U.S. patent application number 13/223604 was filed with the patent office on 2012-02-16 for alanine 2,3-aminomutases and related polynucleotides.
This patent application is currently assigned to CODEXIS, INC.. Invention is credited to Svetlana Balatskaya, Birthe Borup, Ranjini Chatterjee, Ish Dhawan, Richard J. Fox, Kenneth W. Mitchell, Emily C. Mundorff, Les Partridge, Matthew Tobin.
Application Number | 20120040437 13/223604 |
Document ID | / |
Family ID | 37963249 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040437 |
Kind Code |
A1 |
Balatskaya; Svetlana ; et
al. |
February 16, 2012 |
ALANINE 2,3-AMINOMUTASES AND RELATED POLYNUCLEOTIDES
Abstract
The present disclosure relates generally to polypeptides having
improved alanine 2,3-aminomutase (AAM) activity, the
polynucleotides encoding the AAM polypeptides, and expression
vectors and host cells for expressing the AAM polypeptides.
Inventors: |
Balatskaya; Svetlana;
(Fremont, CA) ; Borup; Birthe; (Redwood City,
CA) ; Chatterjee; Ranjini; (Belmont, CA) ;
Dhawan; Ish; (Foster City, CA) ; Fox; Richard J.;
(Kirkwood, MO) ; Mitchell; Kenneth W.; (Sunnyvale,
CA) ; Mundorff; Emily C.; (Poughkeepsie, NY) ;
Partridge; Les; (San Francisco, CA) ; Tobin;
Matthew; (San Carlos, CA) |
Assignee: |
CODEXIS, INC.
Redwood City
CA
|
Family ID: |
37963249 |
Appl. No.: |
13/223604 |
Filed: |
September 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11581946 |
Oct 16, 2006 |
8030051 |
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13223604 |
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60726967 |
Oct 14, 2005 |
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Current U.S.
Class: |
435/233 |
Current CPC
Class: |
C12N 9/90 20130101; C12P
13/06 20130101 |
Class at
Publication: |
435/233 |
International
Class: |
C12N 9/90 20060101
C12N009/90 |
Claims
1. An engineered alanine-2,3-aminomutase polypeptide with improved
enzyme activity as compared to enzyme activity of the polypeptide
of SEQ ID NO:4 and having at least about 80% sequence identity with
SEQ ID NO:4.
2. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
in which the engineered alanine-2,3-aminomutase polypeptide has at
least about 90% sequence identity with SEQ ID NO:4.
3. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
in which the enzyme activity is improved at least about 1.5 times
to about 30 times or more the enzyme activity of the polypeptide of
SEQ ID NO:4.
4. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
in which the enzyme activity is improved at least about 2 time to
about 30 times or more the enzyme activity of the polypeptide of
SEQ ID NO:4.
5. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
in which the enzyme activity is improved at least about 10 times to
about 30 times or more enzyme activity of the polypeptide of SEQ ID
NO:4.
6. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which has an amino acid sequence corresponding to the amino acid
sequence of SEQ ID NO:4 and which includes one or more
substitutions selected from: Y8H; D20N; L26I; 33P; K36E; E51G;
N74K; Q85R or K; Q86R; A91S; Q95M; L99V; T111P; C130S; R1355; 138Y;
A139T; Q141R; K142R; E149V; D156E; T163A; 1921; 5202T; T204A;
E244V; E247G; N251Y or S; R275G; M283I or V; V292A; L297M or F;
G300R; I313T; N315S; G331H; I339V; V341L or I; T342A; H353R; V355I;
V362A; I363T; N371D; E379V; A383V; G384D; K385E; K387R; A392T;
G395E; L399M; I401L; S404P; K410E; K412R; and F413L or S.
7. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which is a polypeptide having at least 97% identity to a
polypeptide selected from SEQ ID NO:295, 2068 and 2150.
8. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which is a polypeptide having at least 98% identity to a
polypeptide selected from SEQ ID NO: 258, 316, 350, 356, 790, 1006,
and 1250.
9. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which is a polypeptide having at least 99% identify to a
polypeptide selected from SEQ ID NO:14, 24, 58, 86, 100, 312, 336,
380, 318, 404, 444, 490, 550 or 774.
10. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which has an amino acid sequence corresponding to the amino acid
sequence of SEQ ID NO:4 and which includes one or more
substitutions selected from: S4R; K7E; D15N; N19C; D20N; L26I or T;
D33P or G; T40A; L41P; E44G; E47G; K50Q; S51G; Y63H; Y64C; S66C;
T68L or M; D69N; N74K or H; A81T; Q85R or K; A91S; Q95M or V; P98S;
L99V; R113C; C130S; H132R; R135S; R137H; Q141R; I155V; T163A; S178G
or C; Y184C; I185T; K187R or E; E190C; I197T or V; S202T; V206I or
A; I212M; D218G; V226A; H230A; E244G or V; E247G; R248G, K, or M;
M249I; A250T or V; A252T; D268G; H278Y; M283I or V; L297M or F;
I299M; R303H; E311G; I313N, V, S, or T; S321L; Y323F; G336A; V341L,
I, or A; V355I; V362A or I; I363T; T367A; E368G; Y372C; K385E;
Q397R; Q398R; I400V; P403S, G, or T; and P413L, G, or S.
11. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which has an amino acid sequence corresponding to the amino acid
sequence of SEQ ID NO:4 and which includes one or more
substitutions selected from: A2S or T; S4C or N; Y8H; Y9F; F10W;
E16K; Q17K; Y19H; H22R; 129V; K30R; T31A or S; L32V; K36A; K37E;
V49I; 168M; C75D; 177V; R78C; 182V; Q85K; Q86R; P106S; F120L; I122T
or V, K142R, S145P, E149V, D156E, N160T, V164I, V177L, S178C,
E180G, K187E, E191G, V192I, E196A or G, I197V, V198I, I200V, T204A,
V206A, Q215H or R, V217A, K222R, Y223H, L228V, F232S, V238I, R248K
or M, A250V, N251S, I254V, T260S, I266V, R275K, L279K, K282N,
M2831, I290M or V, G300R or S, F302L, I310T or V, I313V, N315S,
F328Y, P333A, V341A, V347I, Y359F, V362I, E370D, E374G, D377G,
E379V, C381S, R382H, A383V, G384D, H386Q, K387A or R, E388I, V390I,
A392E or T, S394L, G395S, G396S, I401M, E402V, P403G, I, L, M, or
T, S404P, D405N, L406S, A407E, K409E or M, K410D or E, K412R, P413G
or S, D414G or V, K415E or R, and N416A.
12. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which is a polypeptide having at least 99% identity to a
polypeptide selected from SEQ ID NOS: 2226, 2240, 2268, 2286, 2282,
2312, 2316, 2324, 2338, 2346, and 2348.
13. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which is a polypeptide having at least 96% identity to a
polypeptide selected from SEQ ID NOS: 2380 and 2378.
14. The engineered alanine-2,3-aminomutase polypeptide of claim 1,
which is a polypeptide having at least 95% identity to a
polypeptide selected from SEQ ID NO: 2670, 2666, and 4586.
15. A process for preparing beta-alanine, comprising contacting
alpha-alanine with an engineered alanine-2,3-aminomutase of claim
1, under conditions suitable for conversion of alpha-alanine to
beta-alanine.
16. The process of claim 15, which is carried out with whole cells
that express the engineered alanine-2,3-aminomutase, or an extract
or lysate of such cells.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 11/581,946, filed Oct. 16, 2006, which claims
benefit under 35 U.S.C. .sctn.119(e), to U.S. Provisional Patent
Application Ser. No. 60/726,967, filed Oct. 14, 2005, now
abandoned, the contents of each of which are incorporated herein by
reference.
2. REFERENCE TO SEQUENCE LISTING
[0002] The "Sequence Listing" submitted concurrently herewith on
compact disc under 37 C.F.R. .sctn..sctn.1.821(c) and the computer
readable form (CRF) of the Sequence Listing submitted under 37
C.F.R. .sctn..sctn.1.821(e) are incorporated by reference herein in
its entirety. Two copies of the Sequence Listing, one on each of
two compact discs labeled "Copy-1" and "Copy-2," are provided. Each
electronic copy of the Sequence Listing was created on Oct. 16,
2005 with a file size of 18,633 KB. The file names are as follows:
Copy-1--377804-008_CRF.txt and Copy-2--377804-008SRF.txt. The
"Sequence Listing" incorporated herein by reference is the same as
the CRF of the Sequence Listing being submitted concurrently
herewith.
3. TECHNICAL FIELD
[0003] The disclosure relates to compositions and methods involving
biocatalysts for the efficient production of useful chemical
products.
4. BACKGROUND
[0004] Organic chemicals such as organic acids, esters, and polyols
can be used to synthesize plastic materials and other products. To
meet the increasing demand for organic chemicals, more efficient
and cost-effective production methods are being developed which
utilize raw materials based on carbohydrates rather than
hydrocarbons. For example, certain bacteria have been used to
produce large quantities of lactic acid used in the production of
polylactic acid. 3-hydroxypropionic acid (3-HP) is an organic acid.
Several chemical synthesis routes have been described to produce
3-HP, and biocatalytic routes have also been disclosed (WO
01/116346). 3-HP has utility for specialty synthesis and can be
converted to commercially important intermediates by known methods
in the chemical industry, e.g., acrylic acid by dehydration,
malonic acid by oxidation, esters by esterification reactions with
alcohols, and 1,3-propanediol by reduction. The 3-HP compound can
be produced biocatalytically from phosphoenolpyruvate or pyruvate
through a key beta-alanine (.beta.-alanine) intermediate (FIG. 1).
.beta.-alanine can be synthesized in cells from carnosine,
.beta.-alanyl arginine, .beta.-alanyl lysine, uracil via
5,6-dihydrouracil and N-carbamoyl-.beta.-alanine,
N-acetyl-.beta.-alanine, anserine, or aspartate (FIG. 2). However,
these routes may not be cost-effective because they require rare
precursors or starting compounds that are more costly than 3-HP.
Therefore, production of 3-HP using biocatalytic routes would be
more efficient if alpha alanine (.alpha.-alanine) could be
efficiently converted to .beta.-alanine directly (FIG. 1). A
naturally occurring enzyme that inter-converts .alpha.-alanine to
.beta.-alanine has not yet been identified. It would be
advantageous if enzymatic activities that carry out the efficient
conversion of .alpha.-alanine to .beta.-alanine were identified,
such as an alanine 2,3-aminomutase (AAM). Enzymes having very low
levels of AAM have been evolved in the laboratory (WO
03/1062173).
[0005] There are naturally occurring enzymes known to interconvert
lysine to .beta.-lysine. Lysine 2,3-aminomutase (KAM) was first
described by Barker in Clostridium SB4 (now C. subterminale) as
catalyzing the first step in the fermentation of lysine. KAM has
been purified from C. subterminale, the corresponding gene cloned
and expressed in E. coli (e.g., U.S. Pat. No. 6,248,874,
incorporated herein by reference). The specific activity of
purified KAM from C. subterminale SB4 cells has been reported as
30-40 units/mg (Lieder et. al., 1998, Biochemistry 37:2578), where
a unit is defined as .mu.moles lysine/min. The corresponding
recombinant KAM had equivalent enzyme activity (34.5.+-.1.6
.mu.moles lysine/min/mg protein). See U.S. Patent Application
Publication No. 2003/0113882, the whole of which is incorporated
herein by reference.
[0006] Based upon the sequence of the KAM from C. subterminale, KAM
genes have been annotated in the genomes of other organisms.
However, in most cases, the enzymatic activities of the
polypeptides encoded by these genes have not been confirmed.
Exceptions are the B. subtilis gene (Chen et al., 2000, Biochem. J.
348:539-549), and the Porphyromonas gingivalis and F. nucleatum
genes. The B. subtilis KAM, encoded by the yodO gene, is more
resistant to O.sub.2 than the C. subterminale KAM, but it is
markedly less active. As reported in U.S. Patent Application
Publication No. 200310113882, the B. subtilis KAM has a specific
KAM activity of only 0.62 U/mg.
[0007] C. subterminale SB4 KAM has been reported to have some
cross-reactivity with L-alanine, converting it into .beta.-alanine.
See U.S. Patent Application Publication No. 2003/0113882 A1. The
publications WO 03/062173 and WO 02/42418 disclose the first
reports of AAM activity based upon modification of kam genes. In
these applications, the synthetic aam genes had AAM activity as
detected by the complementation of an E. coli .DELTA.panD strain.
However, because alanine is not the natural substrate for this
enzyme, the activity for this conversion is substantially less than
the activity for conversion of lysine, its natural substrate. Such
low AAM activity would not provide a cost-effective route to
converting .alpha.-alanine to .beta.-alanine.
[0008] The polynucleotide for the wild-type lysine aminomutase
(KAM) of Porphyromonas gingivalis, designated as Pgaam, has the
polynucleotide sequence of SEQ ID NO:1 (GenBank Accession No.
AE017175) and encodes the 416 amino acid residue polypeptide of SEQ
ID NO:2.
[0009] The polynucleotide (SEQ ID NO: 1) encoding this molecule was
modified (SEQ ID NO: 3) as described in WO 03/106217, to produce a
polypeptide (SEQ ID NO:4), designated as Pgaam2, having a
detectable AAM activity. The polypeptide of SEQ ID NO:4, which
exhibits a detectible AAM activity, differs from wild-type P.
gingivalis Pgaam by having the following seven (7) amino acid
substitutions: N19Y, E30K, L53P, H85Q, 1192V, D331G, and M342T.
[0010] The percent homology between the amino acid sequences of the
various KAMs from B. subtilis, C. stricklandii, F. nucleatum, and
P. gingivalis are shown in Table 1 below.
TABLE-US-00001 TABLE 1 B subtilis C. stricklandii F. nucleatum P.
gingivalis B subtilis 100% 53% 50% 52% C. stricklandii 53% 100% 69%
72% F. nucleatum 50% 69% 100% 70% P. gingivalis 52% 72% 70%
100%
[0011] The present disclosure is directed to engineered alanine
2,3-aminomutases having improved activity over those AAM enzymes
known in the art.
5. SUMMARY
[0012] The present disclosure relates to engineered polypeptides
and polynucleotides encoding the polypeptides having improved
alanine 2,3-aminomutase (AAM) activity relative to the wild-type
polypeptide from P. gingivalis of SEQ ID NO: 2 and the polypeptide
of SEQ ID NO: 4, which is derived from the P. gingivalis
polypeptide of SEQ ID NO:2. In some embodiments, the AAM
polypeptides with improved enzyme activity has at least about 80%
sequence identity with the amino acid sequence of SEQ ID NO:4.
[0013] The engineered AAM polypeptides can have from 1.5 times to
30 time or more times greater enzyme activity as compared to the
AAM activity of the polypeptide of SEQ ID NO:4, or other engineered
AAM polypeptides disclosed herein. In some embodiments, the
improved enzyme activity may have 1.5 times the enzymatic activity
of the corresponding reference AAM enzyme, to as much as 2 times, 5
times, 10 times, 20 times, 25 times, 30 times, up to 50 times or
more times enzymatic activity than the reference enzyme. In some
embodiments, the engineered AAM polypeptides can have from 2 times
to about 30 times more activity than the polypeptide of SEQ ID
NO:4. In some embodiments, the engineered AAM polypeptides can have
AAM activity that is from about 2 to about 10 times greater
activity than the AAM polypeptide of SEQ ID NO:36.
[0014] In some embodiments, the engineered AAM polypeptides has an
amino acid sequence corresponding to the amino acid sequence of SEQ
ID NO:4 and includes one or more substitutions selected from the
groups of substitutions provided in the descriptions, below. Thus,
In some embodiments, the engineered AAM polypeptide with improved
enzyme activity is a polypeptide having at least 97% homology to a
polypeptide of SEQ ID NO: 295, 2068 or 2150. In some embodiments,
the engineered AAM polypeptide has at least 98% homology to a
polypeptide of SEQ ID NO: 258, 316, 350, 356, 790, 1006, or 1250.
In some embodiments, the engineered AAM polypeptide has at least
99% homology to a polypeptide of SEQ ID NO: 14, 24, 58, 86, 100,
312, 336, 380, 318, 404, 444, 490, 550 or 774.
[0015] In some embodiments, the engineered AAM polypeptide with
improved enzyme activity is a polypeptide having at least 99%
identity to a polypeptide of SEQ ID NO: 2230, 2348, 2286, 2312,
2282, 2346, 2338, 2226, 2268, 2324, or 2240. In some embodiments,
the AAM polypeptide has at least 96% identity to a polypeptide of
SEQ ID NO: 2380 or 2378. In some embodiments, the AAM polypeptide
has at least 95% identity to a polypeptide of SEQ ID NO: 2670 or
2666. In some embodiments, the engineered AAM has at least 93%
sequence identity to SEQ ID NO. 4288. In some embodiments, the
engineered AAM polypeptide has at least 95% sequence identity to
SEQ ID NO. 4586.
[0016] In some embodiments, the AAM polypeptide of the present
disclosure has an amino acid sequence selected from the
polypeptides listed in the Sequence Listing, beginning from SEQ ID
NO:6. In some embodiments, the AAM polypeptides are encoded by a
polynucleotide selected from the polynucleotides listed in the
Sequence Listing, beginning from SEQ ID NO:5. In some embodiments,
the AAM polypeptides are encoded by a polynucleotide which
hybridizes under high stringency conditions with the complement of
the polynucleotide sequences in the Sequence Listing beginning from
SEQ ID NO:5, and where the encoded polypeptide has greater AAM
activity than the polypeptide of SEQ ID NO: 4.
[0017] In addition to the above, the present disclosure also
provides variants of the AAM polypeptides listed in the Sequence
Listing, beginning from SEQ ID NO:6, comprising a substitution,
deletion, and/or insertion of one or more amino acids and having
greater AAM activity than the polypeptide of SEQ ID NO: 4, SEQ ID
NO: 36, or in some embodiments, SEQ ID NO:909. In some embodiments,
the variants of the AAM polypeptides comprise conservative amino
acid substitutions of the specific AAM polypeptides in the Sequence
Listing, beginning from SEQ ID NO:6.
[0018] Further provided herein are polynucleotides encoding the
engineered AAM polypeptides, corresponding expression vectors, and
host cells comprising the polynucleotides operably linked to
control sequences for the expression of the AAM polypeptides. The
polynucleotide and expression vectors can be used in vivo or in
vitro to produce the AAM polypeptides. In some embodiments, the
polynucleotide encoding the engineered AAM enzyme is selected from
the polynucleotides listed in the Sequence Listing, beginning from
SEQ ID NO:5. As described above, in some embodiments, the
polynucleotides encoding the AAM polypeptides with improved AAM
activity can comprise polynucleotides that hybridize under highly
stringent conditions to a polynucleotide selected from the
polynucleotides listed in the Sequence Listing, beginning from SEQ
ID NO:5. The polynucleotides, expression vectors, and host cells
can be used in methods to express the engineered polypeptide, for
example, by cultivating the host cell comprising the expression
vector under conditions suitable for expression of the AAM
polypeptide.
