Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM)

Article abstract of DOI:10.1021/ja035742h

Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L^-1 d^-1. Copyright

Full text of DOI:10.1021/ja035742h

Published on Web 08/26/2003
Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site
Saturation Mutagenesis (GSSM)
Grace DeSantis,* Kelvin Wong, Bob Farwell, Kelly Chatman, Zoulin Zhu, Geoff Tomlinson,
Hongjun Huang, Xuqiu Tan, Lisa Bibbs, Pei Chen, Keith Kretz, and Mark J. Burk*
DiVersa Corporation, 4955 Directors Place, San Diego, California 92121
Received April 22, 2003; E-mail: gdesantis@diversa.com; mburk@diversa.com
Despite the successful development of biocatalysts for a variety
of important transformations, enzymes historically have suffered
from numerous shortcomings that have limited their utility in
chemical, agrochemical, and pharmaceutical applications.1 At-
tributes such as low specific activity, inadequate substrate scope,
low or undesired enantiomer specificity, intolerance to organic
solvents, and low volumetric productivity are challenges often
encountered when implementing biocatalytic processes. By screen-
ing genomic libraries prepared from environmental samples col-
lected around the globe, we recently discovered over 200 unique
nitrilases that allow mild and selective hydrolysis of organonitriles
Scheme 1. Synthesis (R)-4-Cyano-3-hydroxybutyric Acid
Scheme 2. High-Throughput Screen for Improved Selectivity
,2
for each amino acid residue of the WT enzyme. Subsequent use of
standard methods for DNA replication generates a library of genes
3
to carboxylic acid derivatives. Several enzymes were shown to be
effective for enantioselective hydrolysis of hydroxynitriles to
produce R- and â-hydroxy acids on small reaction scale. Of
particular commercial interest was the nitrilase-catalyzed desym-
metrization of 3-hydroxyglutaryl nitrile (1) to afford (R)-4-cyano-
possessing all codon variations required for saturation mutagenesis
of the original gene.4 Transformation and expression of this
collection of genes in a host organism, such as Escherichia coli,
5
furnishes a comprehensive library of single-site enzyme mutants.
3
-hydroxybutyric acid (2), the ethyl ester of which is an intermediate
The GSSM technique was employed to create a comprehensive
library from our best wild-type nitrilase, which is 330 amino acids
in length. The nitrilase GSSM library accesses every single amino
acid variant and contained 10,528 genetic variants. It was screened
with three times oversampling to ensure complete coverage of the
3
to the cholesterol-lowering drug Lipitor (Scheme 1).
Unfortunately, efforts to develop a larger-scale process for
economic production of this intermediate were plagued by lower
enantiomeric excess (ee) as the substrate concentration was
increased. We now report the application of a novel directed-
evolution technique, the gene site saturation mutagenesis (GSSM)4
method, to address this challenge. This led to a single amino acid
nitrilase variant that has ideal properties for the synthesis of this
important pharmaceutical intermediate, (R)-4-cyano-3-hydroxy-
butyric acid.
6
library. Hence, 31,584 clones were screened in an attempt to
identify mutant enzymes with improved enantioselectivity.
Success in identification of the requisite phenotype in gene
evolution experiments relies on the development of methods to
7
rapidly evaluate libraries containing large numbers of mutants. To
efficiently probe our GSSM library of nitrilases for improved
enantioselectivity, we developed a novel high-throughput screen
based on mass spectrometric detection of isotopically differentiated
The most effective nitrilase identified in discovery efforts
catalyzed the complete conversion of 1 to 2 in 24 h with a product
3
8
ee of 95% at 100 mM substrate concentration. However, attempts
products (Scheme 2). When developing an assay it is imperative
to operate this transformation under more practical conditions
revealed a steady decrease in enantioselectivity with increasing
concentration; at [1] ) 0.5, 1, 2, and 3 M, enantiomeric excesses
were 92.1, 90.7, 89.2, and 87.6%, respectively, determined at
reaction completion.
that the surrogate substrate be chemically similar to the actual
substrate to ensure that any variants identified will perform the
desired transformation effectively. Since substrate 1 is symmetric,
a method was required to distinguish the two nitrile groups and
1
5
detect the site of hydrolysis. Accordingly, we synthesized the N-
labeled analogue 15N-(R)-1. Use of the chiral N-(R)-1 substrate
is important since the labeled atom is removed from the product
upon hydrolysis with a (R)-selective enzyme, whereas the label
remains when hydrolyzed with a (S)-selective enzyme. The hy-
drolysis products differ by one mass unit and can be distinguished
and quantified by mass spectrometry. In this assay the screening
substrate and target substrate differ only by an isotope label,
therefore ensuring that the identified nitrilase will perform equally
well on the native unlabeled substrate. For the WT enzyme, it was
15
To develop a practical process, the overall objective was to
identify a nitrilase that would catalyze the hydrolysis of 1 to (R)-2
with high enantioselectivities at substrate concentrations up to 3
M. Enzymatic reactions rarely operate well at such high concentra-
tions of organic substrate (33 wt %) due to substrate inhibition,
product inhibition, enzyme instability, and lower activity and
selectivity.1 Our desire to develop a viable process for production
