic
boundary conditions.52ꢀ53 For WT KSI, the crystal structure of KSI with bound 6 (PDB: 1OHS) was
used, with substrate 3 built in using the coordinates of the corresponding ligand 6 atoms as the
starting point. For KSI variant V88I/L99V/V101A/D103S, ligand 3 was placed in the active site of
the crystal structure (PDB: 1OPY) by aligning the protein to the wild type KSI model. The energy
minimization parameters are described in detail in Supporting Information Section S6.
RESULTS AND DISCUSSION
Identification of KSI as a (+)ꢀcisꢀisopulegone isomerase. Prior studies with the partially purified
native M. piperita isomerase suggested its mechanism resembled that of the bacterial ∆5ꢀ3ꢀ
ketosteroid isomerase family (EC 5.3.3.1).26 These enzymes catalyze the isomerization of ∆5ꢀ3ꢀ
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ketosteroids, such as 5(10)estreneꢀ3,17ꢀdione, to the equivalent ∆4ꢀ3ꢀketosteroid by intramolecular
transfer of the C4β proton to the C6β position.54 Prior studies showed that KSI from P. putida reacts
with truncated substrate 3ꢀcyclohexenꢀ1ꢀone, forming the more stable 2ꢀcyclohexenꢀ1ꢀone.55
Therefore we postulated that KSI might react with the structurally related compound 3 to generate
(R)ꢀ2 using a similar mechanism (Scheme 1B).
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A Cꢀterminally His6ꢀtagged version of wildꢀtype KSI from P. putida was generated, and
biotransformations with 3 showed 2 was formed (35% yield). Chiral GC analysis confirmed the
product generated was (R)ꢀ2, the enantiomeric form required for 1 biosynthesis (Table S5).2 An
additional minor quantity (<1% yield) of side product (–)ꢀtransꢀisopulegone (7) was also detected.
As the (R)ꢀ2 yields were judged to be too low for biotechnological applications, we employed a
directed evolution strategy to engineer KSI for improved synthesis of (R)ꢀ2.
High throughput directed evolution of KSI. Comparative studies of KSI with single and multiple
ring substrates showed that while the specificity (kcat/Km) is 27,000 fold higher for steroid
substrates, the reaction rates were within twoꢀfold.55 This suggests that remote binding interactions
with steroid substrates have a marked impact on the strength of substrate binding, and singleꢀring
substrates like 3 may have poor binding affinity. Therefore, our initial KSI mutagenesis strategy
was to increase binding of 3 by targeting every amino acid known or predicted to interact with
steroid substrates. An examination of the coꢀcrystal structure of wildꢀtype KSI bound to the reaction
intermediate analogue equilenin (PDB 1OH0)34 showed that sixteen residues were located within 5
Å of the ligand. Given the overall apolar nature of the KSI active site and 3, maintaining this
environment would likely maximise our search for optimised mutations whilst minimising the size
of the variant libraries and subsequent screening effort. Reducing the number of screening assays
was critical given the limited availability of the inꢀhouse synthesised substrate 3.
Twelve of these residues were targeted for individual limited randomizing mutagenesis, where the
general physicochemical properties of the amino acids were conserved (Table S1). Residues Y16
and Y57 are both known to partake in Hꢀbonding with the substrate carbonyl moiety, therefore
these residues were mutated combinatorially, using an ambiguous codon (WVC) that encodes other
residues capable of Hꢀbonding. Similarly, residues V88 and L99 were selected for combinatorial
mutations using an ambiguous codon (DTK) for hydrophobic residues, given their close proximity
to the substrate analogue.34 Variant sequences were created by gene synthesis using ambiguous
codons (Table S2) and constructed using the SpeedyGenes protocols.37ꢀ38 In total, 158 variants were
created and screened for improvement in (R)ꢀ2 production using high throughput robotic culturing
and protein purification protocols (summarized in Figure S2). Comparative biotransformations were
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performed with 3, with quantitation and product identification by GCꢀMS, to identify variants with
3
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higher activity than wild type KSI.
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Table 1. Biotransformations of wild type and variant KSI with 3.