[0019] In other embodiments, the present disclosure is directed to
various compositions comprising an engineered AAM polypeptide, such
as a whole cell preparation, as a crude preparation, or as an
isolated polypeptide, and a suitable carrier, typically a buffer,
such as an aqueous buffer solution having a pH from about 6.0 to
about 8.0. In some embodiments, the AAM polypeptides can be
provided in a lyophilized form, wherein the composition can be
reconstituted by the addition of an aqueous based composition. In
some embodiments, the AAM polypeptide can be prepared in a
substantially non-oxidized form, while in other embodiments, the
polypeptides can be provided in its oxidized form, which can be
reduced by the use of a suitable reducing agent.
[0020] The present disclosure is also directed to a method of
producing .beta.-alanine, the method comprising contacting
.alpha.-alanine with an engineered AAM polypeptide described herein
under conditions suitable for conversion of the .alpha.-alanine to
.beta.-alanine. The process can be carried out with, among others,
whole cells that express the engineered AAM polypeptide, or an
extract or lysate of such cells. The .beta.-alanine may be
optionally recovered from the cells.
6. BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows the reversible reaction between alpha-alanine
(i.e., .alpha.-alanine, L-alanine, or 2-aminopropionic acid) and
beta-alanine (.beta.-alanine or 3-aminopropionic acid) that is
catalyzed by alanine 2,3-aminomutase.
[0022] FIG. 2 is a pathway for synthesis of 3-hydroxypropionic acid
(3-HP) from .alpha.-alanine, via .beta.-alanine as an
intermediate.
[0023] FIG. 3 is a 4036 by expression vector (pCK110900-I Bla)
comprising a P15A origin of replication (PISA ori), a lac1
repressor, a CA binding site, a lac promoter (lac), a T7 ribosomal
binding site (T7g10 RBS), and a chloramphenicol resistance gene
(camR).
[0024] FIG. 4 shows the pCK110900 I-Bla vector with the synthetic
operon of Pgaam-Baat-PpmmsB-Spaat genes used to screen for AAM
polypeptides with improved enzymatic activity based on synthesis of
3-HP (see Example 5).
[0025] The foregoing summary, as well as the following detailed
description of certain embodiments of the present disclosure, will
be better understood when read in conjunction with the appended
drawings. For the purpose of illustrating the disclosure, there is
shown in the drawings, certain embodiments. It should be
understood, however, that the present disclosure is not limited to
the arrangements and instrumentality shown in the attached
drawings.
7. DETAILED DESCRIPTION
[0026] The present disclosure provides engineered alanine
2,3-aminomutases (AAM) capable of converting .alpha.-alanine to
.beta.-alanine. The enzymatic product .beta.-alanine is a substrate
in the pathway for synthesis of a number of different compounds,
such as acrylyl-CoA, 3-hydroxypropionic acid (3-HP),
1,3-propanediol, and panthothenic acid, which have important
commercial uses. For example, the compounds can be used to
synthesize polyacrylates, acrylate esters, polymerized 3-HP,
co-polymers of 3-HP and other compounds such as butyrates and
valerates, esters of 3-HP, and malonic acid and its esters. In
particular, 3-HP has biologically and commercially important uses,
for example as a food additive or as a preservative. Hydrogenation
of 3-HP results in 1,3-propanediol, a valuable polyester monomer.
The engineered AAM polypeptides described herein are useful in
efficiently generating the key intermediate .beta.-alanine. For the
description of the various embodiments herein, the technical and
scientific terms used will have the meanings commonly understood by
one of ordinary skill in the art, unless specifically defined
otherwise.
7.1 Definitions
[0027] As used herein, the following terms are intended to have the
following meanings:
[0028] "Alanine 2,3-aminomutase" or "AAM" refers to an enzyme which
can convert .alpha.-alanine to .beta.-alanine. The term also
includes any alanine 2,3-aminomutase gene, cDNA, RNA, or protein
from any organism, such as a prokaryote, or an engineered
polypeptide with AAM activity. For example, an AAM can be a mutated
lysine 2,3-aminomutase or a mutated lysine 5,6-aminomutase which
has alanine 2,3-aminomutase activity. Lysine 2,3-aminomutases (or
genes annotated in genetic databases as lysine 2,3 aminomutase) can
be obtained from any organism, such as a prokaryote, for example
Bacillus subtilis, Deinococcus radiodurans, Clostridium
subterminale, Porphyromonas gingivalis or E. coli, and mutated
using any method known in the art.
[0029] "Alanine 2,3-aminomutase activity" as used herein is meant
that a polypeptide catalyzes the reaction of FIG. 1, whereby
.alpha.-alanine is converted to .beta.-alanine. This reaction can
occur in a cell or in vitro.
[0030] "Coding sequence" refers to that portion of a nucleic acid
(e.g., a gene) that encodes an amino acid sequence of a
protein.
[0031] "Naturally-occurring" or "wild-type" refers to the form
found in nature. For example, a naturally occurring or wild-type
polypeptide or polynucleotide sequence is a sequence present in an
organism that can be isolated from a source in nature and which has
not been intentionally modified by human manipulation.
[0032] "Reference sequence" refers to a defined sequence used as a
basis for a sequence comparison. A reference sequence may be a
subset of a larger sequence, for example, a segment of a
full-length gene or polypeptide sequence. Generally, a reference
sequence is at least 20 nucleotide or amino acid residues in
length, at least 25 residues in length, at least 50 residues in
length, or the full length of the nucleic acid or polypeptide.
Since two polynucleotides or polypeptides may each (1) comprise a
sequence (i.e., a portion of the complete sequence) that is similar
between the two sequences, and (2) may further comprise a sequence
that is divergent between the two sequences, sequence comparisons
between two (or more) polynucleotides or polypeptide are typically
performed by comparing sequences of the two polynucleotides over a
"comparison window" to identify and compare local regions of
sequence similarity.
[0033] "Substantial identity" refers to a polynucleotide or
polypeptide sequence that has at least 80 percent sequence
identity, at least 85 percent sequence identity, about 89 to 95
percent sequence identity, and more usually at least 99 percent
sequence identity as compared to a reference sequence over a
comparison window of at least 20 residue positions, frequently over
a window of at least 30-50 residues, wherein the percentage of
sequence identity is calculated by comparing the reference sequence
to a sequence that includes deletions or additions which total 20
percent or less of the reference sequence over the window of
comparison. In specific embodiments applied to polypeptides, the
term "substantial identity" means that two polypeptide sequences,
when optimally aligned, such as by the programs GAP or BESTFIT
using default gap weights, share at least 80 percent sequence
identity, preferably at least 89 percent sequence identity, at
least 95 percent sequence identity or more (e.g., 99 percent
sequence identity). In some embodiments, residue positions which
are not identical differ by conservative amino acid
substitutions.
[0034] "Percentage of sequence identity" and "percentage homology"
are used interchangeably herein to refer to comparisons among
polynucleotides and polypeptides, which are determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide or polypeptide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage may be calculated by determining the number of
positions at which the identical nucleic acid base or amino acid
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison and multiplying the
result by 100 to yield the percentage of sequence identity.
Alternatively, the percentage may be calculated by determining the
number of positions at which either the identical nucleic acid base
or amino acid residue occurs in both sequences or a nucleic acid
base or amino acid residue is aligned with a gap to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the window of
comparison and multiplying the result by 100 to yield the
percentage of sequence identity. Those of skill in the art
appreciate that there are many established algorithms available to
align two sequences. Optimal alignment of sequences for comparison
can be conducted, e.g., by the local homology algorithm of Smith
and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology
alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.
48:443, by the search for similarity method of Pearson and Lipman,
1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized
implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and
TFASTA in the GCG Wisconsin Software Package), or by visual
inspection (see generally, Current Protocols in Molecular Biology,
F. M. Ausubel et al., eds., Greene Publishing Associates, Inc. and
John Wiley & Sons, Inc., (1995 Supplement)). Examples of
algorithms that are suitable for determining percent sequence
identity and sequence similarity are the BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., 1990, J. Mol.
Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res.
3389-3402, respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information website. This algorithm involves first identifying high
scoring sequence pairs (HSPs) by identifying short words of length
W in the query sequence, which either match or satisfy some
positive-valued threshold score T when aligned with a word of the
same length in a database sequence. T is referred to as, the
neighborhood word score threshold (Altschul et al, supra). These
initial neighborhood word hits act as seeds for initiating searches
to find longer HSPs containing them. The word hits are then
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci
USA 89:10915). Exemplary determination of sequence alignment and %
sequence identity can employ the BESTFIT or GAP programs in the GCG
Wisconsin Software package (Accelrys, Madison Wis.), using default
parameters provided, or the ClustalW multiple alignment program
(available from the European Bioinformatics Institute, Cambridge,
UK), using, in some embodiments, the parameters below.
[0035] For purposes of the present disclosure, in some embodiments,
the degree of percent amino acid sequence identity can be obtained
by ClustalW analysis (version W 1.8), counting the number of
identical matches in the alignment and dividing such number of
identical matches by the length of the reference sequence, and
using the following default ClustalW parameters to achieve
slow/accurate pairwise optimal alignments--Gap Open Penalty: 10;
Gap Extension Penalty: 0.10; Protein weight matrix: Gonnet series;
DNA weight matrix: IUB; Toggle Slow/Fast pairwise alignments=SLOW
or FULL Alignment.
[0036] "Comparison window" refers to a conceptual segment of at
least about 20 contiguous nucleotide positions or amino acids
residues wherein a sequence may be compared to a reference sequence
of at least 20 contiguous nucleotides or amino acids and wherein
the portion of the sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20 percent or less as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The comparison window can be longer than 20 contiguous residues,
and includes, optionally 30, 40, 50, 100, or longer windows.
[0037] "Recombinant" when used with reference to, e.g., a cell,
nucleic acid, or polypeptide, refers to a material, or a material
corresponding to the natural or native form of the material, that
has been modified in a manner that would not otherwise exist in
nature, or is identical thereto but produced or derived from
synthetic materials and/or by manipulation using recombinant
techniques. Non-limiting examples include, among others,
recombinant cells expressing genes that are not found within the
native (non-recombinant) form of the cell or express native genes
that are otherwise expressed at a different level.
7.2 Engineered Alanine 2,3-aminomutases
[0038] The present disclosure is directed to engineered
polypeptides that have improved (i.e., increased) alanine
2,3-aminomutase (AAM) activity relative to the wild-type
polypeptide from P. gingivalis of SEQ ID NO: 2 and the mutated
polypeptide of SEQ ID NO: 4. Because the polypeptide of SEQ ID NO:
4 has greater AAM activity than the wild-type enzyme from P.
gingivalis, it is sufficient for the purposes herein to compare the
AAM polypeptides of the present disclosure to the polypeptide of
SEQ ID NO: 4. In some embodiments, the engineered polypeptides
display an improved AAM activity than the reference AAM polypeptide
of SEQ ID NO: 36, which has substantially increased AAM activity
relative to SEQ ID NO: 4. In some embodiments, the engineered
polypeptides may display an improved AAM activity than the
reference polypeptide of SEQ ID NO:909.
[0039] In some embodiments herein, the engineered AAM with improved
enzyme activity can be obtained by mutating the genetic material
encoding the enzymes and identifying polynucleotides that express
engineered enzymes with the improved activity. These non-naturally
occurring AAMs can be generated by various well-known techniques,
such as in vitro mutagenesis and/or directed evolution. In some
embodiments, directed evolution is an attractive method for
generating engineered enzymes because of the relative ease of
generating mutations throughout the whole of the gene coding for
the polypeptide, as well as providing the ability to take
previously mutated polynucleotides and subjecting them to
additional cycles of mutagenesis and/or recombination to obtain
further improvements in a selected enzyme property. Subjecting the
whole gene to mutagenesis can reduce the bias that may result from
restricting the changes to a limited region of the gene. It can
also enhance generation of enzymes affected in different enzyme
properties since distantly spaced parts of the enzyme may play a
role in various aspects of enzyme function. Mutagenesis and
directed evolution techniques useful for the purposes herein are
amply described in the literature: Ling et al., 1997, "Approaches
to DNA mutagenesis: an overview," Anal Biochem. 254(2):157-78; Dale
et al., 1996, "Oligonucleotide-directed random mutagenesis using
the phosphorothioate method," Methods Mol. Biol. 57:369-74; Smith,
1985, "In vitro mutagenesis," 1985, Ann Rev Genet. 19:423-462;
Botstein, et al., 1985, "Strategies and applications of in vitro
mutagenesis," Science 229:1193-1201; Carter, 1986, "Site-directed
mutagenesis," Biochem J. 237:1-7; Kramer et al., 1984, "Point
Mismatch Repair," Cell 38:879-887; Wells et al., 1985, "Cassette
mutagenesis: an efficient method for generation of multiple
mutations at defined sites," Gene 34:315-323; Minshull et al.,
1999, "Protein evolution by molecular breeding," Curr Opin Chem.
Biol. 3:284-290; Christians et al., 1999, "Directed evolution of
thymidine kinase for AZT phosphorylation using DNA family
shuffling," Nat. Biotech. 17:259-264; Crameri et al., 1998, "DNA
shuffling of a family of genes from diverse species accelerates
directed evolution," Nature 391:288-291; Crameri et al., 1997,
"Molecular evolution of an arsenate detoxification pathway by DNA
shuffling," Nat. Biotech. 15:436-438; Zhang et al., 1997, "Directed
evolution of an effective fructosidase from a galactosidase by DNA
shuffling and screening," Proc Natl Acad Sci USA 94:45-4-4509;
Crameri et al., 1996, "Improved green fluorescent protein by
molecular evolution using DNA shuffling,` Nat Biotech. 14:315-319;
Stemmer, 1994, "Rapid evolution of a protein in vitro by DNA
shuffling," Nature 370:389-391; Stemmer, 1994, "DNA shuffling by
random fragmentation and reassembly: In vitro recombination for
molecular evolution," Proc Natl Acad Sci USA 91:10747-10751; WO
95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO
01/75767 and U.S. Pat. No. 6,537,746. All publications are
incorporated herein by reference.
[0040] Without being bound by theory, the improvements in enzyme
properties may arise from introduction of modifications into a
polypeptide chain that may, in effect, perturbs the
structure-function of the enzyme and/or interaction with another
molecule (e.g., substrate). Some regions of the polypeptide may be
critical to enzyme activity, for example amino acids involved in
catalysis and substrate binding domains, such that small
perturbations to these regions will have significant effects on
enzyme function. Some amino acid residues may be at important
positions for maintaining the secondary or tertiary structure of
the enzyme, and thus also produce noticeable changes in enzyme
properties when modified. Thus, in some embodiments, the number of
modifications to the reference parent polypeptide that produces an
improved AAM property may comprise one or more amino acids, 2 or
more amino acids, 5 or more amino acids, 10 or more amino acids, or
20 or more amino acids, up to 10% of the total number of amino
acids, up to 20% of the total number of amino acids, or up to 30%
of the total number of amino acids making up the reference
enzyme.
[0041] In some embodiments, improvements in enzyme activity can
arise from substitutions at one amino acid residue, 2 or more amino
acid residues, 5 or more amino acid residues, 10 or more amino acid
residue, 15 or more amino acid residues, 20 or more amino acid
residues, up to 10% of the total number of amino acids, up to 20%
of the total number of amino acids, or up to 30% of the total
number of amino acids making up the reference parent enzyme.
[0042] As will be apparent to the skilled artisan, an analysis of
amino acid substitutions for a large number of engineered AAM
polypeptides derived from SEQ ID NO:4 or another reference AAM
polypeptide, shows that the substitutions recur at certain amino
acid residues in the AAM polypeptide as well as a bias towards
certain types of amino acids. This recurrence of substitutions at
certain defined positions within the polypeptide may reflect the
effect of amino acid residue on that particular enzyme property and
its retention by the continued imposition of selection for the
particular improved enzyme property. The recurrence of
substitutions as well as the bias of the types of substituted amino
acids can be grouped to describe the types of substitutions
allowable for generating the improved AAM. Typically, genetically
encoded amino acids can be grouped into the following classes based
on their side chain properties:
[0043] "Hydrophilic Amino Acid or Residue" refers to an amino acid
or residue having a side chain exhibiting a hydrophobicity of less
than zero according to the normalized consensus hydrophobicity
scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142.
Genetically encoded hydrophilic amino acids include L-Thr (T),
L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (O), L-Asp (D),
L-Lys (K) and L-Arg (R).
[0044] "Acidic Amino Acid or Residue" refers to a hydrophilic amino
acid or residue having a side chain exhibiting a pK value of less
than about 6 when the amino acid is included in a peptide or
polypeptide. Acidic amino acids typically have negatively charged
side chains at physiological pH due to loss of a hydrogen ion.
Genetically encoded acidic amino acids include L-Glu (E) and L-Asp
(D).
[0045] "Basic Amino Acid or Residue" refers to a hydrophilic amino
acid or residue having a side chain exhibiting a pK value of
greater than about 6 when the amino acid is included in a peptide
or polypeptide. Basic amino acids typically have positively charged
side chains at physiological pH due to association with hydronium
ion. Genetically encoded basic amino acids include L-His (H), L-Arg
(R) and L-Lys (K).
[0046] "Polar Amino Acid or Residue" refers to a hydrophilic amino
acid or residue having a side chain that is uncharged at
physiological pH, but which has at least one bond in which the pair
of electrons shared in common by two atoms is held more closely by
one of the atoms. Genetically encoded polar amino acids include
L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).
[0047] "Hydrophobic Amino Acid or Residue" refers to an amino acid
or residue having a side chain exhibiting a hydrophobicity of
greater than zero according to the normalized consensus
hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.
179:125-142. Genetically encoded hydrophobic amino acids include
L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W),
L-Met (M), L-Ala (A) and L-Tyr (Y).
[0048] "Aromatic Amino Acid or Residue" refers to a hydrophilic or
hydrophobic amino acid or residue having a side chain that includes
at least one aromatic or heteroaromatic ring. Genetically encoded
aromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).
Although owing to the pKa of its heteroaromatic nitrogen atom L-His
(H) is classified above as a basic residue, as its side chain
includes a heteroaromatic ring; it may also be classified as an
aromatic residue.