of 2 which yields high enantiomeric excess at high volumetric
productivities led us to employ our GSSM technology for directed
evolution of a nitrilase enzyme.
,2
1
5
shown that the observed ee is the same regardless of whether N-
(R)-1 or 15N-(S)-1 is used.
The GSSM technology is a unique method for rapid laboratory
evolution of proteins whereby each amino acid of a protein is
replaced with each of the other 19 naturally occurring amino acids.
This is accomplished at the genetic level through the use of
degenerate primer sets, comprising either 32 or 64 codon variants,
Screening the nitrilase GSSM library by the method outlined in
Scheme 2 was performed using 10 mM 15N-(R)-1 substrate in 100
mM phosphate buffer at 37 °C. Amino acid changes at 17 different
residues led to enhanced enantioselectivity over the wild-type
11476
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J. AM. CHEM. SOC. 2003, 125, 11476-11477
10.1021/ja035742h CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
Table 1. GSSM Mutants with Enhanced Enantioselectivity
substrate concentration. HGN (1 g, 9 mmol) was dissolved in
phosphate buffer (2.03 mL), and A190H nitrilase (30 mg) was
added. The reaction, was stirred at 20 °C for 15 h to furnish the
a
b
nitrilase
[HGN] 100 mM, % ee
[HGN] 2.25 M, % ee
time, h
WT
Ala55Gly
Ile60Glu
94.5 ( 0.1
96.5 ( 0.4
96.5 ( 0.5
97.9 ( 0.1
96.8 ( 0.2
96.5 ( 0.2
95.8 ( 0.5
97.9 ( 0.1
97.9 ( 0.1
97.9 ( 0.1
97.9 ( 0.1
97.9 ( 0.1
97.9 ( 0.1
87.8 ( 0.2
nd
24
>160
>160
15
-
1
-1
desired acid (R)-2 (1.1 g, 96%, 98.5% ee) with a 619 g L
d
nd
volumetric productivity. This demonstrates that an efficient process
achieving both high product enantiomeric excess and volumetric
productivity may be accessed using the evolved nitrilase.
The benefits of the GSSM evolution strategy were demonstrated
through identification and development of an improved nitrilase
that provides practical access to a valuable intermediate for the
drug Lipitor. Changing Ala to His provided a 10% increase in the
enantiomeric excess at the commercially relevant 3 M substrate
reaction concentration. Furthermore, error-prone PCR or gene
recombination of this enzyme with known nitrilases would not have
furnished the improved A190H variant enzyme. We currently are
attempting to solve the structure of this variant as well as the wild-
type enzyme to gain an understanding of the molecular interactions
responsible the improvements.
Ala190His
Ala190Ser
Ala190Thr
Asn111Ser
Phe191Leu
Phe191Thr
Phe191Met
Phe191Val
Met199Glu
Met199Leu
98.1 ( 0.1
95.5 ( 0.7
96.6 ( 0.4
96.1 ( 0.9
40
40
>160
>160
>160
>160
>160
>160
160
c
nd
nd
nd
nd
nd
95.4 ( 0.1
a
1
00 mM reactions were performed with nitrilase expressed from E.
b
coli in whole cell format and were complete within 36 h. 2.25 M reactions
were performed with nitrilase as lyophilized clarified cell lysate. All % ee
data were determined at the indicated time and are reported as the average
of three measurements with standard deviation of the mean. Time for
c
reaction completion established by TLC. nd ) not determined.
Acknowledgment. We are grateful to Diversa Sequencing for
sequence confirmation and J. Macomber, J. Verruto, and J. Poland
for subcloning and library construction, A. Solbak and N. Cloar
for colony picking, T. Todaro and M. Lafferty for robotics support,
E. O’Donoghue, A. Flordeliza, and C. Cowden, for protein analysis
support, M. Podar, M. Wall, and T. Richardson for bioinformatics
support, and D. Robertson, B. Morgan, D. Weiner, J. Chaplin, and
J. M Short are gratefully acknowledged for guidance.
enzyme. Secondary characterization of primary hits was performed
by GC analysis using 100 mM of unlabeled substrate 1. These
studies showed that residues Ala190 and Phe191 were enantio-
selectivity “hot spots” with several mutants affording product 2
with higher ee (Table 1). Interestingly, in all identified up-mutations
at position 190, alanine was replaced with a residue that has
hydrogen-bonding potential.
Since the overall objective was to identify a nitrilase that would
catalyze the hydrolysis of 1 to (R)-2 with high enantioselectivity
at >1 M substrate concentration, each of these up-mutant enzymes
was evaluated at 2.25 M substrate concentration. Many of the
variants did not perform well at this higher substrate loading, and
reactions were not substantially complete after 6 days. However,
for each of the serine, histidine, and threonine 190 variants,
significant ee enhancement was observed. The Ala190His mutant
is the most selective and most active of the GSSM mutants allowing
complete conversion of 1 to (R)-2 in 98% ee within 15 h. This is
a dramatic improvement relative to WT, which yields (R)-2 in only
Supporting Information Available: Materials and methods, in-
cluding procedures for the preparation of all reagents, analytical details,
library construction, and screening methods. (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(
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5) Saturation mutagenesis and unbiased access to all 32 (NNK) codons and
6
access to all codon variations. Random mutagenesis techniques
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do not have His in position 190.
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process for synthesis of the Lipitor intermediate (R)-2 at 3 M
(
6) See Supporting Information for details about construction and screening
of the GSSM library.
(
7) (a) Wahler, D.; Reymond, J.-L. Curr. Opin. Chem. Biol. 2001, 5, 152.
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