Specific Activity (ꢀM/ꢀM/d)a
Round
Variant
Relative Activity (fold)
9
ꢀ
Wild type
13.88 ± 0.08
1.0
1.4
1.7
1.3
1.4
1.9
1.8
1.9
4.5
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D103S
20.09 ± 1.16
L99I
23.58 ± 0.10
L99V
18.46 ± 0.48
V88I/L99V
V88I/L99V/D103S
L99V/D103S
L99I/D103S
V88I/L99V/V101A/D103S
19.39 ± 0.57
2
3
26.47 ±1.59
25.51 ± 0.83
25.91 ± 0.58
63.10 ± 0.61
Duplicate reactions (200 ꢂL) were performed in 50 mM Tris pH 7.0 containing 1 mM 3 and 10 ꢀM KSI. After a
24 h incubation at 30 °C (180 rpm), reactions were extracted with 180 ꢂL ethyl acetate containing 0.01% secꢀ
butylbenzene and dried with anhydrous MgSO4. Products were analysed by GCꢀMS using a DBꢀWAX column.
aSpecific activity is expressed as ꢀM (R)ꢀ2 produced per ꢀM KSI in 24 hours.
Four enzyme variants were identified as having minor increases in (R)ꢀ2 yield (1.4ꢀ1.7 fold), three
of which contained mutations of residue L99 (L99I, L99V and V88I/L99V; Table 1). Interestingly,
both L99I and L99V involve mutations to a smaller side chain, suggesting these changes may
impact on 3 binding by reducing steric hindrance. The fourth improved variant D103S also
contained a smaller side chain, however there was also a change from a negatively charged moiety
to a polar functionality. As a serine hydroxyl group can participate in Hꢀbonding, this mutation
might maintain the native enzyme hydrogen bonding interaction with the substrate carbonyl group.
Second round: compiling mutations. To investigate the combinatorial effect of the initial screen
variants, the L99Xꢀcontaining single and double mutants were each combined with D103S using
asymmetric PCR mutagenesis.39ꢀ40 Each variant was purified and underwent biotransformations to
determine any improvement in (R)ꢀ2 yield by GCꢀMS (Table 1). Unfortunately, these double and
triple variants showed only moderate improvements in (R)ꢀ2 yield compared to wild type enzyme
(1.8ꢀ1.9 fold).
Third round: Structureꢀguided mutation. To ensure the mutations in the active site and
surrounding residues did not affect the overall fold of KSI, we solved the Xꢀray crystal structures of
four key variants D103S, V88I/L99V, V88I/L99V/D103S and V88I/L99V/V101A/D103S (Table
S4). Each of these structures showed backbone conformations virtually identical to the coꢀcrystal
structure of wildꢀtype KSI bound to reaction intermediate analogue equilenin (Figure S3),34 with
RMSD values of 0.427, 0.418, 0.443 and 0.357, respectively. All the mutations could be clearly
identified in Fo-Fc difference maps, and were incorporated without any major displacement of the
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backbone atoms or surrounding residues. We did note minor rearrangements of backbone atoms
within the loop region 90ꢀ98 (results not shown). This loop is exposed to the bulk solvent and
shows above average B factors compared to surrounding residues, implying the observed
differences are down to inherently flexibility, rather than any mutational changes to neighbouring
residues.
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Inspecting the spatial arrangement of these mutations showed residue V101 was positioned directly
between L99V and D103S, highlighting it as another potential target for mutagenesis (Figure 1B
inset). Given that most of the existing variants resulted in a shortening of the amino acid side chain,
the triple variant was modified to incorporate the mutation V101A. Biotransformations were
performed on the KSI V88I/L99V/V101A/D103S variant, which showed a more dramatic increase
in (R)ꢀ2 production over wild type enzyme (4.5ꢀfold; Table 1). Therefore, the modification of V101
to an alanine residue has resulted in over a 2ꢀfold increase in (R)ꢀ2 formation over the triple variant.