[0049] "Non-polar Amino Acid or Residue" refers to a hydrophobic
amino acid or residue having a side chain that is uncharged at
physiological pH and which has bonds in which the pair of electrons
shared in common by two atoms is generally held equally by each of
the two atoms (i.e., the side chain is not polar). Genetically
encoded non-polar amino acids include L-Leu (L), L-Val (V), L-Ile
(I), L-Met (M) and L-Ala (A).
[0050] "Aliphatic Amino Acid or Residue" refers to a hydrophobic
amino acid or residue having an aliphatic hydrocarbon side chain.
Genetically encoded aliphatic amino acids include L-Ala (A), L-Val
(V), L-Leu (L) and L-Ile (I).
[0051] The amino acid L-Cys (C) is unusual in that it can form
disulfide bridges with other L-Cys (C) amino acids or other
sulfanyl- or sulfhydryl-containing amino acids. The "cysteine-like
residues" include cysteine and other amino acids that contain
sulfhydryl moieties that are available for formation of disulfide
bridges. The ability of L-Cys (C) (and other amino acids with --SH
containing side chains) to exist in a peptide in either the reduced
free --SH or oxidized disulfide-bridged form affects whether L-Cys
(C) contributes net hydrophobic or hydrophilic character to a
peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29
according to the normalized consensus scale of Eisenberg (Eisenberg
et al., 1984, supra), it is to be understood that for purposes of
the present disclosure L-Cys (C) is categorized as a polar
hydrophilic amino acid, notwithstanding the general classifications
defined above.
[0052] The amino acid Gly (G) is also unusual in that it bears no
side chain on its .alpha.-carbon and, as a consequence, contributes
only a peptide bond to a particular peptide sequence. Moreover,
owing to the lack of a side chain, it is the only
genetically-encoded amino acid having an achiral .alpha.-carbon.
Although Gly (G) exhibits a hydrophobicity of 0.48 according to the
normalized consensus scale of Eisenberg (Eisenberg et al., 1984,
supra), for purposes of the present disclosure, Gly is categorized
as an aliphatic amino acid or residue.
[0053] "Small Amino Acid or Residue" refers to an amino acid or
residue having a side chain that is composed of a total three or
fewer carbon and/or heteroatoms (excluding the .alpha.-carbon and
hydrogens). The small amino acids or residues may be further
categorized as aliphatic, non-polar, polar or acidic small amino
acids or residues, in accordance with the above definitions.
Genetically-encoded small amino acids include Gly, L-Ala (A), L-Val
(V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).
[0054] "Hydroxyl-containing Amino Acid or Residue" refers to an
amino acid containing a hydroxyl (--OH) moiety. Genetically-encoded
hydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and
L-Tyr (Y).
[0055] As will be appreciated by those of skill in the art, the
above-defined categories are not mutually exclusive. Indeed, the
delineated category of small amino acids includes amino acids from
all of the other delineated categories except the aromatic
category. Thus, amino acids having side chains exhibiting two or
more physico-chemical properties can be included in multiple
categories. As a specific example, amino acid side chains having
heteroaromatic moieties that include ionizable heteroatoms, such as
His, may exhibit both aromatic properties and basic properties, and
can therefore be included in both the aromatic and basic
categories. The appropriate classification of any amino acid or
residue will be apparent to those of skill in the art, especially
in light of the detailed disclosure provided herein.
[0056] In some embodiments, the substitution in the engineered AAM
may be a conservative substitution. The term "conservative amino
acid substitution" refers to the interchangeability of residues
having similar side chains, and thus typically involves
substitution of the amino acid in the polypeptide with amino acids
within the same or similar defined class of amino acids. By way of
example and not limitation, an amino acid with an aliphatic side
chain may be substituted with another aliphatic amino acid, e.g.,
alanine, valine, leucine, and isoleucine; an amino acid with
hydroxyl side chain is substituted with another amino acid with a
hydroxyl side chain, e.g., serine and threonine; an amino acid
having aromatic side chains is substituted with another amino acid
having an aromatic side chain, e.g., phenylalanine, tyrosine,
tryptophan, and histidine; an amino acid with a basic side chain is
substituted with another amino acid with a basic side chain, e.g.,
lysine, arginine, and histidine; an amino acid with an acidic side
chain is substituted with another amino acid with an acidic side
chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or
hydrophilic amino acid is replaced with another hydrophobic or
hydrophilic amino acid, respectively Amino acid substitutions,
which do not generally alter the protein activity, are known in the
art and are described, for example, Neurath and Hill, 1979, In The
Proteins, Academic Press, New York. Commonly occurring conservative
exchanges include, for example, Ala/Ser; Val/Ile; Asp/Glu; Thr/Ser;
Ala/Gly; Ala/Thr, Ser/Asn; Ala/Val; Ser/Gly; Tyr/Phe; Ala/Pro;
Lys/Arg; Asp/Asn; Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as
these in reverse.
[0057] In some embodiments, the substitution in the engineered AAM
may be a non-conservative substitution. The term "non-conservative
substitution" refers to substitution of an amino acid in the
polypeptide with an amino acid with significantly differing side
chain properties. Non-conservative substitutions may use amino
acids between, rather than within, the defined groups and affects
(a) the structure of the peptide backbone in the area of the
substitution (e.g., proline for glycine) (b) the charge or
hydrophobicity, or (c) the bulk of the side chain. By way of
example and not limitation, an exemplary non-conservative
substitution can be an acidic amino acid substituted with a basic
or aliphatic amino acid; an aromatic amino acid substituted with a
small amino acid; and a hydrophilic amino acid substituted with a
hydrophobic amino acid. Thus, in various embodiments, the
substitutions for generating an improved AAM can comprise
conservative substitutions, non-conservative substitutions, as well
as combinations of conservative and non-conservative
substitutions.
[0058] In some embodiments, the improved engineered AAM enzymes can
comprise deletions of the naturally occurring AAM polypeptides as
well as deletions of other improved AAM polypeptides. The term
"deletion" refers to modification to the polypeptide by removal of
one or more amino acids from the reference polypeptide. Deletions
can comprise removal of 1 or more amino acids, 2 or more amino
acids, 5 or more amino acids, 10 or more amino acids, 15 or more
amino acids, or 20 or more amino acids, up to 10% of the total
number of amino acids, or up to 20% of the total number of amino
acids making up the reference enzyme while retaining enzymatic
activity and/or retaining the improved properties of an engineered
AAM enzyme. Deletions can be directed to the internal portions
and/or terminal portions of the polypeptide. In various
embodiments, the deletion can comprise a continuous segment or can
be discontinuous.
[0059] In other embodiments, the improved engineered AAM enzymes
comprise insertions of one or more amino acids to the reference AAM
polypeptide, including insertions of one or more amino acids to
other improved reference AAM polypeptides. Insertions can be in the
internal portions of the polypeptide, or to the carboxy or amino
terminus Insertions as used herein include fusion proteins as is
known in the art. The insertion can be a contiguous segment of
amino acids or be separated by one or more of the amino acids in
the reference parent polypeptide.
[0060] As described above, the various modifications introduced
into the reference, parent polypeptide to generate an engineered
AAM enzyme can be targeted to a specific property of the enzyme.
Thus, in some embodiments, the improved property of the engineered
AAM polypeptides is an increased enzymatic activity, typically
represented by an increase in specific activity (e.g., product
produced/time/weight protein) as compared to the reference AAM
enzyme (e.g., the alanine 2,3-aminomutase activity of SEQ ID NO:4
of Porphyromonas gingivalis). Exemplary methods to determine enzyme
activity are provided in the Examples. Any property relating to
enzyme activity may be affected, including the classical enzyme
properties of K.sub.M, V.sub.max or k.sub.cat, changes of which can
lead to increased enzymatic activity. It is understood by the
skilled artisan that the activity of any enzyme is diffusion
limited such that the catalytic turnover rate cannot exceed the
diffusion rate of the substrate, including any required cofactors.
The theoretical maximum of the diffusion limit, or
k.sub.cat/K.sub.M, is generally about 10.sup.8 to 10.sup.9
(M.sup.-1 s.sup.-1). Hence, any improvements in the enzyme activity
of the AAM will have an upper limit related to the diffusion rate
of the substrates acted on by the AAM enzyme. Alanine
2,3-aminomutase activity can be measured by any one of a number of
assays used for measuring AAM activity, such as the assays
described in the Examples herein. Comparisons of enzyme activities
can be made using a defined preparation of enzyme and a defined
assay under a set condition, as further described in detail herein.
Generally, when lysates are compared, the numbers of cells and the
amount of protein assayed are determined as well as use of
identical expression systems, identical host cells to minimize
variations in amount of enzyme produced by the host cells and
present in the lysates.
[0061] In various embodiments, the improvements in enzyme activity
can be from 1.5 times the enzymatic activity of the corresponding
reference AAM enzyme, to as much as 2 times. 5 times, 10 times, 20
times, 25 times, 30 times, up to 50 times or more times enzymatic
activity than the reference enzyme. In some embodiments, the
engineered AAM can exhibit improved enzymatic activity in the range
of 1.5 to 5 times; 1.5 to 10 times; 1.5 to 20 times; 1.5 to 30
times; 1.5 to 40 times, 1.5 to 50 times greater than that of the
AAM enzyme from which it was derived. Thus, the AAM polypeptides of
the present disclosure can have significantly enhanced AAM activity
relative to the parent KAM polypeptide of SEQ ID NO: 4 from which
they are ultimately derived. In some embodiments, the AAM
polypeptides of the present disclosure have AAM activity that is
from about 2 to about 30 times greater than the AAM polypeptide of
SEQ ID NO: 4 as measured in the assay of Example 4. In other
embodiments, the AAM polypeptides of the present disclosure have
AAM activity that is from about 2 to about 10 times greater than
the AAM polypeptide of SEQ ID NO: 36 as measured in the assay of
Example 4.
[0062] In various embodiments, the engineered AAMs with improved
enzyme activity comprise engineered polypeptides derived from
Porphyromonas gingivalis alanine 2,3-aminomutase of SEQ ID NO:4. In
some embodiments, the amino acid residues (identified herein as
X.sub.n, where n represents the position of the amino acid residue
in the polypeptide chain) of the naturally occurring Porphyromonas
gingivalis SEQ ID NO:2 or the AAM enzyme of SEQ ID NO:4 that can be
modified to generate engineered polypeptides with improved enzyme
activity are indicated below.
TABLE-US-00002 Residue Amino Acid X.sub.2 hydroxyl containing amino
acid X.sub.4 basic or polar amino acid X.sub.7 charged amino acid
X.sub.8 aromatic amino acid X.sub.9 aromatic amino acid X.sub.10
aromatic amino acid X.sub.15 polar amino acid X.sub.16 charged
amino acid X.sub.17 basic amino acid X.sub.19 basic or polar amino
acid X.sub.20 acid or polar amino acid X.sub.22 basic amino acid
X.sub.26 aliphatic amino acid X.sub.27 basic or polar amino acid
X.sub.29 aliphatic amino acid X.sub.30 charged amino acid X.sub.31
aliphatic or hydroxyl containing amino acid X.sub.32 aliphatic
amino acid X.sub.33 small or conformationally constrained amino
acid X.sub.36 acidic or small amino acid X.sub.37 charge amino acid
X.sub.40 small amino acid X.sub.41 constrained amino acid X.sub.44
aliphatic amino acid X.sub.47 small or aliphatic amino acid
X.sub.49 aliphatic amino acid X.sub.50 hydrophilic amino acid
X.sub.51 small or aliphatic amino acid X.sub.63 aromatic amino acid
X.sub.64 hydrophilic amino acid X.sub.66 hydrophilic amino acid
X.sub.68 hydrophobic amino acid X.sub.69 polar amino acid X.sub.74
basic amino acid X.sub.75 acidic amino acid X.sub.77 aliphatic
amino acid X.sub.78 hydrophilic amino acid X.sub.81 small amino
acid X.sub.82 aliphatic amino acid X.sub.85 basic amino acid
X.sub.86 hydrophilic amino acid X.sub.91 small amino acid X.sub.95
hydrophobic amino acid X.sub.98 small amino acid X.sub.99 aliphatic
amino acid X.sub.106 small amino acid X.sub.111 hydrophobic amino
acid X.sub.113 polar amino acid X.sub.120 hydrophobic amino acid
X.sub.122 small amino acid X.sub.130 polar amino acid X.sub.132
basic amino acid X.sub.135 hydroxyl containing amino acid X.sub.137
basic amino acid X.sub.138 aromatic amino acid X.sub.139 small
amino acid X.sub.141 basic or polar amino acid X.sub.142 basic
amino acid X.sub.145 hydrophobic amino acid X.sub.149 aliphatic
amino acid X.sub.155 aliphatic amino acid X.sub.156 acidic amino
acid X.sub.160 hydroxyl containing amino acid X.sub.163 small amino
acid X.sub.164 aliphatic amino acid X.sub.177 aliphatic amino acid
X.sub.178 small amino acid; X.sub.180 small amino acid X.sub.184
hydrophobic or constrained amino acid X.sub.185 hydroxyl containing
amino acid X.sub.187 charged amino acid X.sub.188 basic amino acid
X.sub.190 polar amino acid X.sub.191 small amino acid X.sub.192
aliphatic amino acid X.sub.192 small or aliphatic amino acid
X.sub.197 small amino acid X.sub.198 aliphatic amino acid X.sub.200
aliphatic amino acid X.sub.202 hydroxyl containing amino acid
X.sub.204 small amino acid X.sub.206 aliphatic amino acid X.sub.212
non-polar amino acid X.sub.215 basic amino acid X.sub.217 aliphatic
amino acid X.sub.218 small amino acid X.sub.222 basic amino acid
X.sub.223 aromatic amino acid X.sub.226 aliphatic amino acid
X.sub.228 hydrophobic amino acid X.sub.230 small amino acid
X.sub.232 small amino acid X.sub.238 aliphatic amino acid X.sub.244
small amino acid X.sub.247 small amino acid X.sub.248 basic or
hydrophobic amino acid X.sub.249 hydrophobic amino acid X.sub.250
hydroxyl containing or aliphatic amino acid X.sub.251 aromatic or
hydroxyl containing amino acid X.sub.252 hydroxyl containing amino
acid X.sub.254 hydrophobic amino acid X.sub.260 hydroxyl containing
amino acid X.sub.266 aliphatic amino acid X.sub.268 small amino
acid X.sub.275 basic or small amino acid X.sub.278 aromatic amino
acid X.sub.279 basic amino acid X.sub.282 charged amino acid
X.sub.283 aliphatic amino acid X.sub.290 hydrophobic amino acid
X.sub.292 aliphatic amino acid X.sub.297 hydrophobic amino acid
X.sub.299 hydrophobic amino acid X.sub.300 charged or polar amino
acid X.sub.302 hydrophobic amino acid X.sub.303 basic amino acid
X.sub.310 aliphatic or hydroxyl containing amino acid X.sub.311
small amino acid X.sub.313 polar or aliphatic amino acid X.sub.315
hydroxyl containing amino acid X.sub.321 aliphatic amino acid
X.sub.323 aromatic amino acid X.sub.328 aromatic amino acid
X.sub.331 charged or small amino acid X.sub.333 aliphatic amino
acid X.sub.336 aliphatic amino acid X.sub.339 aliphatic amino acid
X.sub.341 hydrophobic amino acid X.sub.342 hydrophobic amino acid
X.sub.347 aliphatic amino acid X.sub.353 basic amino acid X.sub.355
aliphatic amino acid X.sub.359 aromatic amino acid X.sub.362
aliphatic amino acid X.sub.363 hydroxyl containing amino acid
X.sub.367 small amino acid X.sub.368 small amino acid X.sub.370
acidic amino acid X.sub.371 charge or polar amino acid X.sub.372
charged or polar amino acid X.sub.374 small amino acid X.sub.377
small amino acid X.sub.379 small amino acid X.sub.381 polar amino
acid X.sub.382 basic amino acid X.sub.383 aliphatic amino acid
X.sub.384 acidic amino acid X.sub.385 charged amino acid X.sub.386
basic or polar amino acid X.sub.387 basic or small amino acid
residue X.sub.388 aliphatic amino acid X.sub.390 aliphatic amino
acid X.sub.392 charged or polar amino acid X.sub.394 aliphatic
amino acid X.sub.395 acidic or polar amino acid X.sub.396 hydroxyl
containing amino acid X.sub.397 basic or polar amino acid X.sub.398
basic or polar amino acid X.sub.399 hydrophobic amino acid
X.sub.400 aliphatic amino acid X.sub.401 hydrophobic amino acid
X.sub.402 small amino acid X.sub.403 hydrophobic or small amino
acid X.sub.404 hydropobic amino acid X.sub.405 acidic or polar
amino acid X.sub.406 hydroxyl containing amino acid X.sub.407
acidic or polar amino acid X.sub.409 charged or hydrophobic amino
acid X.sub.410 acidic amino acid X.sub.412 basic amino acid
X.sub.413 hydrophobic or small amino acid X.sub.414 aliphatic or
small amino acid X.sub.415 charged amino acid X.sub.416 aliphatic
amino acid
[0063] In some embodiments, the amino acid residues can be
substituted with one or more of the specific amino acid residues
below to obtain AAMs with improved enzymatic activity:
TABLE-US-00003 Residue Amino Acid X.sub.2 S or T X.sub.4 R, C or N
X.sub.7 E X.sub.8 H X.sub.9 F X.sub.10 W X.sub.15 N X.sub.16 K
X.sub.17 K X.sub.19 C or H X.sub.20 N X.sub.22 R X.sub.26 I or T
X.sub.27 H X.sub.29 V X.sub.30 R X.sub.31 A or S X.sub.32 V
X.sub.33 P or G X.sub.36 E or A X.sub.37 E X.sub.40 A X.sub.41 P
X.sub.44 G X.sub.47 G X.sub.49 I X.sub.50 Q X.sub.51 G X.sub.63 H
X.sub.64 C X.sub.66 C X.sub.68 L or M X.sub.69 N X.sub.74 K or H
X.sub.75 D X.sub.77 V X.sub.78 C X.sub.81 T X.sub.82 V X.sub.85 R
or K X.sub.86 R X.sub.91 S X.sub.95 M or V X.sub.98 S X.sub.99 V
X.sub.106 S X.sub.111 P X.sub.113 C X.sub.120 L X.sub.122 T or V
X.sub.130 S X.sub.132 R X.sub.135 S X.sub.137 H X.sub.138 Y
X.sub.139 T X.sub.141 R X.sub.142 R X.sub.145 P X.sub.149 V
X.sub.155 V X.sub.156 E X.sub.160 T X.sub.163 A X.sub.164 I
X.sub.177 L X.sub.178 G or C X.sub.180 G X.sub.184 C X.sub.185 T
X.sub.187 R or E X.sub.188 H X.sub.190 C X.sub.191 G X.sub.192 I
X.sub.196 A or G X.sub.197 T or V X.sub.198 I X.sub.200 V X.sub.202
T X.sub.204 A X.sub.206 I or A X.sub.212 M X.sub.215 H or R
X.sub.217 A X.sub.218 G X.sub.222 R X.sub.223 H X.sub.226 A
X.sub.228 V X.sub.230 A X.sub.232 S X.sub.238 I X.sub.244 G or V
X.sub.247 G X.sub.248 G, K, or M X.sub.249 I X.sub.250 T or V
X.sub.251 Y or S X.sub.252 T X.sub.254 V X.sub.260 S X.sub.266 V
X.sub.268 G X.sub.275 G or K X.sub.278 Y X.sub.279 K X.sub.282 N
X.sub.283 I or V X.sub.290 M or V X.sub.292 A X.sub.297 M or F
X.sub.299 M X.sub.300 R or S X.sub.302 L X.sub.303 H X.sub.310 T or
V X.sub.311 G X.sub.313 N, V, S, or T X.sub.315 S X.sub.321 L
X.sub.323 F X.sub.328 Y X.sub.331 H X.sub.333 A X.sub.336 A
X.sub.339 V X.sub.341 L, I, or A X.sub.342 A X.sub.347 I X.sub.353
R X.sub.355 I X.sub.359 F X.sub.362 A or I X.sub.363 T X.sub.367 A
X.sub.368 G X.sub.370 D X.sub.371 D X.sub.372 C X.sub.374 G
X.sub.377 G X.sub.379 V X.sub.381 S X.sub.382 H X.sub.383 V
X.sub.384 D or G X.sub.385 E X.sub.386 Q X.sub.387 R or A X.sub.388
I X.sub.390 I X.sub.392 T or E X.sub.394 L X.sub.395 E or S
X.sub.396 S X.sub.397 R X.sub.398 R X.sub.399 M X.sub.400 V
X.sub.401 L or M X.sub.402 V X.sub.403 S, G, I, L, M, or T
X.sub.404 P X.sub.405 N X.sub.406 S X.sub.407 E X.sub.409 E or M
X.sub.410 E or D X.sub.412 R X.sub.413 L, G, or S X.sub.414 G or V
X.sub.415 E or R X.sub.416 A
[0064] In some embodiments, the engineered AAMs with improved
enzyme activity can have modifications to a subset of the residues
above. In some of these embodiments, the amino acid residues of the
naturally occurring AAM polypeptide of SEQ ID NO:2 or the AAM
polypeptide of SEQ ID NO:4 that can be substituted are as
follows.
TABLE-US-00004 Residue Amino Acid X.sub.8 aromatic amino acid
X.sub.20 acid or polar amino acid X.sub.26 aliphatic amino acid
X.sub.33 small or conformationally constrained amino acid X.sub.36
acidic or small amino acid X.sub.51 small or aliphatic amino acid
X.sub.74 basic amino acid; X.sub.85 basic amino acid X.sub.86
hydrophilic amino acid X.sub.91 small amino acid X.sub.95
hydrophobic amino acid X.sub.99 aliphatic amino acid X.sub.111
hydrophobic amino acid X.sub.130 polar amino acid X.sub.135
hydroxyl containing amino acid X.sub.138 aromatic amino acid
X.sub.139 small amino acid X.sub.141 basic or polar amino acid
X.sub.142 basic amino acid X.sub.149 aliphatic amino acid X.sub.155
aliphatic amino acid X.sub.156 acidic amino acid X.sub.163 small
amino acid X.sub.192 aliphatic amino acid X.sub.202 hydroxyl
containing amino acid X.sub.204 small amino acid X.sub.244 small
amino acid X.sub.247 small amino acid X.sub.251 aromatic or
hydroxyl containing amino acid X.sub.275 basic or small amino acid
X.sub.283 aliphatic amino acid X.sub.292 aliphatic amino acid
X.sub.297 hydrophobic amino acid X.sub.300 charged or polar amino
acid X.sub.313 polar or aliphatic amino acid X.sub.315 hydroxyl
containing amino acid X.sub.331 charged or small amino acid
X.sub.339 aliphatic amino acid X.sub.341 hydrophobic amino acid
X.sub.342 hydrophobic amino acid X.sub.353 basic amino acid
X.sub.355 aliphatic amino acid X.sub.362 aliphatic amino acid
X.sub.363 hydroxyl containing amino acid X.sub.371 charge or polar
amino acid X.sub.379 small amino acid X.sub.383 aliphatic amino
acid X.sub.384 acidic amino acid X.sub.385 charged amino acid
X.sub.387 basic or small amino acid residue X.sub.392 charged or
polar amino acid X.sub.395 acidic or polar amino acid X.sub.399
hydrophobic amino acid X.sub.401 hydrophobic amino acid X.sub.404
hydropobic amino acid X.sub.410 acidic amino acid X.sub.412 basic
amino acid X.sub.413 hydrophobic or small amino acid
[0065] In some embodiments, the subset of the amino acid residues
above can be substituted with one or more of the specific amino
acid residues below to obtain AAMs with improved enzymatic
activity:
TABLE-US-00005 Residue Amino Acid X.sub.8 H X.sub.20 N X.sub.26 I
X.sub.33 P X.sub.36 E X.sub.51 G X.sub.74 K X.sub.85 R or K
X.sub.86 R X.sub.91 S X.sub.95 M X.sub.99 V X.sub.111 P X.sub.130 S
X.sub.135 S X.sub.138 Y X.sub.139 T X.sub.141 R X.sub.142 R
X.sub.149 V X.sub.156 E X.sub.163 A X.sub.192 I X.sub.202 T
X.sub.204 A X.sub.244 V X.sub.247 G X.sub.251 Y or S X.sub.275 G
X.sub.283 I or V X.sub.292 A X.sub.297 M or F X.sub.300 R X.sub.313
T X.sub.315 S X.sub.331 H X.sub.339 V X.sub.341 L or I X.sub.342 A
X.sub.353 R X.sub.355 I X.sub.362 A X.sub.363 T X.sub.371 D
X.sub.379 V X.sub.383 V X.sub.384 D X.sub.385 E X.sub.387 R
X.sub.392 T X.sub.395 E X.sub.399 M X.sub.401 L X.sub.404 P
X.sub.410 E X.sub.412 R X.sub.413 L or S X.sub.341 L or I X.sub.342
A X.sub.353 R X.sub.355 I X.sub.362 A X.sub.363 T X.sub.371 D
X.sub.379 V X.sub.383 V X.sub.384 D X.sub.385 E X.sub.387 R
X.sub.392 T X.sub.395 E X.sub.399 M X.sub.401 L X.sub.404 P
X.sub.410 E X.sub.412 R X.sub.413 L or S
[0066] In some embodiments, the engineered AAM with improved enzyme
activity can have modifications to a subset of the residues above
based on a reference polypeptide of another engineered AAM. Thus,
in some embodiments, the modifications can be based on the
reference engineered AAM of SEQ ID NO:909, with the residues that
can be substituted with reference to SEQ ID NO:4 as indicated
below.
TABLE-US-00006 Residue Amino Acid X.sub.4 basic or polar amino acid
X.sub.7 charged amino acid X.sub.15 polar amino acid X.sub.19 basic
or polar amino acid X.sub.20 acid or polar amino acid X.sub.26
aliphatic amino acid X.sub.33 small or conformationally constrained
amino acid X.sub.40 small amino acid X.sub.41 contrained amino acid
X.sub.44 aliphatic amino acid; X.sub.47 small or aliphatic amino
acid X.sub.50 hydrophilic amino acid X.sub.51 small or aliphatic
amino acid; X.sub.63 aromatic amino acid X.sub.64 hydrophilic amino
acid; X.sub.66 hydrophilic amino acid X.sub.68 hydrophobic amino
acid X.sub.69 polar amino acid X.sub.74 basic amino acid X.sub.75
acidic amino acid X.sub.81 small amino acid X.sub.85 basic amino
acid X.sub.91 small amino acid X.sub.95 hydrophobic amino acid
X.sub.98 small amino acid X.sub.99 aliphatic amino acid X.sub.113
polar amino acid X.sub.130 polar amino acid X.sub.132 basic amino
acid X.sub.135 hydroxyl containing amino acid X.sub.137 basic amino
acid X.sub.141 basic or polar amino acid X.sub.155 aliphatic amino
acid X.sub.156 acidic amino acid X.sub.163 small amino acid
X.sub.178 small amino acid; X.sub.180 small amino acid X.sub.184
hydrophobic or constrained amino acid X.sub.185 hydroxyl containing
amino acid X.sub.187 charged amino acid X.sub.188 basic amino acid
X.sub.180 small amino acid X.sub.184 hydrophobic or constrained
amino acid X.sub.185 hydroxyl containing amino acid X.sub.190 polar
amino acid X.sub.191 small amino acid X.sub.197 small amino acid
X.sub.202 hydroxyl containing amino acid X.sub.206 aliphatic amino
acid X.sub.212 non-polar amino acid X.sub.218 small amino acid
X.sub.226 aliphatic amino acid X.sub.230 small amino acid X.sub.244
small amino acid X.sub.247 small amino acid X.sub.248 basic or
hydrophobic amino acid X.sub.249 hydrophobic amino acid X.sub.250
hydroxyl containing or aliphatic amino acid X.sub.252 hydroxyl
containing amino acid X.sub.268 small amino acid X.sub.278 aromatic
amino acid X.sub.283 aliphatic amino acid X.sub.297 hydrophobic
amino acid X.sub.299 hydrophobic amino acid X.sub.303 basic amino
acid X.sub.310 aliphatic or hydroxyl containing amino acid
X.sub.311 small amino acid X.sub.313 polar or aliphatic amino acid
X.sub.315 hydroxyl containing amino acid X.sub.321 aliphatic amino
acid X.sub.323 aromatic amino acid X.sub.336 aliphatic amino acid
X.sub.341 hydrophobic amino acid X.sub.355 aliphatic amino acid
X.sub.362 aliphatic amino acid X.sub.363 hydroxyl containing amino
acid X.sub.367 small amino acid X.sub.368 small amino acid
X.sub.372 charged or polar amino acid X.sub.385 charged amino acid
X.sub.397 basic or polar amino acid X.sub.398 basic or polar amino
acid X.sub.400 aliphatic amino acid X.sub.403 hydrophobic or small
amino acid X.sub.413 hydrophobic or small amino acid
[0067] In some embodiments, the substitutions based on the
reference sequence of SEQ ID NO:909 can be selected from one or
more of the specific amino acids as follows:
TABLE-US-00007 Residue Amino Acid X.sub.4 R X.sub.7 E X.sub.15 N
X.sub.19 C X.sub.20 N X.sub.26 I or T X.sub.33 P or G X.sub.40 A
X.sub.41 P X.sub.44 G X.sub.47 G X.sub.50 Q X.sub.51 G X.sub.63 H
X.sub.64 C X.sub.66 C X.sub.68 L or M; X.sub.69 N X.sub.74 K or H
X.sub.81 T X.sub.85 R or K X.sub.91 S X.sub.95 M or V X.sub.98 S
X.sub.99 V X.sub.113 C X.sub.130 S X.sub.132 R X.sub.135 S
X.sub.137 H X.sub.141 R X.sub.155 V X.sub.163 A X.sub.178 G or C
X.sub.184 C X.sub.185 T X.sub.187 R or E X.sub.190 C X.sub.197 T or
V X.sub.202 T X.sub.206 I or A X.sub.212 M X.sub.218 G X.sub.226 A
X.sub.230 A X.sub.244 G or V X.sub.247 G X.sub.248 G, K, or M
X.sub.249 I X.sub.250 T or V X.sub.252 T X.sub.268 G X.sub.278 Y
X.sub.283 I or V X.sub.297 M or F X.sub.299 M X.sub.303 H X.sub.311
G X.sub.313 N, V, S, or T X.sub.321 L X.sub.323 F X.sub.336 A
X.sub.341 L, I, or A X.sub.355 I X.sub.362 A or I X.sub.363 T
X.sub.367 A X.sub.368 G X.sub.372 C X.sub.385 E X.sub.397 R
X.sub.398 R X.sub.400 V; X.sub.403 S, G, or T X.sub.413 L, G, or
S
[0068] In other embodiments, the subset of modifications to the
engineered alanine 2,3-aminomutase of SEQ ID NO:909 can include the
following substitutions with respect to SEQ ID NO:4:
TABLE-US-00008 Residue Amino Acid X.sub.2 hydroxyl containing amino
acid X.sub.4 basic or polar amino acid X.sub.8 aromatic amino acid
X.sub.9 aromatic amino acid X.sub.10 aromatic amino acid X.sub.16
charged amino acid X.sub.17 basic amino acid X.sub.19 basic or
polar amino acid X.sub.22 basic amino acid X.sub.29 aliphatic amino
acid X.sub.30 charged amino acid X.sub.31 aliphatic or hydroxyl
containing amino acid X.sub.32 aliphatic amino acid X.sub.36 acidic
or small amino acid X.sub.37 charge amino acid X.sub.49 aliphatic
amino acid X.sub.68 hydrophobic amino acid X.sub.75 acidic amino
acid X.sub.77 aliphatic amino acid X.sub.78 hydrophilic amino acid
X.sub.82 aliphatic amino acid X.sub.85 basic amino acid X.sub.86
hydrophilic amino acid X.sub.106 small amino acid X.sub.120
hydrophobic amino acid X.sub.122 small amino acid X.sub.142 basic
amino acid X.sub.145 hydrophobic amino acid X.sub.149 aliphatic
amino acid X.sub.160 hydroxyl containing amino acid X.sub.164
aliphatic amino acid X.sub.177 aliphatic amino acid X.sub.178 small
amino acid X.sub.180 small amino acid X.sub.187 charged amino acid
X.sub.191 small amino acid X.sub.192 aliphatic amino acid X.sub.196
small or aliphatic amino acid X.sub.197 small amino acid X.sub.198
aliphatic amino acid X.sub.200 aliphatic amino acid X.sub.204 small
amino acid X.sub.206 aliphatic amino acid X.sub.215 basic amino
acid X.sub.217 aliphatic amino acid X.sub.222 basic amino acid
X.sub.223 aromatic amino acid X.sub.228 hydrophobic amino acid
X.sub.232 small amino acid X.sub.238 aliphatic amino acid X.sub.248
basic or hydrophobic amino acid X.sub.250 hydroxyl containing or
aliphatic amino acid X.sub.251 aromatic or hydroxyl containing
amino acid X.sub.254 hydrophobic amino acid X.sub.260 hydroxyl
containing amino acid X.sub.266 aliphatic amino acid X.sub.275
basic or small amino acid X.sub.279 basic amino acid X.sub.282
charged amino acid X.sub.283 aliphatic amino acid X.sub.290
hydrophobic amino acid X.sub.300 charged or polar amino acid
X.sub.302 hydrophobic amino acid X.sub.310 aliphatic or hydroxyl
containing amino acid X.sub.313 polar or aliphatic amino acid
X.sub.315 hydroxyl containing amino acid X.sub.328 aromatic amino
acid X.sub.333 aliphatic amino acid X.sub.339 aliphatic amino acid
X.sub.341 hydrophobic amino acid X.sub.342 hydrophobic amino acid
X.sub.347 aliphatic amino acid X.sub.359 aromatic amino acid
X.sub.362 aliphatic amino acid X.sub.370 acidic amino acid
X.sub.374 small amino acid X.sub.377 small amino acid X.sub.379
small amino acid X.sub.381 polar amino acid X.sub.382 basic amino
acid X.sub.383 aliphatic amino acid X.sub.384 acidic amino acid
X.sub.386 basic or polar amino acid X.sub.387 basic or small amino
acid residue X.sub.388 aliphatic amino acid X.sub.390 aliphatic
amino acid X.sub.392 charged or polar amino acid X.sub.394
aliphatic amino acid X.sub.395 acidic or polar amino acid X.sub.396
hydroxyl containing amino acid X.sub.401 hydrophobic amino acid
X.sub.402 small amino acid X.sub.403 hydrophobic or small amino
acid X.sub.404 hydropobic amino acid X.sub.405 acidic or polar
amino acid X.sub.406 hydroxyl containing amino acid X.sub.407
acidic or polar amino acid X.sub.409 charged or hydrophobic amino
acid X.sub.410 acidic amino acid X.sub.412 basic amino acid
X.sub.413 hydrophobic or small amino acid X.sub.414 aliphatic or
small amino acid X.sub.415 charged amino acid X.sub.416 aliphatic
amino acid
[0069] In some embodiments, the subset of the amino acid residues
that can be modified based on the reference AAM polypeptide of SEQ
ID NO:909 include substitutions with one or more specific amino
acids below with respect to SEQ ID NO:4:
TABLE-US-00009 Residue Amino Acid X.sub.2 S or T X.sub.4 R, C or N
X.sub.8 H X.sub.9 F X.sub.10 W X.sub.16 K X.sub.17 K X.sub.19 C or
H X.sub.22 R X.sub.29 V X.sub.30 R X.sub.31 A or S X.sub.32 V
X.sub.36 E or A X.sub.37 E X.sub.49 I X.sub.68 L or M X.sub.75 D
X.sub.77 V X.sub.78 C X.sub.82 V X.sub.85 R or K X.sub.86 R
X.sub.106 S X.sub.120 L X.sub.122 T or V X.sub.142 R X.sub.145
X.sub.149 V X.sub.156 E X.sub.160 T X.sub.164 I X.sub.177 L
X.sub.178 G or C X.sub.180 G X.sub.187 R or E X.sub.191 G X.sub.192
I X.sub.196 A or G X.sub.197 T or V X.sub.198 I X.sub.200 V
X.sub.204 A X.sub.206 I or A X.sub.215 H or R X.sub.217 A X.sub.222
R X.sub.223 H X.sub.228 V X.sub.232 S X.sub.238 I X.sub.248 G, K,
or M X.sub.250 T or V X.sub.251 Y or S X.sub.254 V X.sub.260 S
X.sub.266 V X.sub.275 G or K X.sub.279 K X.sub.282 N X.sub.283 I or
V X.sub.290 M or V X.sub.300 R or S X.sub.302 L X.sub.310 T or V
X.sub.313 N, V, S, or T X.sub.315 S X.sub.321 L X.sub.328 Y
X.sub.333 A X.sub.339 V X.sub.341 L, I, or A X.sub.342 A X.sub.347
I X.sub.359 F X.sub.362 A or I X.sub.370 D X.sub.374 G X.sub.377 G
X.sub.379 V X.sub.381 S X.sub.382 H X.sub.383 V X.sub.384 D or G
X.sub.386 Q X.sub.387 R or A X.sub.388 I X.sub.390 I X.sub.392 T or
E X.sub.394 L X.sub.395 E or S X.sub.396 S X.sub.401 L or M
X.sub.402 V X.sub.403 S, G, I, L, M, or T X.sub.404 P X.sub.405 N
X.sub.406 S X.sub.407 E X.sub.409 E or M X.sub.410 E or D X.sub.412
R X.sub.413 L, G, or S X.sub.414 G or V X.sub.415 E or R X.sub.416
A
[0070] It is to be understood that the forgoing description is not
to be limiting but is presented to provide guidance to the skilled
artisan for identifying the amino acid residue positions for
modification and the type of amino acid substitutions that can be
used to obtain the engineered AAM enzymes of the present
disclosure.