Figure 1. Location of key active site mutations implicated in improving KSI activity towards 3. A) Residues
located in the equileninꢀbinding region of wild type KSI from P. putida (PDB: 1OH0).34 The residues and
equilenin are shown as atom coloured sticks with yellow and green carbons, respectively. Interactions are
shown as red dotted lines. The backbone is shown as a grey cartoon. B) Comparative steady state activity of
wild type and variant KSI enzymes. Reactions (100 ꢂL) were composed of 50 mM Tris pH 7.0 containing 1
mM 3. The absorbance was monitored at 260 nm for 1 h at 20 °C. Inset: The location of the variant residues
in KSI V881/L99V/D103S. Backbone and mutations are shown as grey ribbons and balls, respectively (blue
balls for V101).
To investigate the impact of the mutations further, the specific activity of the variants with substrate
3 was determined under pseudoꢀsteady state conditions (1 h reactions). This method takes
advantage of the enolization of 3 to (R)ꢀ2, which results in the development of a peak around 260
nm (Figure S1). First generation variants showed minor rate increases (around 1.3ꢀfold), while a
near 2ꢀfold improvement was seen with triple variant V88I/L99V/D103S (Figure 1B). The addition
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of mutation V101A generated a 4.3ꢀfold increase in specific activity compared to the WT (61.17
ꢂmol/min/mg compared to 14.16 ꢂmol/min/mg; Figure 1B). Therefore, the increases in the overall
reaction rate mirror the increased product yields detected in the 24 h biotransformations.
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Modeling and molecular dynamics simulations for KSI variants. We performed comparative
DFT modeling and MD simulations between wild type KSI and variant V88I/L99V/V101A/D103S
to investigate the origin(s) of activity rate changes. The DFT optimized model for wild type KSI
suggests substrate 3 carbonyl group hydrogen bonds with D103 (< 2.0 Å), but the orientation is
nonꢀideal for catalysis due to a less favourable interaction distance for the proton abstraction step
(Figure 2 and Figure S4; Scheme 1B). Additionally, residue L99 has reoriented outwards from the
active site, causing a twist in the βꢀhairpin from residue 88 to 99 (Figure S2).
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B
A
Figure 2. Overlay of energy minimised structure (green) and representative structure from MD simulations
(brown) for (A) wildꢀtype KSI and (B) variant V88I/L99V/V101A/D103S modelled with 3 in the active site.
The solvent accessible surface areas for the substrate and residues 88, 99 and 101 from the representative and
minimised structures, respectively, are shown as transparent surfaces. The dotted lines illustrate potential
steric clashes between the substrate and enzyme. The MD simulations were performed using Gaussian09
revision D.01.49
MD simulations of variant KSI with 3 restrained in a reactive conformation suggests the decrease in
bulk of variant residues I88, V99 and A101 allows the substrate to comfortably adopt this position
(Figure 2B). The position of 3 looks more ideal, with a D40ꢀsubstrate hydrogenꢀoxygen distance of
2.04 Å in the DFT model. Therefore, the decrease in bulk of variant residues I88, V99 and A101
has allowed this reorienting of the substrate, whilst still maintaining a (slightly longer) hydrogen
bonding interaction with residue S103 (D103 in wild type). MD simulations with the variant model
showed no repositioning of the hairpin loop from 88ꢀ99 was necessary, as 3 fitted more easily
within the active site. Overall, molecular simulations suggest these four mutations have improved
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binding of
3
in a functional conformation within the active site. Therefore, the
3
4
V88I/L99V/V101A/D103S variant was selected for further studies to see if it can be used in
cascading reactions for the biosynthetic production of 1 from 3.