[0071] To illustrate the generation of engineered AAM enzymes, the
AAM polynucleotide of SEQ ID NO:3 was subjected to mutagenesis by
directed evolution, and the mutated polynucleotides expressed and
then screened in accordance with the protocol described in Example
4 to identify those having improved AAM activity. In the process of
Example 4, the screening of clones from the expression libraries
was performed by measuring the conversion of .alpha.-alanine to
.beta.-alanine using liquid chromatography and mass spectrometry.
Based upon the screening results, a set of the AAM polypeptides of
the present disclosure are listed in Table 3 below along with their
residue changes relative to the parental AAM polypeptide, i.e., the
polypeptide of SEQ ID NO: 4, as well as their AAM activity (i.e.,
rate of production of .beta.-alanine in .mu.M/hour).
[0072] Enzymatic activities of exemplary engineered alanine
2,3-aminomutases are shown in Table 2, which shows the total .mu.M
of .beta.-alanine produced after 17 hours and the rate of
production of .beta.-alanine in units of .mu.M/hr.
TABLE-US-00010 TABLE 2 uM .beta.-alanine Rate of .beta.-alanine
.mu.M SEQ ID NO. produced at t = 17 hrs produced/hr 36 24.99 1.47
2218 25.35 1.49 794 24.1 1.42 804 42.11 2.48 910 106.28 6.25 2220
70.79 4.16 1282 88.52 5.21 2222 104 6.12 1696 107.12 6.3 1884
131.43 7.73 1886 119.0 7.00 2052 138 8.12 2162 157 9.23
[0073] Shown in Table 3 are exemplary AAMs with defined amino acid
changes, which result in improved enzymatic activity. The set of
changes in each enzyme, in addition to other such enzymes in the
descriptions herein, provides the skilled artisan with guidance on
the types and sets of amino acids that can be changed to obtain AAM
polypeptides with improved enzymatic activity.
TABLE-US-00011 Rate of .beta.-alanine SEQ ID Residue Changes
Relative to produced: NOS. Parent SEQ ID NO: 4 .mu.M/hr 36 I339V
1.47 794 L26I; D33P; Q85K; Q95M; V192I; G300R; 1.42 G331H; I339V;
L399M 804 L26I; K36E; Q85K; Q95M; F138Y; G331H; 2.48 I339V; L399M
910 L261I; K36E Q95M; V292A G331H; I339V; 6.25 V341L; T342A; N371D;
I401L 1282 L261I; K36E Q85K; Q95M; V292A; G331H; 5.21 I339V; T342A;
N371D; K387R; I401L 1696 L261I; K36E; Q95M; V292A; G331H; N371D;
6.3 I401L; S404P 1884 L261I; K36E Q85K; Q95M; A139T; K142R; 7.73
V292A; G331H; I339V; T342A; N371D; E379V; K387R; I401L 1886 L261I;
K36E Q85K; Q95M; A139T; V292A; 7.00 G331H; I339V; T342A; N371D;
K387R; I401L 2052 L261I; K36E; Q95M; D156E; T204A; M283I; 8.12
V292A; G331H; I339V; N371D; G384D; I401L; S404P 2162 L261I; K36E;
Q85K; Q95M; A139T; S202T; 9.23 V292A; G331H; I339V; V341L; T342A;
N371D; I401L 2218 L26I; K36E; Q95M; G300R; G331H 1.49 2220 L261I;
K36E; Q95M; V292A; G331H; N371D; 4.16 I401L 2222 L261I; K36E; Q95M;
D156E; M283I; V292A; 6.12 G331H; N371D; G384D; I401L; S404P
[0074] As show in the exemplary engineered AAM above, the
polypeptides, can have in some embodiments from 1 to 14 residue
differences relative to SEQ ID NO: 4, and display significant AAM
activity based on production of .beta.-alanine in the assay of
Example 4. As comparison, very little .beta.-alanine productivity
is detected for SEQ ID NO: 4 under the assay conditions used to
test the AAM variants. Thus, in some embodiments, the engineered
AAM polypeptide can have about 1-14 amino acid residue
substitutions, 2-14 amino acid residue substitutions, 8-14 amino
acid residue substitutions, or 12-14 amino acid residue
substitutions.
[0075] In some embodiments, the amino acid substitutions of the
above groupings are selected from the group of substitutions
comprising Y8H, D20N, L26I, K36E, E51G, N74K, Q85R or K, Q86R,
A91S, Q95M, L99V, T111P, C130S, R135S, A139T, Q141R, K142R, E149V,
D156E, T163A, S202T, T204A, E244V, E247G, N251Y or S, R275G, M283I
or V, V292A, L297M or F, I313T, N315S, G331H, I339V, V341L or I,
T342A, H353R, V355I, V362A, I363T, N371D, A383V, G384D, K385E,
K387R, A392T, G395E, I401L, S404P, K410E, K412R, or F413L or S.
[0076] In some embodiments, the polypeptide having improved alanine
2,3-aminomutase activity is selected from the amino acid sequences
of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,
204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,
230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,
256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,
282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,
308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,
334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,
360, 362, 364, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,
388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412,
414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438,
440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464,
466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,
518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542,
544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568,
570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594,
596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620,
622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646,
648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672,
674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698,
700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724,
726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750,
752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776,
778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802,
804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828,
830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854,
856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880,
882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906,
908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932,
934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958,
960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984,
986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008,
1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030,
1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052,
1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074,
1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096,
1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118,
1170, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140,
1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162,
1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184,
1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206,
1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228,
1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250,
1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272,
1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294,
1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316,
1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338,
1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360,
1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382,
1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404,
1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426,
1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448,
1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470,
1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492,
1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,
1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536,
1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558,
1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580,
1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602,
1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1670, 1622, 1624,
1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646,
1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668,
1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690,
1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712,
1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734,
1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756,
1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778,
1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800,
1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822,
1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844,
1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866,
1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888,
1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910,
1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932,
1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954,
1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976,
1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998,
2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020,
2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042,
2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064,
2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086,
2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108,
2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130,
3132, 2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152,
2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174,
2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196,
2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218,
2220, or 2222.
[0077] In some embodiments, the polypeptide with improved AAM
enzyme activity is a polypeptide encoded by a polynucleotide
sequence selected from, or which hybridizes under high stringency
conditions, with the polynucleotide sequence or with a complement
of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,
179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,
205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,
231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,
257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,
283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,
309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,
335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,
361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,
387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411,
413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,
439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463,
465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489,
491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,
517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541,
543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567,
569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593,
595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619,
621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645,
647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,
673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697,
699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723,
725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,
751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,
777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,
803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827,
829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853,
855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879,
881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905,
907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931,
933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957,
959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983,
985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007,
1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029,
1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051,
1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073,
1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095,
1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117,
1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139,
1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161,
1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183,
1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205,
1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227,
1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249,
1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271,
1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293,
1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315,
1317, 1319, 1321, 1323, 1325, 1027, 1029, 1331, 1333, 1335, 1337,
1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1057, 1059,
1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381,
1383, 1385, 1087, 1089, 1391, 1393, 1395, 1397, 1399, 1401, 1403,
1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425,
1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447,
1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469,
1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491,
1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513,
1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535,
1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555, 1557,
1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579,
1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601,
1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623,
1625, 1627, 1029, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645,
1647, 1649, 1651, 1653, 1655, 1657, 1059, 1661, 1663, 1665, 1667,
1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1089,
1691, 1693, 1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711,
1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733,
1735, 1737, 1739, 1741, 1743, 1745, 1747, 1749, 1751, 1753, 1755,
1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777,
1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799,
1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821,
1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843,
1845, 1848, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865,
1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885, 1887,
1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909,
1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931,
1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953,
1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975,
1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997,
1999, 2001, 2003, 2005, 2007, 2009, 2011, 2013, 2015, 2017, 2019,
2021, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041,
2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063,
2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2083, 2085,
2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107,
2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129,
2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149, 2151,
2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173,
2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195,
2197, 2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215, 2217,
2219 or 2221, wherein the encoded polypeptide has greater alanine
2,3-aminomutase (AAM) activity than the polypeptide of SEQ ID NO:
4.
[0078] In some embodiments, the AAM polypeptides is selected from
the amino acid sequences of SEQ ID NO: 36, 794, 804, 910, 1282,
1696, 1884, 1886, 2052, 2162, 2218, 2220, and 2222. In some
embodiments, the AAM is selected from the amino acid sequences of
SEQ ID NO: 910, 1262, 1696, 1884, 1886, 2052 and 2162. In some
embodiments, the AMM has an amino acid sequence of SEQ ID NO: 2052
or 2162.
[0079] In some embodiments, the amino acid substitutions of the
groupings are selected from one or more substitutions comprising:
S4R; K7E; D15N; N19C; D20N; L26I or T; D33P or G; T40A; L41P; E44G;
E47G; K50Q; S51G; Y63H; Y64C; S66C; T68L or M; D69N; N74K or H;
A81T; Q85R or K; A91S; Q95M or V; P98S; L99V; R113C; C130S; H132R;
R135S; R137H; Q141R; I155V; T163A; S178G or C; Y184C; I185T; K187R
or E; E190C; I197T or V; S202T; V206I or A; I212M; D218G; V226A;
H230A; E244G or V; E247G; R248G, K, or M; M249I; A250T or V; A252T;
D268G; H278Y; M283I or V; L297M or F; I299M; R303H; E311G; I313N,
V, S, or T; S321L; Y323F; G336A; V341L, I, or A; V355I; V362A or I;
I363T; T367A; E368G; Y372C; K385E; Q397R; Q398R; I400V; P403S, G,
or T; and P413L, G, or S.
[0080] In some embodiments, the amino acid substitutions of the
groupings are selected from one or more substitutions comprising
SEQ ID NO:4 and which includes one or more substitutions selected
from: A2S or T; S4C or N; Y8H; Y9F; F10W; E16K; Q17K; Y19H; H22R;
129V; K30R; T31A or S; L32V; K36A; K37E; V49I; 168M; C75D; 177V;
R78C; 182V; Q85K; Q86R; P106S; F120L; I122T or V, K142R, S145P,
E149V, D156E, N160T, V164I, V177L, S178C, E180G, K187E, E191G,
V192I, E196A or G, I197V, V198I, I200V, T204A, V206A, Q215H or R,
V217A, K222R, Y223H, L228V, F232S, V238I, R248K or M, A250V, N251S,
I254V, T260S, I266V, R275K, L279K, K282N, M283I, I290M or V, G300R
or S, F302L, I310T or V, I313V, N315S, F328Y, P333A, V341A, V347I,
Y359F, V362I, E370D, E374G, D377G, E379V, C381S, R382H, A383V,
G384D, H386Q, K387A or R, E388I, V390I, A392E or T, S394L, G395S,
G396S, I401M, E402V, P403G, I, L, M, or T, S404P, D405N, L406S,
A407E, K409E or M, K410D or E, K412R, P413G or S, D414G or V, K415E
or R, and N416A.
[0081] In some embodiments, the polypeptide having improved AAM
activity is selected from the amino acid sequences of SEQ ID NO:
2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242, 2244,
2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266,
2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288,
2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310,
2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332,
2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354,
2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376,
2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398,
2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420,
2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442,
2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464,
2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486,
2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508,
2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530,
2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552,
2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574,
2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594, 2596,