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Oneꢀpot (−)ꢀmenthol production with purified enzymes. Initial cascading reactions were
performed with purified enzymes to eliminate any interference from competing E. coli enzymes
present in whole cells and cell extracts (Table 2). Interestingly, reactions with KSI variant
V88I/L99V/V101A/D103S showed the formation of ~ 10% of by products, such as transꢀ
isopulegone (7). This suggests the presence of the 4 mutations has altered the range of possible
binding conformations of 3, leading to additional isomer formation. The pathway from 3 to 1 also
requires the presence of two Mentha enzymes, namely pulegone reductase (MpPGR)29 and (–)ꢀ
menthone:(–)ꢀmenthol reductase (MMR) to generate (–)ꢀmenthone (8) and 1, respectively (Scheme
1A).2 Initial biotransformations of purified MpPGR with (R)ꢀ2 showed it produced near equivalent
yields of 8 and (+)ꢀisomenthone (9) (ratio 49:51; Table 2). This differs slightly from previous
studies of a nonꢀHis6ꢀtagged recombinant form of MpPGR, which showed a 8 to 9 ratio of 55:45.29
However this enzyme showed a significantly higher yield of 8 than seen with the homologous
enzyme from Nicotiana tabacum (NtDBR) performed under the same reaction conditions.56
Similarly, purified MMRꢀHis6 demonstrated similar activity towards 8 as in previous studies,5
showing primarily 1 production (Table 2).
7
8
9
10
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Table 2. Biotransformations of individual enzymes and cascading reactions with Mentha monoterpenoids.
Substrate remaining/Product yield (ꢀM)
Biocatalyst
(R)ꢀ2
3
8
9
3
5
Individual enzymes
KSI WT
361.7 ± 11.5
40.7 ± 0.7
N/A
210.4 ± 6.0
436.3 ± 10.8
10.0 ± 1.1
N/A
N/A
N/A
153.3 ± 5.8
8.0 ± 2.0
N/A
N/A
160.1 ± 4.5
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
KSI variantaꢀb
MpPGR
MMR
N/A
280.7 ± 10.3
Cascading reactions
Cascadeb
Cell extractb
Whole cellsb
16.7 ± 1.2
17.1 ± 0.1
32.5 ± 6.7
10.3 ± 0.1
11.5 ± 0.5
1.0 ± 2.1
25.2 ± 0.9
60.6 ± 1.9
44.7 ± 10.3
16.6 ± 0.5
34.8 ± 0.3
98.6 ± 2.4
63.4 ± 3.8
52.8 ± 0.9
158.8 ± 0.4
11.1 ± 0.6
18.6 ± 0.8
19.6 ± 0.1
Duplicate reactions (200 ꢂL) were performed in 50 mM Tris pH 7.0 containing 1 mM monoterpenoid substrate,
enzyme(s) and cofactor recycling system (10 U Sigma glucose dehydrogenase, 10 ꢂM NADP+ and 15 mM Dꢀglucose).
After a 24 h incubation at 30 °C (180 rpm), reactions were extracted with 180 ꢂL ethyl acetate containing 0.01% secꢀ
butylbenzene and dried with anhydrous MgSO4. Products were analysed by GCꢀMS using a DBꢀWAX column (Figure
S7). Reactions with individual enzymes had enzyme concentrations of 10 ꢀM. The in vitro cascading reaction had
enzyme concentrations of 10, 2 and 0.3 ꢀM for KSI variant, MpPGR and MMR, respectively. Control
biotransformations of each compound in the absence of enzymes (data not shown) showed losses occurred over 24 h
due to nonꢀenzymatic degradation. Therefore the product yields are likely an underestimation of the true yields. The cell
extract and whole cell slurry volumes in the cascading reactions were 50 ꢀL. aKSI variant
=
V88I/L99V/V101A/D103S; bAdditional products were detected that were not quantified (e.g. 7 and (+)ꢀneoisoꢀ
isopulegol (10)). Substrates are shown in bold. N/A = not applicable.
Initial
cascading
biotransformations
of
purified
KSI
wildꢀtype
and
variant
V88I/L99V/V101A/D103S with MpPGR and MMR with 3 showed the major product was an
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isomer of isopulegol (results not shown). Chiral GC analysis of the reaction showed the main
isomer formed was (+)ꢀneoisoꢀisopulegol (10), with a minor proportion of (–)ꢀisopulegol generated
(Figure S6).57 However product 1 was also detected, with 5.1ꢀfold higher yields obtained for
5
6
7
reactions containing V88I/L99V/V101A/D103S variant KSI (46 ꢀM vs 9 ꢀM).