2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618,
2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640,
2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662,
2664, 2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684,
2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704, 2706,
2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728,
2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750,
2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772,
2774, 2776, 2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792, 2794,
2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816,
2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, 2836, 2838,
2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854, 2856, 2858, 2860,
2862, 2864, 2866, 2868, 2870, 2872, 2874, 2876, 2878, 2880, 2882,
2884, 2886, 2888, 2890, 2892, 2894, 2896, 2898, 2900, 2902, 2904,
2906, 2908, 2910, 2912, 2914, 2916, 2918, 2920, 2922, 2924, 2926,
2928, 2930, 2932, 2934, 2936, 2938, 2940, 2942, 2944, 2946, 2948,
2950, 2952, 2954, 2956, 2958, 2960, 2962, 2964, 2966, 2968, 2970,
2972, 2974, 2976, 2978, 2980, 2982, 2984, 2986, 2988, 2990, 2992,
2994, 2996, 2998, 3000, 3002, 3004, 3006, 3008, 3010, 3012, 3014,
3016, 3018, 3020, 3022, 3024, 3026, 3028, 3030, 3032, 3034, 3036,
3038, 3040, 3042, 3044, 3046, 3048, 3050, 3052, 3054, 3056, 3058,
3060, 3062, 3064, 3066, 3068, 3070, 3072, 3074, 3076, 3078, 3080,
3082, 3084, 3086, 3088, 3090, 3092, 3094, 3096, 3098, 3100, 3102,
3104, 3106, 3108, 3110, 3112, 3114, 3116, 3118, 3120, 3122, 3124,
3126, 3128, 3130, 3132, 3134, 3136, 3138, 3140, 3142, 3144, 3146,
3148, 3150, 3152, 3154, 3156, 3158, 3160, 3162, 3164, 3166, 3168,
3170, 3172, 3174, 3176, 3178, 3180, 3182, 3184, 3186, 3188, 3190,
3192, 3194, 3196, 3198, 3200, 3202, 3204, 3206, 3208, 3210, 3212,
3214, 3216, 3218, 3220, 3222, 3224, 3226, 3228, 3230, 3232, 3234,
3236, 3238, 3240, 3242, 3244, 3246, 3248, 3250, 3252, 3254, 3256,
3258, 3260, 3262, 3264, 3266, 3268, 3270, 3272, 3274, 3276, 3278,
3280, 3282, 3284, 3286, 3288, 3290, 3292, 3294, 3296, 3298, 3300,
3302, 3304, 3306, 3308, 3310, 3312, 3314, 3316, 3318, 3320, 3322,
3324, 3326, 3328, 3330, 3332, 3334, 3336, 3338, 3340, 3342, 3344,
3346, 3348, 3350, 3352, 3354, 3356, 3358, 3360, 3362, 3364, 3366,
3368, 3370, 3372, 3374, 3376, 3378, 3380, 3382, 3384, 3386, 3388,
3390, 3392, 3394, 3396, 3398, 3400, 3402, 3404, 3406, 3408, 3410,
3412, 3414, 3416, 3418, 3420, 3422, 3424, 3426, 3428, 3430, 3432,
3434, 3436, 3438, 3440, 3442, 3444, 3446, 3448, 3450, 3452, 3454,
3456, 3458, 3460, 3462, 3464, 3466, 3468, 3470, 3472, 3474, 3476,
3478, 3480, 3482, 3484, 3486, 3488, 3490, 3492, 3494, 3496, 3498,
3500, 3502, 3504, 3506, 3508, 3510, 3512, 3514, 3516, 3518, 3520,
3522, 3524, 3526, 3528, 3530, 3532, 3534, 3536, 3538, 3540, 3542,
3544, 3546, 3548, 3550, 3552, 3554, 3556, 3558, 3560, 3562, 3564,
3566, 3568, 3570, 3572, 3574, 3576, 3578, 3580, 3582, 3584, 3586,
3588, 3590, 3592, 3594, 3596, 3598, 3600, 3602, 3604, 3606, 3608,
3610, 3612, 3614, 3616, 3618, 3620, 3622, 3624, 3626, 3628, 3630,
3632, 3634, 3636, 3638, 3640, 3642, 3644, 3646, 3648, 3650, 3652,
3654, 3656, 3658, 3660, 3662, 3664, 3666, 3668, 3670, 3672, 3674,
3676, 3678, 3680, 3682, 3684, 3686, 3688, 3690, 3692, 3694, 3696,
3698, 3700, 3702, 3704, 3706, 3708, 3710, 3712, 3714, 3716, 3718,
3720, 3722, 3724, 3726, 3728, 3730, 3732, 3734, 3736, 3738, 3740,
3742, 3744, 3746, 3748, 3750, 3752, 3754, 3756, 3758, 3760, 3762,
3764, 3766, 3768, 3770, 3772, 3774, 3776, 3778, 3780, 3782, 3784,
3786, 3788, 3790, 3792, 3794, 3796, 3798, 3800, 3802, 3804, 3806,
3808, 3810, 3812, 3814, 3816, 3818, 3820, 3822, 3824, 3826, 3828,
3830, 3832, 3834, 3836, 3838, 3840, 3842, 3844, 3846, 3848, 3850,
3852, 3854, 3856, 3858, 3860, 3862, 3864, 3866, 3868, 3870, 3872,
3874, 3876, 3878, 3880, 3882, 3884, 3886, 3888, 3890, 3892, 3894,
3896, 3898, 3900, 3902, 3904, 3906, 3908, 3910, 3912, 3914, 3916,
3918, 3920, 3922, 3924, 3926, 3928, 3930, 3932, 3934, 3936, 3938,
3940, 3942, 3944, 3946, 3948, 3950, 3952, 3954, 3956, 3958, 3960,
3962, 3964, 3966, 3968, 3970, 3972, 3974, 3976, 3978, 3980, 3982,
3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004,
4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, 4022, 4024, 4026,
4028, 4030, 4032, 4034, 4036, 4038, 4040, 4042, 4044, 4046, 4048,
4050, 4052, 4054, 4056, 4058, 4060, 4062, 4064, 4066, 4068, 4070,
4072, 4074, 4076, 4078, 4080, 4082, 4084, 4086, 4088, 4090, 4092,
4094, 4096, 4098, 4100, 4102, 4104, 4106, 4108, 4110, 4112, 4114,
4116, 4118, 4120, 4122, 4124, 4126, 4128, 4130, 4132, 4134, 4136,
4138, 4140, 4142, 4144, 4146, 4148, 4150, 4152, 4154, 4156, 4158,
4160, 4162, 4164, 4166, 4168, 4170, 4172, 4174, 4176, 4178, 4180,
4182, 4184, 4186, 4188, 4190, 4192, 4194, 4196, 4198, 4200, 4202,
4204, 4206, 4208, 4210, 4212, 4214, 4216, 4218, 4220, 4222, 4224,
4226, 4228, 4230, 4232, 4234, 4236, 4238, 4240, 4242, 4244, 4246,
4248, 4250, 4252, 4254, 4256, 4258, 4260, 4262, 4264, 4266, 4268,
4270, 4272, 4274, 4276, 4278, 4280, 4282, 4284, 4286, 4288, 4290,
4292, 4294, 4296, 4298, 4300, 4302, 4304, 4306, 4308, 4310, 4312,
4314, 4316, 4318, 4320, 4322, 4324, 4326, 4328, 4330, 4332, 4334,
4336, 4338, 4340, 4342, 4344, 4346, 4348,4350, 4352, 4354, 4356,
4358, 4360, 4362, 4364, 4366, 4368, 4370, 4372, 4374, 4376, 4378,
4380, 4382, 4384, 4386, 4388, 4390, 4392, 4394, 4396, 4398, 4400,
4402, 4404, 4406, 4408, 4410, 4412, 4414, 4416, 4418, 4420, 4422,
4424, 4426, 4428, 4430, 4432, 4434, 4436, 4438, 4440, 4442, 4444,
4446, 4448,4450, 4452, 4454, 4456, 4458, 4460, 4462, 4464, 4466,
4468, 4470, 4472, 4474, 4476, 4478, 4480, 4482, 4484, 4486, 4488,
4490, 4492, 4494, 4496, 4498, 4500, 4502, 4504, 4506, 4508, 4510,
4512, 4514, 4516, 4518, 4520, 4522, 4524, 4526, 4528, 4530, 4532,
4534, 4536, 4538, 4540, 4542, 4544, 4546, 4548,4550, 4552, 4554,
4556, 4558, 4560, 4562, 4564, 4566, 4568, 4570, 4572, 4574, 4576,
4578, 4580, 4582, 4584, 4586, 4588, 4590, 4592, 4594, 4596, 4598,
4600, 4602, 4604, 4606, 4608, 4610, 4612, 4614, 4616, 4618, 4620,
4622, 4624, 4626, 4628, 4630, 4632, 4634, 4636,4638, 4640, 4642,
4644, 4646, 4648,4650, 4652, 4654, 4656, 4658, 4660, 4662, 4664,
4666, 4668, 4670, 4672, 4674, 4676, 4678, 4680, 4682, 4684, 4686,
4688, 4690, 4692, 4694, 4696, 4698, 4700, 4702, 4704, 4706, 4708,
4710, 4712, 4714, 4716, 4718, 4720, 4722, 4724, 4726, 4728, 4730,
4732, 4734, 4736, 4738, 4740, 4742, 4744, 4746, 4748, 4750, 4752,
4754, 4756, 4758, 4760, 4762, 4764, 4766, 4768,4770, 4772, 4774,
4776, 4778, 4780, 4782, 4784, 4786, 4788, 4790, 4792, 4794, 4796,
4798, 4800, 4802, 4804, 4806, 4808, 4810, 4812, 4814, 4816, 4818,
4820, 4822, 4824, 4826, 4828, 4830, 4832, 4834, 4836,4838, 4840,
4842, 4844, 4846, 4848, 4850, 4852, 4854, 4856, 4858, 4860, 4862,
4864, 4866, 4868, 4870, 4872, 4874, 4876, 4878, 4880, 4882, 4884,
4886, 4888, 4890, 4892, 4894, 4896, 4898, 4900, 4902, 4904, 4906,
4908, 4910, 4912, 4914, 4916, 4918, 4920, 4922, 4924, 4926, 4928,
4930, 4932, 4934, 4936, 4938, 4940, 4942, 4944, 4946, 4948, 4950,
4952, 4954, 4956, 4958, 4960, 4962, 4964, 4966, 4968, 4970, 4972,
4974, 4976, 4978, 4980, 4982, 4984, 4986, 4988, 4990, 4992, 4994,
4996, 4998, 5000, 5002, 5004, 5006, 5008, 5010, 5012, 5014, 5016,
5018, 5020, 5022, 5024, 5026, 5028, 5030, 5032, 5034, 5036, 5038,
5040, 5042, 5044, 5046, 5048, 5050, 5052, 5054, 5056, 5058, 5060,
5062, 5064, 5066, 5068, 5070, 5072, 5074, 5076, 5078, 5080, 5082,
5084, 5086, 5088, 5090, 5092, 5094, 5096, 5098, 5100, 5102, 5104,
5106, 5108, 5110, 5112, 5114, 5116, 5118, 5120, 5122, 5124, 5126,
5128, 5130, 5132, 5134, 5136, 5138, 5140, 5142, 5144, 5146, 5148,
5150, 5152, 5154, 5156, 5158, 5160, 5162, 5164, 5166, 5168, 5170,
5172, 5174, 5176, 5178, 5180, 5182, 5184, 5186, 5188, 5190, 5192,
5194, 5196, 5198, 5200, 5202, 5204, 5206, 5208, 5210, 5212, 5214,
5216, 5218, 5220, 5222, 5224, 5226, 5228, 5230, 5232, 5234, 5236,
5238, 5240, 5242, 5244, 5246, 5248, 5250, 5252, 5254, 5256, 5258,
5260, 5262, 5264, 5266, 5268, 5270, 5272, 5274, 5276, 5278, 5280,
5282, 5284, 5286, 5288, 5290, 5292, 5294, 5296, 5298, 5300, 5302,
5304, 5306, 5308, 5310, 5312, 5314, 5316, 5318, 5320, 5322, 5324,
5326, 5328, 5330, 5332, 5334, 5336, 5338, 5340, 5342, 5344, 5346,
5348, 5350, 5352, 5354, 5356, 5358, 5360, 5362, 5364, 5366, 5368,
5370, 5372, 5374, 5376, 5378, 5380, 5382, 5384, 5386, 5388, 5390,
5392, 5394, 5396, 5398, 5400, 5402, 5404, 5406, 5408, 5410, 5412,
5414, 5416, 5418, and 5420.
[0082] In some embodiments, the polypeptide with improved enzyme
activity is a polypeptide encoded by a polynucleotide sequence
selected from, or which hybridizes under high stringency conditions
with the nucleotide sequence or a complement of SEQ ID NO: 2223,
2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245,
2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267,
2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289,
2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311,
2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333,
2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355,
2357, 2359, 2361, 2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377,
2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399,
2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, 2421,
2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443,
2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465,
2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 2487,
2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509,
2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531,
2533, 2535, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553,
2555, 2557, 2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575,
2577, 2579, 2581, 2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597,
2599, 2601, 2603, 2605, 2607, 2609, 2611, 2613, 2615, 2617, 2619,
2621, 2623, 2625, 2627, 2629, 2631, 2633, 2635, 2637, 2639, 2641,
2643, 2645, 2647, 2649, 2651, 2653, 2655, 2657, 2659, 2661, 2663,
2665, 2667, 2669, 2671, 2673, 2675, 2677, 2679, 2681, 2683, 2685,
2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701, 2703, 2705, 2707,
2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725, 2727, 2729,
2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749, 2751,
2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773,
2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795,
2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817,
2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839,
2841, 2843, 2845, 2847, 2849, 2851, 2853, 2855, 2857, 2859, 2861,
2863, 2865, 2867, 2869, 2871, 2873, 2875, 2877, 2879, 2881, 2883,
2885, 2887, 2889, 2891, 2893, 2895, 2897, 2899, 2901, 2903, 2905,
2907, 2909, 2911, 2913, 2915, 2917, 2919, 2921, 2923, 2925, 2927,
2929, 2931, 2933, 2935, 2937, 2939, 2941, 2943, 2945, 2947, 2949,
2951, 2953, 2955, 2957, 2959, 2961, 2963, 2965, 2967, 2969, 2971,
2973, 2975, 2977, 2979, 2981, 2983, 2985, 2987, 2989, 2991, 2993,
2995, 2997, 2999, 3001, 3003, 3005, 3007, 3009, 3011, 3013, 3015,
3017, 3019, 3021, 3023, 3025, 3027, 3029, 3031, 3033, 3035, 3037,
3039, 3041, 3043, 3045, 3047, 3049, 3051, 3053, 3055, 3057, 3059,
3061, 3063, 3065, 3067, 3069, 3071, 3073, 3075, 3077, 3079, 3081,
3083, 3085, 3087, 3089, 3091, 3093, 3095, 3097, 3099, 3101, 3103,
3105, 3107, 3109, 3111, 3113, 3115, 3117, 3119, 3121, 3123, 3125,
3127, 3129, 3131, 3133, 3135, 3137, 3139, 3141, 3143, 3145, 3147,
3149, 3151, 3153, 3155, 3157, 3159, 3161, 3163, 3165, 3167, 3169,
3171, 3173, 3175, 3177, 3179, 3181, 3183, 3185, 3187, 3189, 3191,
3193, 3195, 3197, 3199, 3201, 3203, 3205, 3207, 3209, 3211, 3213,
3215, 3217, 3219, 3221, 3223, 3225, 3227, 3229, 3231, 3233, 3235,
3237, 3239, 3241, 3243, 3245, 3247, 3249, 3251, 3253, 3255, 3257,
3259, 3261, 3263, 3265, 3267, 3269, 3271, 3273, 3275, 3277, 3279,
3281, 3283, 3285, 3287, 3289, 3291, 3293, 3295, 3297, 3299, 3301,
3303, 3305, 3307, 3309, 3311, 3313, 3315, 3317, 3319, 3321, 3323,
3325, 3327, 3329, 3331, 3333, 3335, 3337, 3339, 3341, 3343, 3345,
3347, 3349, 3351, 3353, 3355, 3357, 3359, 3361, 3363, 3365, 3367,
3369, 3371, 3373, 3375, 3377, 3379, 3381, 3383, 3385, 3387, 3389,
3391, 3393, 3395, 3397, 3399, 3401, 3403, 3405, 3407, 3409, 3411,
3413, 3415, 3417, 3419, 3421, 3423, 3425, 3427, 3429, 3431, 3433,
3435, 3437, 3439, 3441, 3443, 3445, 3447, 3449, 3451, 3453, 3455,
3457, 3459, 3461, 3463, 3465, 3467, 3469, 3471, 3473, 3475, 3477,
3479, 3481, 3483, 3485, 3487, 3489, 3491, 3493, 3495, 3497, 3499,
3501, 3503, 3505, 3507, 3509, 3511, 3513, 3515, 3517, 3519, 3521,
3523, 3525, 3527, 3529, 3531, 3533, 3535, 3537, 3539, 3541, 3543,
3545, 3547, 3549, 3551, 3553, 3555, 3557, 3559, 3561, 3563, 3565,
3567, 3569, 3571, 3573, 3575, 3577, 3579, 3581, 3583, 3585, 3587,
3589, 3591, 3593, 3595, 3597, 3599, 3601, 3603, 3605, 3607, 3609,
3611, 3613, 3615, 3617, 3619, 3621, 3623, 3625, 3627, 3629, 3631,
3633, 3635, 3637, 3639, 3641, 3643, 3645, 3647, 3649, 3651, 3653,
3655, 3657, 3659, 3661, 3663, 3665, 3667, 3669, 3671, 3673, 3675,
3677, 3679, 3681, 3683, 3685, 3687, 3689, 3691, 3693, 3695, 3697,
3699, 3701, 3703, 3705, 3707, 3709, 3711, 3713, 3715, 3717, 3719,
3721, 3723, 3725, 3727, 3729, 3731, 3733, 3735, 3737, 3739, 3741,
3743, 3745, 3747, 3749, 3751, 3753, 3755, 3757, 3759, 3761, 3763,
3765, 3767, 3769, 3771, 3773, 3775, 3777, 3779, 3781, 3783, 3785,
3787, 3789, 3791, 3793, 3795, 3797, 3799, 3801, 3803, 3805, 3807,
3809, 3811, 3813, 3815, 3817, 3819, 3821, 3823, 3825, 3827, 3829,
3831, 3833, 3835, 3837, 3839, 3841, 3843, 3845, 3847, 3849, 3851,
3853, 3855, 3857, 3859, 3861, 3863, 3865, 3867, 3869, 3871, 3873,
3875, 3877, 3879, 3881, 3883, 3885, 3887, 3889, 3891, 3893, 3895,
3897, 3899, 3901, 3903, 3905, 3907, 3909, 3911, 3913, 3915, 3917,
3919, 3921, 3923, 3925, 3927, 3929, 3931, 3933, 3935, 3937, 3939,
3941, 3943, 3945, 3947, 3949, 3951, 3953, 3955, 3957, 3959, 3961,
3963, 3965, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983,
3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005,
4007, 4009, 4011, 4013, 4015, 4017, 4019, 4021, 4023, 4025, 4027,
4029, 4031, 4033, 4035, 4037, 4039, 4041, 4043, 4045, 4047,
4049,4051,4053, 4055, 4057, 4059, 4061, 4063, 4065, 4067, 4069,
4071, 4073, 4075, 4077, 4079, 4081, 4083, 4085, 4087, 4089, 4091,
4093, 4095, 4097, 4099, 4101, 4103, 4105, 4107, 4109, 4111, 4113,
4115, 4117, 4119, 4121, 4123, 4125, 4127, 4129, 4131, 4133, 4135,
4137,4139, 4141, 4143, 4145, 4147, 4149, 4151, 4153, 4155, 4157,
4159, 4161, 4163, 4165, 4167, 4169, 4171, 4173, 4175, 4177, 4179,
4181, 4183, 4185, 4187, 4189, 4191, 4193, 4195, 4197, 4199, 4201,
4203, 4205, 4207, 4209, 4211, 4213, 4215, 4217, 4219, 4221, 4223,
4225, 4227, 4229, 4231, 4233, 4235, 4237, 4239, 4241, 4243, 4245,
4247, 4249, 4251, 4253, 4255, 4257, 4259, 4261, 4263, 4265, 4267,
4269, 4271, 4273, 4275, 4277, 4279, 4281, 4283, 4285, 4287, 4289,
4291, 4293, 4295, 4297, 4299, 4301, 4303, 4305, 4307, 4309, 4311,
4313, 4315, 4317, 4319, 4321, 4323, 4325, 4327, 4329, 4331, 4333,
4335, 4337, 4339, 4341, 4343, 4345, 4347, 4349, 4351, 4353, 4355,
4357, 4359, 4361, 4363, 4365, 4367, 4369, 4371, 4373, 4375, 4377,
4379, 4381, 4383, 4385, 4387, 4389, 4391, 4393, 4395, 4397, 4399,
4401, 4403, 4405, 4407, 4409, 4411, 4413, 4415, 4417, 4419, 4421,
4423, 4425, 4427, 4429, 4431, 4433, 4435, 4437, 4439, 4441, 4443,
4445, 4447, 4449, 4451, 4453, 4455, 4457, 4459, 4461, 4463, 4465,
4467, 4469, 4471, 4473, 4475, 4477, 4479, 4481, 4483, 4485, 4487,
4489, 4491, 4493, 4495, 4497, 4499, 4501, 4503, 4505, 4507, 4509,
4511, 4513, 4515, 4517, 4519, 4521, 4523, 4525, 4527, 4529, 4531,
4533, 4535, 4537, 4539, 4541, 4543, 4545, 4547, 4549, 4551, 4553,
4555, 4557, 4559, 4561, 4563, 4565, 4567, 4569, 4571, 4573, 4575,
4577, 4579, 4581, 4583, 4585, 4587, 4589, 4591, 4593, 4595, 4597,
4599, 4601, 4603, 4605, 4607, 4609, 4611, 4613, 4615, 4617, 4619,
4621, 4623, 4625, 4627, 4629, 4631, 4633, 4635, 4637, 4639, 4641,
4643, 4645, 4647, 4649, 4651, 4653, 4655, 4657, 4659, 4661, 4663,
4665, 4667, 4669, 4671, 4673, 4675, 4677, 4679, 4681, 4683, 4685,
4687, 4689, 4691, 4693, 4695, 4697, 4699, 4701, 4703, 4705, 4707,
4709, 4711, 4713, 4715, 4717, 4719, 4721, 4723, 4725, 4727, 4729,
4731, 4733, 4735, 4737, 4739, 4741, 4743, 4745, 4747, 4749, 4751,
4753, 4755, 4757, 4759, 4761, 4763, 4765, 4767, 4769, 4771, 4773,
4775, 4777, 4779, 4781, 4783, 4785, 4787, 4789, 4791, 4793, 4795,
4797, 4799, 4801, 4803, 4805, 4807, 4809, 4811, 4813, 4815, 4817,
4819, 4821, 4823, 4825, 4827, 4829, 4831, 4833, 4835, 4837, 4839,
4841, 4843, 4845, 4847, 4849, 4851, 4853, 4855, 4857, 4859, 4861,
4863, 4865, 4867, 4869, 4871, 4873, 4875, 4877, 4879, 4881, 4883,
4885, 4887, 4889, 4891, 4893, 4895, 4897, 4899, 4901, 4903, 4905,
4907, 4909, 4911, 4913, 4915, 4917, 4919, 4921, 4923, 4925, 4927,
4929, 4931, 4933, 4935, 4937, 4939, 4941, 4943, 4945, 4947, 4949,
4951, 4953, 4955, 4957, 4959, 4961, 4963, 4965, 4967, 4969, 4971,
4973, 4975, 4977, 4979, 4981, 4983, 4985, 4987, 4989, 4991, 4993,
4995, 4997, 4999, 5001, 5003, 5005, 5007, 5009, 5011, 5013, 5015,
5017, 5019, 5021, 5023, 5025, 5027, 5029, 5031, 5033, 5035, 5037,
5039, 5041, 5043, 5045, 5047, 5049, 5051, 5053, 5055, 5057, 5059,
5061, 5063, 5065, 5067, 5069, 5071, 5073, 5075, 5077, 5079, 5081,
5083, 5085, 5087, 5089, 5091, 5093, 5095, 5097, 5099, 5101, 5103,
5105, 5107, 5109, 5111, 5113, 5115, 5117, 5119, 5121, 5123, 5125,
5127, 5129, 5131, 5133, 5135, 5137, 5139, 5141, 5143, 5145, 5147,
5149, 5151, 5153, 5155, 5157, 5159, 5161, 5163, 5165, 5167, 5169,
5171, 5173, 5175, 5177, 5179, 5181, 5183, 5185, 5187, 5189, 5191,
5193, 5195, 5197, 5199, 5201, 5203, 5205, 5207, 5209, 5211, 5213,
5215, 5217, 5219, 5221, 5223, 5225, 5227, 5229, 5231, 5233, 5235,
5237, 5239, 5241, 5243, 5245, 5247, 5249, 5251, 5253, 5255, 5257,
5259, 5261, 5263, 5265, 5267, 5269, 5271, 5273, 5275, 5277, 5279,
5281, 5283, 5285, 5287, 5289, 5291, 5293, 5295, 5297, 5299, 5301,
5303, 5305, 5307, 5309, 5311, 5313, 5315, 5317, 5319, 5321, 5323,
5325, 5327, 5329, 5331, 5333, 5335, 5337, 5339, 5341, 5343, 5345,
5347, 5349, 5351, 5353, 5355, 5357, 5359, 5361, 5363, 5365, 5367,
5369, 5371, 5373, 5375, 5377, 5379, 5381, 5383, 5385, 5387, 5389,
5391, 5393, 5395, 5397, 5399, 5401, 5403, 5405, 5407, 5409, 5411,
5413, 5415, 5417, and 5419.