8
9
Subsequent reactions with each individually purified biocatalyst showed this side product was
obtained by the action of MMR on 3, which differs from earlier studies.5 However, this apparent
disparity is likely due to the 144ꢀfold reduction in the reaction time of the earlier study compared to
this work (6ꢀ9 min vs 24 h).5 It is likely that the mechanism of action of MMR with 3 proceeds in a
similar manner to the Short Chain Dehydrogenase/Reductase ketoreduction mechanism described
previously for MMR and MNMR with substrates 8 and 9 (Scheme 2).58 Unfortunately, the
formation of 10 by MMR appeared to dominate over the reaction with 8, likely due to the lack of
the latter substrate at the start of the reaction.
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CONH2
H
CONH2
NADPH
NADP+
H
O
O
H
R
R
N
N
10
3
HO
HO
O
H
MMR
OH
H
H
O
H
H
HO
N
H
H
N
H
H
H
H
O
O
H
H
O
O
O
O
S188
S188
K248
K248
H
H
Y244
Y244
Scheme 2. Proposed mechanism of action of the NADPHꢀdependent MMRꢀcatalysed reduction of 3 to 10.
This is adapted from the proposed SDR ketoreductase mechanism of MNMR with 8.58
Subsequent cascading reactions were performed with a 30ꢀfold molar excess of KSI over MMR, to
allow KSI activity with 3 to predominate. Using this approach, we dramatically increased 1 titres
(63.4 ꢂM; 14.1% conversion; Table 2) relative to 10, producing a near 1:1 ratio of products. In
addition, a surprising yet advantageous observation was that relatively lower levels of 9 (and
subsequently 5) were produced than expected for MMR, with a combined ratio of 76:24 (8+1: 9+5).
Therefore, these studies have demonstrated that KSI variant V88I/L99V/V101A/D103S can be
utilised in cascading reactions as the first step in generating 1 from 3.
Cascading production of 3 using cellꢀfree extracts and whole cells. We have previously reported
the biosynthesis of menthol isomers from (R)ꢀ2, using E. coli cell extracts expressing the enzymes
NtDBR and MMR.23 To further extend this approach; we developed a single expression construct in
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pET21b containing KSI variant V88I/L99V/V101A/D103S, MpPGR and MMR, with each gene
under the control of one T7 promoter (Table S3). This enabled the IPTGꢀdependent coꢀexpression
of all three genes in E. coli. Biotransformations were performed using cell extracts with 3 in the
presence of a cofactor recycling system, the latter to supply MpPGR and MMR with NADPH. As
expected, the major product obtained was 10, due to the promiscuous activity of MMR (results not
shown).
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To mimic the effect of increasing the KSI:MMR concentration ratio in vivo, we modified the RBS
sequence upstream of MMR to three alternative ones, each with predicted translation initiation rates
(TIR) less than the existing one of 4100 (1000, 500 and 200; Table S3).59ꢀ60 Of the three constructs,
only the one containing an RBS with a predicted TIR of 500 showed any MMR activity, leading to
the production of both 1 and 10 (Table 2). Cell extracts of this construct yielded 53 ꢂM of 1, similar
to that obtained with the purified enzyme oneꢀpot biotransformations. The higher levels of 8 and 9
detected reflects the lower in vivo concentration of MMR within this construct. Reactions were also
conducted with whole cells, producing nearly 3ꢀfold higher levels of 1 than cell extract
biotransformations (159 ꢂM, 27% total yield; Table 2) with proportionally less 10.
These studies have demonstrated that these three enzymes have great potential in an overall strategy
for in vivo production of 1 in E. coli. Ideally KSI V88I/L99V/V101A/D103S would be incorporated
into an E. coli construct containing all the remaining enzymes required for 1 production from (S)ꢀ4
(Scheme 1A). This would enable oneꢀpot biotransformations or in vivo fermentations to be
performed using the inexpensive and renewable starting material (S)ꢀ4. Ultimately, the inclusion of
an (S)ꢀ4 production pathway would enable E. coli to produce 1 in vivo from simple sugars.61 This
would avoid the addition of high concentrations of toxic monoterpenoid precursors and eliminate
the need for exogenous cofactor recycling systems. Additionally, the low in vivo concentration of 3
would likely minimise pathway hijacking to form 10, increasing the overall yields of 1.