[0083] In some embodiments, the engineered AAM polypeptide is
selected from the polypeptide sequences of SEQ ID NO: 2348, 2286,
2230, 2312, 2282, 2346, 2338, 2226, 2268, 2324, and 2240.
[0084] In some embodiments, the engineered alanine-2,3-aminomutase
polypeptide is selected from the polypeptide sequences of SEQ ID
NO: 2306, 2294, 2250, 2300, 2256, 2244, 2242, 2232, 2358, and
2238.
[0085] It is to be understood that the engineered AAMs are not
limited to those identified directly by mutagenesis or other gene
evolution techniques, but can also include variants or analogs of
the improved engineered AAM polypeptides. The term "variant" and
"analog" are used interchangeably herein to refer to polypeptides
which are comprised of a segment having AAM activity, with or
without retention of the improved property, and has substantial
identity to a portion or the whole of the engineered AAM enzyme. In
some embodiments, variant AAM polypeptides can comprise a
conservative amino acid substitution, or addition or deletion of
one or more amino acid residues with respect to the engineered
sequence. Variants typically are at least an enzymatically active
fragment and typically as long as the full-length
naturally-occurring reference polypeptide. Thus, in some
embodiments, the variants herein include AAM polypeptides with
conservative substitutions in each of the AAM polypeptides in the
Sequence Listing, where the substitutions are in addition to the
substitutions present in the specific AAM polypeptide.
[0086] In some embodiments, the variant or analog of the engineered
AAM can comprise an enzymatically active AAM polypeptide that has
at least about 70% or more amino acid identity, at least about 80%
or more amino acid identity, at least about 90% or more amino acid
identity, at least about 95% or more amino acid identity, at least
about 97% or more amino acid identity, at least about 98% or more
amino acid identity, or at least about 99% or more amino acid
identity to the wild type Porphyromonas gingivalis enzyme (i.e.,
SEQ ID NO:2) or to a reference engineered AAM or an enzymatically
active fragment of an AAM disclosed herein, such as, for example,
SEQ ID NO:4, SEQ ID NO:36, and SEQ ID NO:909, and each of the amino
acid sequences of the engineered AAMs listed in the Sequence
Listing of this disclosure, beginning form SEQ ID NO:6.
[0087] In such embodiments, as noted above, the level of
improvements in enzyme activity can be from 1.5 times the enzymatic
activity of the corresponding reference AAM enzyme, to as much as 2
times, 5 times, 10 times, 20 times, 25 times, 30 times, up to 50
times or more times enzymatic activity than the reference enzyme.
In some embodiments, the engineered AAM can have improved enzymatic
activity in the range of 1.5 to 5 times; 1.5 to 10 times; 1.5 to 20
times; 1.5 to 30 times; 1.5 to 40 times, or 1.5 to 50 or more times
greater than that of the AAM enzyme from which it was derived.
Thus, in some embodiments, the variants of the engineered AAM
enzymes can have from about 1.5 to 30 times greater, from 2 to
about 30 time greater, or 5 to about 30 times greater AAM activity
as compared to the AAM polypeptide of SEQ ID NO:4. In some
embodiments, variants may have from about 1.5 to about 10 or more
times greater AAM activity compared to the AAM polypeptide of SEQ
ID NO:36.
[0088] In some embodiments, the engineered polypeptide having
improved AAM activity is a polypeptide having at least 97% identity
to a polypeptide selected from SEQ ID NO: 295, 2068 or 2150.
[0089] In some embodiments, the engineered polypeptide having
improved AAM activity is a polypeptide having at least 98% identity
to a polypeptide selected from SEQ ID NO: 258, 316, 350, 356, 790,
1006, and 1250
[0090] In some embodiments, the engineered polypeptide having
improved enzymatic activity is a polypeptide having at least 99%
identity to a polypeptide selected from SEQ ID NO: 14, 24, 58, 86,
100, 312, 336, 380, 318, 404, 444, 490, 550 and 774.
[0091] In some embodiments, the engineered AAM polypeptide having
improved enzymatic activity is a polypeptide having at least 93%
identity to the polypeptide of SEQ ID NO:4288.
[0092] In some embodiments, the engineered alanine-2,3-aminomutase
polypeptide having improved enzymatic activity is polypeptide
having at least about 95% sequence identity to a polypeptide
selected from SEQ ID NO: 2670, 2666, 4288, and 4586.
[0093] In some embodiments, the engineered AAM polypeptide having
improved AAM activity is a polypeptide having at least 96% identity
to a polypeptide selected from SEQ ID NO: 2378 and 2380.
[0094] In some embodiments, the engineered AAM polypeptide having
improved enzymatic activity is a polypeptide having at least 99%
identity to a polypeptide selected from SEQ ID NO: 2226, 2240,
2268, 2286, 2282, 2312, 2316, 2324, 2338, 2346, and 2348.
[0095] As noted above, these variant polypeptides have greater AAM
activity than the polypeptide of SEQ ID NO:4, as measured in the
assays in the Examples (e.g., Example 4). Some have greater AAM
activity than the polypeptide of SEQ ID NO:36 while others may have
greater AAM activity than the polypeptide of SEQ ID NO:909.
[0096] It is also within the scope of the present disclosure that
the polypeptides described herein, include fragments thereof which
have detectable AAM activity and that are deleted of portions of
the polypeptide as compared to the wild-type or engineered
reference polypeptide, as discussed above. The term "fragment" as
used herein refers to a polypeptide that has an amino-terminal,
carboxy-terminal, or internal deletion, but where the remaining
amino acid sequence is identical to the corresponding positions in
the sequence. Fragments can be at least 14 amino acids long, at
least 20 amino acids long, at least 50 amino acids long or longer,
and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length,
naturally-occurring or other reference AAM polypeptide. In some
embodiments, the deletions may be from 1 to 8 residues from their
N-terminus relative to the parental sequence of SEQ ID NO:4,
typically from 1 to 7 residues, more typically from 1-6 residues,
most typically from 1 to 5 residues. In some embodiments, the
above-described N-terminal truncation can be utilized in
combination with a C-terminal truncation. In some embodiments, AAM
fragments may have deleted from 1 to 45 amino acid residues from
their C-terminus relative to SEQ ID NO:4, typically from 1 to 40
residues, sometimes from 1-35 residues, in some embodiments from 1
to 30 residues, 1 to 25 residues, 1 to 20 residues, 1 to 15
residues 1 to 10 residues, and 1 to 5 residues.
[0097] In some embodiments, segments of the engineered improved AAM
polypeptides can be deleted to generate polypeptide fragments. Thus
in some embodiments, the present disclosure is directed to a
fragment of a polypeptide listed in the Sequence Listing herein,
that has improved enzymatic activity as compared to the AAM
polypeptide of SEQ ID NO: 4. In such embodiments, the fragments of
the engineered AAM enzymes can have from about 1.5 to 30 times
greater, from 2 to about 30 time greater, or 5 to about 30 times
greater AAM activity as compared to the AAM polypeptide of SEQ ID
NO: 4. Some fragments may have from about 1.5 to about 10 or more
times greater AAM activity compared to the AAM polypeptide of SEQ
ID NO: 36.
[0098] The improved AAM enzymes may be present within a cell,
present in the cellular medium, or prepared in various forms, such
as lysates, lyophilizates, or isolated preparations. In some
embodiments, the AAM polypeptides are sensitive to oxygen and
therefore can be maintained and used in an oxygen deficient
environment. If the AAM polypeptide becomes inactivated due to
exposure to oxygen, it can be activated by anaerobic incubation
with a sulfhydryl compound for one hour at 37.degree. C. in
accordance with the method described in Chirpich, et al., 1970, J
Biol. Chem. 245(7):1778-1789, which is incorporated herein by
reference in its entirety. Thus, in some embodiments, the AAM
polypeptides can be used in whole cell form (i.e., as a whole cell
transformed with an AAM polynucleotide that is used under
conditions such that the encoded AAM polypeptide is expressed in
the cell) or alternatively, both isolated and utilized under anoxic
conditions. AAM polypeptides of the present disclosure may be
isolated, and optionally purified, under anaerobic conditions
(e.g., under a nitrogen atmosphere) in accordance with the method
described in Petrovich, et al., 1991, J Biol. Chem.
266(12):7656-7660, which describes the isolation and purification
of lysine-2,3-aminomutase and which is incorporated herein by
reference in its entirety. As used herein, the term "anoxic" refers
to oxygen deficient. In some embodiments, the AAM polypeptides may
be prepared in the oxidized from, and then activated as discussed
above.
[0099] The term "isolated polypeptide" refers to a polypeptide
which is substantially separated from other contaminants that
naturally accompany it, e.g., protein, lipids, and polynucleotides.
The term embraces polypeptides which have been removed or purified
from their naturally-occurring environment or expression system
(e.g., host cell or in vitro synthesis).
[0100] In some embodiments, the isolated improved AAM polypeptide
is a substantially pure polypeptide composition. The term
"substantially pure polypeptide" refers to a composition in which
the polypeptide species is the predominant species present (i.e.,
on a molar or weight basis, it is more abundant than any other
individual macromolecular species in the composition), and is
generally a substantially purified composition when the object
species comprises at least about 50 percent of the macromolecular
species present by mole or % weight. Generally, a substantially
pure AAM composition will comprise about 60% or more, about 70% or
more, about 80% or more, about 90% or more, about 95% or more, and
about 98% or more of all macromolecular species by mole or % weight
present in the composition. In some embodiments, the object species
is purified to essential homogeneity (i.e., contaminant species
cannot be detected in the composition by conventional detection
methods) wherein the composition consists essentially of a single
macromolecular species. Solvent species, small molecules (<500
Daltons), and elemental ion species are not considered
macromolecular species.
[0101] In other embodiments, the engineered AAM can be provided as
a composition comprising an AAM polypeptide as described herein
(e.g., in whole cell form or as an isolated polypeptide) and a
suitable carrier, typically a buffer, more typically an aqueous
buffer solution having a pH from about 6.0 to about 8.0. In some
embodiments, the aqueous buffered composition can be lyophilized to
provide a composition in a lyophilized form, wherein the
composition is reconstituted by the addition of an aqueous based
composition. In other embodiments, the composition comprises an
engineered AAM polypeptide in a glycerol solution. In this latter
embodiment, the composition is capable of being stored at
-20.degree. C. to -40.degree. C. without freezing.
7.3 Polynucleotides Encoding AAM
[0102] The present disclosure further provides polynucleotide
sequences that encode AAM polypeptides with improved enzymatic
activity. The polynucleotides may be operatively linked to one or
more heterologous regulatory sequences that control gene expression
to create a recombinant polynucleotide capable of expressing the
polypeptide. Expression constructs containing a heterologous
polynucleotide encoding the engineered AAM can be introduced into
appropriate host cells to express the corresponding AAM
polypeptide. The term "heterologous" polynucleotide refers to any
polynucleotide that is introduced into a host cell by laboratory
techniques, and includes polynucleotides that are removed from a
host cell, subjected to laboratory manipulation, and then
reintroduced into a host cell.
[0103] Because of the knowledge of the codons corresponding to the
various amino acids, availability of a protein sequence provides a
description of all the polynucleotides capable of encoding the
subject polypeptide. The degeneracy of the genetic code, where the
same amino acids are encoded by alternative or synonymous codons
allows an extremely large number of nucleic acids to be made, all
of which encode the improved AAM enzymes disclosed herein. Thus,
having identified a particular amino acid sequence, those skilled
in the art could make any number of different nucleic acids by
simply modifying the sequence of one or more codons in a way which
does not change the amino acid sequence of the protein. In this
regard, the present disclosure specifically contemplates each and
every possible variation of polynucleotides that could be made by
selecting combinations based on the possible codon choices, and all
such variations are to be considered specifically disclosed for any
polypeptide disclosed herein.
[0104] In some embodiments, the polynucleotides encoding the AAM
enzymes may be codon optimized for optimal production in the host
organism selected for expression. The term "codon optimized" refers
to changes in the codons of the polynucleotide encoding a protein
to those preferentially used in a particular organism such that the
encoded protein is efficiently expressed in the organism of
interest. Although the genetic code is degenerate in that most
amino acids are represented by several codons, called "synonyms" or
"synonymous" codons, it is well known that codon usage by
particular organisms is nonrandom and biased towards particular
codon triplets. The terms "preferred," "optimal," or "high codon
usage bias" codons refer interchangeably to codons that are used at
higher frequency in the protein coding regions than other codons
that code for the same amino acid.
[0105] Codon usage tables are available for a growing list of
organisms (see for example, Wada et al., 1992, Nucleic Acids Res.
20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292;
Duret, et al., supra; Henaut and Danchin, "Escherichia coli and
Salmonella," 1996, Neidhardt, et al. Eds., ASM Press, Washington
D.C., p. 2047-2066. The data source for obtaining codon usage may
rely on any available nucleotide sequence capable of coding for a
protein. These data sets include nucleic acid sequences actually
known to encode expressed proteins (e.g., complete protein coding
sequences-CDS), expressed sequence tags (ESTS), or predicted coding
regions of genomic sequences (see for example, Mount, D.,
Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001;
Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281; and Tiwari
et al., 1997, Comput. Appl. Biosci. 13:263-270).
[0106] In various embodiments, the codons are preferably selected
to fit the host cell in which the protein is being produced. For
example, preferred codons used in bacteria are used to express the
gene in bacteria; preferred codons used in yeast are used for
expression in yeast; and preferred codons used in mammals are used
for expression in mammalian cells. By way of example, the
polynucleotide of SEQ ID NO: 5 has been codon optimized for
expression in E. coli.
[0107] In a some embodiments, the polynucleotide encoding the
engineered AAM polypeptide with improved enzyme activity is
selected from SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,
95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,
123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,
149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,
175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,
201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225,
227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,
253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,
279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303,
305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329,
331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,
357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,
383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407,
409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,
435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,
461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485,
487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511,
513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537,
539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,
565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589,
591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615,
617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641,
643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667,
669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,
695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719,
721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,
747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,
773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797,
799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823,
825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849,
851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875,
877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901,
903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927,
929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953,
955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979,
981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005,
1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027,
1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049,
1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071,
1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,
1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115,
1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137,
1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159,
1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181,
1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203,
1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225,
1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247,
1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269,
1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291,
1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313,
1315, 1317, 1319, 1321, 1323, 1325, 1027, 1029, 1331, 1333, 1335,
1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1057,
1059, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379,
1381, 1383, 1385, 1087, 1089, 1391, 1393, 1395, 1397, 1399, 1401,
1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423,
1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445,
1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467,
1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489,
1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511,
1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533,
1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555,
1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577,
1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599,
1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621,
1623, 1625, 1627, 1029, 1631, 1633, 1635, 1637, 1639, 1641, 1643,
1645, 1647, 1649, 1651, 1653, 1655, 1657, 1059, 1661, 1663, 1665,
1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687,
1089, 1691, 1693, 1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709,
1711, 1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731,
1733, 1735, 1737, 1739, 1741, 1743, 1745, 1747, 1749, 1751, 1753,
1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775,
1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797,
1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819,
1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841,
1843, 1845, 1848, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863,
1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885,
1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907,
1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929,
1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951,
1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973,
1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995,
1997, 1999, 2001, 2003, 2005, 2007, 2009, 2011, 2013, 2015, 2017,
2019, 2021, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039,
2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061,
2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2083,
2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105,
2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127,
2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149,
2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171,
2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193,
2195, 2197, 2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215,
2217, 2219 or 2221.
[0108] In some embodiments, the AAM encoding polynucleotides have
the sequence selected from SEQ ID NO: 35, 793, 803, 909, 1281,
1695, 1883, 1885, 2051 and 2161 or have the sequence selected from
SEQ ID NO: 909, 1261, 1695, 1883, 1885, 2051 and 2161. In some
embodiments, the polynucleotide is SEQ ID NO: 2051 or SEQ ID
NO:2161.