CONCLUDING REMARKS
The implementation of a synthetic biology approach to microbial 1 production has been hampered
by the absence of the sequence for the native Mentha pathway enzyme IPGI, and the lack of
identification of plant proteins exhibiting homology to bacterial ꢁ5ꢀ3ꢀketosteroid isomerases known
to catalyze this reaction on steroid substrates. We took the approach of engineering an existing
bacterial ꢁ5ꢀ3ꢀketosteroid isomerase to improve its activity towards 3, using a structureꢀmodeling
guided, semiꢀrational design to identify target residues. Our implementation of a robotics platform
to ensure rapid and reproducible automated gene assembly, enzyme purification and microꢀscale
biotransformation screening enabled the identification of potential variant biocatalysts with limited
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substrate availability. The incorporation of four targeted mutations into KSI enabled a 4.3ꢀfold
increase in IPGI activity, generating a biotechnologically useful enzyme with significant in vivo
activity. Further engineering studies could potentially improve its functionality, thereby driving the
cascading reactions towards more efficient 1 production.
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This is the first report of a nonꢀMentha enzyme with IPGI activity, thereby identifying the ‘missing
link’ to complete the enzymatic route from (S)ꢀ4 to 1. This will enable for the first time the full
construction of a biosynthetic pathway for microbial production of 1. The demonstration of
successful 1 production from 3 by engineered E. coli adds to a growing number of studies
highlighting the potential usefulness of microbial hosts in monoterpenoid production. It is
anticipated that integrating our existing KSIꢀMpPGRꢀMMR construct into a larger chassis will
enable the production of menthol isomers from inexpensive precursors, providing a sustainable and
renewable production platform to meet the growing global demand for natural menthol products.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information document contains additional experimental details, materials used,
additional tables and figures, results, GC/MS analyses, and discussion of computational and
crystallographic studies. The Supporting Information is available free of charge on the ACS
Publications website at DOI:
AUTHOR INFORMATION
ORCID
Andrew Currin: 0000ꢀ0001ꢀ7845ꢀ8837; Linus O. Johannissen: 0000ꢀ0002ꢀ0916ꢀ9094; Maria
Vinaixa: 0000ꢀ0001ꢀ9804ꢀ0171; Neil Swainston: 0000ꢀ0001ꢀ7020ꢀ1236; Nicholas Rattray: 0000ꢀ
0002ꢀ3528ꢀ6905; John M. Gardiner: 0000ꢀ0003ꢀ1827ꢀ6817; Douglas B. Kell: 0000ꢀ0001ꢀ5838ꢀ
7963; Eriko Takano: 0000ꢀ0002ꢀ6791ꢀ3256; Helen S. Toogood: 0000ꢀ0003ꢀ4797ꢀ0293; Nigel S.
Scrutton: 0000ꢀ0002ꢀ4182ꢀ3500
Author Contributions
We thank Colin Levy for protein crystallisation and data collection, and the wider SYNBIOCHEM
team for their contributions. The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Funding Sources
The UK Catalysis Hub (http://www.ukcatalysishub.co.uk) is kindly thanked for resources and
support. Funding was provided by the Biotechnology and Biological Sciences Research Council
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(BBSRC) and Engineering and Physical Sciences Research Council (EPSRC) under grants
BB/M017702/1 (SYNBIOCHEM), BB/J015512/1 and BB/M017702/1.
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Notes
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8
AC, HT and NS are named inventors on a submitted patent describing the work in this manuscript.
9
ACKNOWLEDGMENTS
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The authors would like to acknowledge the assistance given by IT Services and the use of the
Computational Shared Facility at The University of Manchester.
TOC
Bacterial ketosteroid isomerase replaces peppermint isopulegone isomerase for (ꢀ)ꢀmenthol
biosynthesis in Escherichia coli.
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(3) Leavell, M. D.; McPhee, D. J.; Paddon, C. J., Curr. Opin. Biotechnol. 2016, 37, 114ꢀ119.
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