[0109] In some embodiments, the polynucleotide encoding the
engineered AAM polypeptide is selected from SEQ ID NO: 2223, 2225,
2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247,
2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269,
2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291,
2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313,
2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335,
2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357,
2359, 2361, 2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377, 2379,
2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401,
2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, 2421, 2423,
2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443, 2445,
2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467,
2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 2487, 2489,
2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509, 2511,
2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533,
2535, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555,
2557, 2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575, 2577,
2579, 2581, 2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599,
2601, 2603, 2605, 2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621,
2623, 2625, 2627, 2629, 2631, 2633, 2635, 2637, 2639, 2641, 2643,
2645, 2647, 2649, 2651, 2653, 2655, 2657, 2659, 2661, 2663, 2665,
2667, 2669, 2671, 2673, 2675, 2677, 2679, 2681, 2683, 2685, 2687,
2689, 2691, 2693, 2695, 2697, 2699, 2701, 2703, 2705, 2707, 2709,
2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725, 2727, 2729, 2731,
2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749, 2751, 2753,
2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773, 2775,
2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797,
2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819,
2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841,
2843, 2845, 2847, 2849, 2851, 2853, 2855, 2857, 2859, 2861, 2863,
2865, 2867, 2869, 2871, 2873, 2875, 2877, 2879, 2881, 2883, 2885,
2887, 2889, 2891, 2893, 2895, 2897, 2899, 2901, 2903, 2905, 2907,
2909, 2911, 2913, 2915, 2917, 2919, 2921, 2923, 2925, 2927, 2929,
2931, 2933, 2935, 2937, 2939, 2941, 2943, 2945, 2947, 2949, 2951,
2953, 2955, 2957, 2959, 2961, 2963, 2965, 2967, 2969, 2971, 2973,
2975, 2977, 2979, 2981, 2983, 2985, 2987, 2989, 2991, 2993, 2995,
2997, 2999, 3001, 3003, 3005, 3007, 3009, 3011, 3013, 3015, 3017,
3019, 3021, 3023, 3025, 3027, 3029, 3031, 3033, 3035, 3037, 3039,
3041, 3043, 3045, 3047, 3049, 3051, 3053, 3055, 3057, 3059, 3061,
3063, 3065, 3067, 3069, 3071, 3073, 3075, 3077, 3079, 3081, 3083,
3085, 3087, 3089, 3091, 3093, 3095, 3097, 3099, 3101, 3103, 3105,
3107, 3109, 3111, 3113, 3115, 3117, 3119, 3121, 3123, 3125, 3127,
3129, 3131, 3133, 3135, 3137, 3139, 3141, 3143, 3145, 3147, 3149,
3151, 3153, 3155, 3157, 3159, 3161, 3163, 3165, 3167, 3169, 3171,
3173, 3175, 3177, 3179, 3181, 3183, 3185, 3187, 3189, 3191, 3193,
3195, 3197, 3199, 3201, 3203, 3205, 3207, 3209, 3211, 3213, 3215,
3217, 3219, 3221, 3223, 3225, 3227, 3229, 3231, 3233, 3235, 3237,
3239, 3241, 3243, 3245, 3247, 3249, 3251, 3253, 3255, 3257, 3259,
3261, 3263, 3265, 3267, 3269, 3271, 3273, 3275, 3277, 3279, 3281,
3283, 3285, 3287, 3289, 3291, 3293, 3295, 3297, 3299, 3301, 3303,
3305, 3307, 3309, 3311, 3313, 3315, 3317, 3319, 3321, 3323, 3325,
3327, 3329, 3331, 3333, 3335, 3337, 3339, 3341, 3343, 3345, 3347,
3349, 3351, 3353, 3355, 3357, 3359, 3361, 3363, 3365, 3367, 3369,
3371, 3373, 3375, 3377, 3379, 3381, 3383, 3385, 3387, 3389, 3391,
3393, 3395, 3397, 3399, 3401, 3403, 3405, 3407, 3409, 3411, 3413,
3415, 3417, 3419, 3421, 3423, 3425, 3427, 3429, 3431, 3433, 3435,
3437, 3439, 3441, 3443, 3445, 3447, 3449, 3451, 3453, 3455, 3457,
3459, 3461, 3463, 3465, 3467, 3469, 3471, 3473, 3475, 3477, 3479,
3481, 3483, 3485, 3487, 3489, 3491, 3493, 3495, 3497, 3499, 3501,
3503, 3505, 3507, 3509, 3511, 3513, 3515, 3517, 3519, 3521, 3523,
3525, 3527, 3529, 3531, 3533, 3535, 3537, 3539, 3541, 3543, 3545,
3547, 3549, 3551, 3553, 3555, 3557, 3559, 3561, 3563, 3565, 3567,
3569, 3571, 3573, 3575, 3577, 3579, 3581, 3583, 3585, 3587, 3589,
3591, 3593, 3595, 3597, 3599, 3601, 3603, 3605, 3607, 3609, 3611,
3613, 3615, 3617, 3619, 3621, 3623, 3625, 3627, 3629, 3631, 3633,
3635, 3637, 3639, 3641, 3643, 3645, 3647, 3649, 3651, 3653, 3655,
3657, 3659, 3661, 3663, 3665, 3667, 3669, 3671, 3673, 3675, 3677,
3679, 3681, 3683, 3685, 3687, 3689, 3691, 3693, 3695, 3697, 3699,
3701, 3703, 3705, 3707, 3709, 3711, 3713, 3715, 3717, 3719, 3721,
3723, 3725, 3727, 3729, 3731, 3733, 3735, 3737, 3739, 3741, 3743,
3745, 3747, 3749, 3751, 3753, 3755, 3757, 3759, 3761, 3763, 3765,
3767, 3769, 3771, 3773, 3775, 3777, 3779, 3781, 3783, 3785, 3787,
3789, 3791, 3793, 3795, 3797, 3799, 3801, 3803, 3805, 3807, 3809,
3811, 3813, 3815, 3817, 3819, 3821, 3823, 3825, 3827, 3829, 3831,
3833, 3835, 3837, 3839, 3841, 3843, 3845, 3847, 3849, 3851, 3853,
3855, 3857, 3859, 3861, 3863, 3865, 3867, 3869, 3871, 3873, 3875,
3877, 3879, 3881, 3883, 3885, 3887, 3889, 3891, 3893, 3895, 3897,
3899, 3901, 3903, 3905, 3907, 3909, 3911, 3913, 3915, 3917, 3919,
3921, 3923, 3925, 3927, 3929, 3931, 3933, 3935, 3937, 3939, 3941,
3943, 3945, 3947, 3949, 3951, 3953, 3955, 3957, 3959, 3961, 3963,
3965, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983, 3985,
3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005, 4007,
4009, 4011, 4013, 4015, 4017, 4019, 4021, 4023, 4025, 4027, 4029,
4031, 4033, 4035, 4037, 4039, 4041, 4043, 4045, 4047, 4049, 4051,
4053, 4055, 4057, 4059, 4061, 4063, 4065, 4067, 4069, 4071, 4073,
4075, 4077, 4079, 4081, 4083, 4085, 4087, 4089, 4091, 4093, 4095,
4097, 4099, 4101, 4103, 4105, 4107, 4109, 4111, 4113, 4115, 4117,
4119, 4121, 4123, 4125, 4127, 4129, 4131, 4133, 4135, 4137, 4139,
4141, 4143, 4145, 4147, 4149, 4151, 4153, 4155, 4157, 4159, 4161,
4163, 4165, 4167, 4169, 4171, 4173, 4175, 4177, 4179, 4181, 4183,
4185, 4187, 4189, 4191, 4193, 4195, 4197, 4199, 4201, 4203, 4205,
4207, 4209, 4211, 4213, 4215, 4217, 4219, 4221, 4223, 4225, 4227,
4229, 4231, 4233, 4235, 4237, 4239, 4241, 4243, 4245, 4247, 4249,
4251, 4253, 4255, 4257, 4259, 4261, 4263, 4265, 4267, 4269, 4271,
4273, 4275, 4277, 4279, 4281, 4283, 4285, 4287, 4289, 4291, 4293,
4295, 4297, 4299, 4301, 4303, 4305, 4307, 4309, 4311, 4313, 4315,
4317, 4319, 4321, 4323, 4325, 4327, 4329, 4331, 4333, 4335, 4337,
4339, 4341, 4343, 4345, 4347, 4349, 4351, 4353, 4355, 4357, 4359,
4361, 4363, 4365, 4367, 4369, 4371, 4373, 4375, 4377, 4379, 4381,
4383, 4385, 4387, 4389, 4391, 4393, 4395, 4397, 4399, 4401, 4403,
4405, 4407, 4409, 4411, 4413, 4415, 4417, 4419, 4421, 4423, 4425,
4427, 4429, 4431, 4433, 4435, 4437, 4439, 4441, 4443, 4445, 4447,
4449, 4451, 4453, 4455, 4457, 4459, 4461, 4463, 4465, 4467, 4469,
4471, 4473, 4475, 4477, 4479, 4481, 4483, 4485, 4487, 4489, 4491,
4493, 4495, 4497, 4499, 4501, 4503, 4505, 4507, 4509, 4511, 4513,
4515, 4517, 4519, 4521, 4523, 4525, 4527, 4529, 4531, 4533, 4535,
4537, 4539, 4541, 4543, 4545, 4547, 4549, 4551, 4553, 4555, 4557,
4559, 4561, 4563, 4565, 4567, 4569, 4571, 4573, 4575, 4577, 4579,
4581, 4583, 4585, 4587, 4589, 4591, 4593, 4595, 4597, 4599, 4601,
4603, 4605, 4607, 4609, 4611, 4613, 4615, 4617, 4619, 4621, 4623,
4625, 4627, 4629, 4631, 4633, 4635, 4637, 4639, 4641, 4643, 4645,
4647, 4649, 4651, 4653, 4655, 4657, 4659, 4661, 4663, 4665, 4667,
4669, 4671, 4673, 4675, 4677, 4679, 4681, 4683, 4685, 4687, 4689,
4691, 4693, 4695, 4697, 4699, 4701, 4703, 4705, 4707, 4709, 4711,
4713, 4715, 4717, 4719, 4721, 4723, 4725, 4727, 4729, 4731, 4733,
4735, 4737, 4739, 4741, 4743, 4745, 4747, 4749, 4751, 4753, 4755,
4757, 4759, 4761, 4763, 4765, 4767, 4769, 4771, 4773, 4775, 4777,
4779, 4781, 4783, 4785, 4787, 4789, 4791, 4793, 4795, 4797, 4799,
4801, 4803, 4805, 4807, 4809, 4811, 4813, 4815, 4817, 4819, 4821,
4823, 4825, 4827, 4829, 4831, 4833, 4835, 4837, 4839, 4841, 4843,
4845, 4847, 4849, 4851, 4853, 4855, 4857, 4859, 4861, 4863, 4865,
4867, 4869, 4871, 4873, 4875, 4877, 4879, 4881, 4883, 4885, 4887,
4889, 4891, 4893, 4895, 4897, 4899, 4901, 4903, 4905, 4907, 4909,
4911, 4913, 4915, 4917, 4919, 4921, 4923, 4925, 4927, 4929, 4931,
4933, 4935, 4937, 4939, 4941, 4943, 4945, 4947, 4949, 4951, 4953,
4955, 4957, 4959, 4961, 4963, 4965, 4967, 4969, 4971, 4973, 4975,
4977, 4979, 4981, 4983, 4985, 4987, 4989, 4991, 4993, 4995, 4997,
4999, 5001, 5003, 5005, 5007, 5009, 5011, 5013, 5015, 5017, 5019,
5021, 5023, 5025, 5027, 5029, 5031, 5033, 5035, 5037, 5039, 5041,
5043, 5045, 5047, 5049, 5051, 5053, 5055, 5057, 5059, 5061, 5063,
5065, 5067, 5069, 5071, 5073, 5075, 5077, 5079, 5081, 5083, 5085,
5087, 5089, 5091, 5093, 5095, 5097, 5099, 5101, 5103, 5105, 5107,
5109, 5111, 5113, 5115, 5117, 5119, 5121, 5123, 5125, 5127, 5129,
5131, 5133, 5135, 5137, 5139, 5141, 5143, 5145, 5147, 5149, 5151,
5153, 5155, 5157, 5159, 5161, 5163, 5165, 5167, 5169, 5171, 5173,
5175, 5177, 5179, 5181, 5183, 5185, 5187, 5189, 5191, 5193, 5195,
5197, 5199, 5201, 5203, 5205, 5207, 5209, 5211, 5213, 5215, 5217,
5219, 5221, 5223, 5225, 5227, 5229, 5231, 5233, 5235, 5237, 5239,
5241, 5243, 5245, 5247, 5249, 5251, 5253, 5255, 5257, 5259, 5261,
5263, 5265, 5267, 5269, 5271, 5273, 5275, 5277, 5279, 5281, 5283,
5285, 5287, 5289, 5291, 5293, 5295, 5297, 5299, 5301, 5303, 5305,
5307, 5309, 5311, 5313, 5315, 5317, 5319, 5321, 5323, 5325, 5327,
5329, 5331, 5333, 5335, 5337, 5339, 5341, 5343, 5345, 5347, 5349,
5351, 5353, 5355, 5357, 5359, 5361, 5363, 5365, 5367, 5369, 5371,
5373, 5375, 5377, 5379, 5381, 5383, 5385, 5387, 5389, 5391, 5393,
5395, 5397, 5399, 5401, 5403, 5405, 5407, 5409, 5411, 5413, 5415,
5417, or 5419.
[0110] In some embodiments, the polynucleotide is selected from the
polynucleotide sequences of SEQ ID NO: 2305; 2293; 2249; 2299;
2255; 2243; 2241; 2231; 2357; and 2237.
[0111] In some embodiments, the polynucleotide is selected from the
polynucleotide sequences of SEQ ID NO: 2315; 2379; 2347; 2285;
2311; 2229; 2377; 2669; 2665; 4287; 2281; 2345; 2337; 2225; 2267;
2323; 2239; and 4585.
[0112] In some embodiments, the polynucleotide encodes an
enzymatically active variant AAM polypeptide, where the
polynucleotide has at least 70% sequence identity, at least 80%
sequence identity, at least 85% sequence identity, at least 90%
sequence identity, at least 95% sequence identity, at least 97%
sequence identify, at least 98% sequence identity, or at least 99%
or more sequence identity to a polynucleotide listed in the
Sequence Listing disclosed herein beginning from SEQ ID NO:5.
[0113] In some embodiments, the polynucleotide encodes an
enzymatically active AAM and hybridizes under defined conditions,
such as moderately stringent or highly stringent conditions, to the
polynucleotide sequence encoding an engineered AAM enzyme as
described above, or to a complement of the polynucleotide sequence
encoding the engineered AAM enzyme. The phrase "stringent
hybridization" is used herein to refer to conditions under which
nucleic acid hybrids are stable. As known to those of skill in the
art, the stability of hybrids is reflected in the melting
temperature (T.sub.m) of the hybrids. In general, the stability of
a hybrid is a function of ion strength, temperature, G/C content,
and the presence of chaotropic agents. The T.sub.n, values for
polynucleotides can be calculated using known methods for
predicting melting temperatures (see, e.g., Baldino et al., Methods
Enzymology 168:761-777; Bolton et al., 1962, Proc. Natl. Acad. Sci.
USA 48:1390; Bresslauer et al., 1986, Proc. Natl. Acad. Sci. USA
83:8893-8897; Freier et al., 1986, Proc. Natl. Acad. Sci. USA
83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychlik et
al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, Nucleic
Acids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, In
Developmental Biology Using Purified Genes (Brown et al., eds.),
pp. 683-693, Academic Press; and Wetmur, 1991, Crit. Rev Biochem
Mol Biol 26:227-259. All publications incorporate herein by
reference).
[0114] Generally, hybridization reactions are performed under
conditions of lower stringency, followed by washes of varying but
higher stringency. Typically, reference to "hybridization
stringency" relates to such washing conditions. The term
"moderately stringent hybridization" refers to conditions that
permit target-DNA to bind a complementary nucleic acid that has
about 60% identity, about 75% identity, about 85% identity to the
target DNA; or with greater than about 90% identity to
target-polynucleotide. Exemplary moderately stringent conditions
are conditions equivalent to hybridization in 50% formamide,
5.times.Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.2.times.SSPE, 0.2% SDS, at 42.degree.
C.
[0115] The term "high stringency hybridization" refers generally to
conditions that are about 10.degree. C. or less from the thermal
melting temperature T.sub.m as determined under the solution
condition for a defined polynucleotide sequence. In some
embodiments, a high stringency condition refers to conditions that
permit hybridization of only those nucleic acid sequences that form
stable hybrids in 0.018 M NaCl at 65.degree. C. (i.e., if a hybrid
is not stable in 0.018 M NaCl at 65.degree. C., it will not be
stable under high stringency conditions, as contemplated herein).
High stringency conditions can be provided, for example, by
hybridization in conditions equivalent to 50% formamide,
5.times.Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.1.times.SSPE, and 0.1% SDS at
65.degree. C. In some embodiments, the SSPE can be replaced with
SSC, as provided in Sambrook, supra, for high stringency
hybridization conditions as well as moderately stringent
conditions. For example, for polynucleotides of at least 100
nucleotides in length, the carrier material can be washed with 15
minutes using 2.times.SSC, 0.2% SDS at least at 50.degree. C. (low
stringency), at least at 55.degree. C. (medium stringency), at
least at 60.degree. C. (medium-high stringency), at least at
65.degree. C. (high stringency), and at least at 70.degree. C.
(very high stringency).
[0116] Thus, in some embodiments, the polynucleotide encodes an
enzymatically active AAM and hybridizes under defined conditions,
such as moderately stringent or highly stringent conditions, to the
complement of a polynucleotide sequence encoding an engineered AAM
enzyme selected from the polynucleotides listed in the Sequence
Listing disclosed herein, beginning from SEQ ID NO:5.
[0117] In some embodiments, the present disclosure is directed to
an AAM polypeptide encoded by a polynucleotide sequence which
hybridizes under high stringency conditions with the polynucleotide
sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 41, 43, 45, 47 or 49; or a subsequence
of at least 100 nucleotides of a polynucleotide selected from the
polynucleotides of the Sequence Listing herein, beginning from SEQ
ID NO:5.
[0118] In the embodiments herein, an isolated polynucleotide
encoding an improved alanine 2,3-aminomutase polypeptide can be
manipulated in a variety of ways to provide for expression of the
polypeptide. Manipulation of the isolated polynucleotide prior to
its insertion into a vector may be desirable or necessary depending
on the expression vector. The techniques for modifying
polynucleotides utilizing recombinant DNA methods are well known in
the art. Guidance is provided in Sambrook et al., 2001, Molecular
Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor
Laboratory Press; and Current Protocols in Molecular Biology,
Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2005.
[0119] In some embodiments, the present disclosure is directed to
an expression vector capable of expressing the engineered AAM
polypeptides disclosed herein. To obtain expression of the AAM
polypeptide, the polynucleotide can be operably linked to one or
more heterologous regulatory sequences that control gene expression
to create a nucleic acid construct, such as an expression vector or
expression cassette, as further described below. Thereafter, the
resulting nucleic acid construct can be introduced into an
appropriate host cell for ultimate expression of the encoded AAM
polypeptide. A "nucleic acid construct" or "recombinant nucleic
acid" is used herein refers to a polynucleotide, either single or
double-stranded, which is isolated from a naturally occurring gene
or which has been modified to contain segments of nucleic acid
combined and juxtaposed in a manner that would not otherwise exist
in nature. Thus, in some embodiments, the present disclosure is
directed to a nucleic acid construct comprising a polynucleotide
encoding an AAM polypeptide of the present disclosure.
[0120] The term "control sequence" is defined herein to include all
components which are necessary or advantageous for the expression
of a polypeptide of the present disclosure. Each control sequence
may be native or foreign to the polynucleotide sequence encoding
the polypeptide. Such control sequences include, but are not
limited to, a leader, polyadenylation sequence, propeptide
sequence, promoter sequence, signal peptide sequence, and
transcription terminator sequence. At a minimum, the control
sequences include a promoter, and transcriptional and translational
stop signals. The control sequences may be provided with linkers
for the purpose of introducing specific restriction sites
facilitating ligation of the control sequences with the coding
region of the nucleic acid sequence encoding a polypeptide.
[0121] The term "operably linked" is defined herein as a
configuration in which a control sequence is appropriately placed
at a position relative to the coding sequence of the DNA sequence
such that the control sequence directs the expression of a
polynucleotide and/or polypeptide.
[0122] The control sequence may be an appropriate promoter
sequence. A "promoter sequence" refers to a nucleic acid sequence
that is recognized by a host cell for expression of a
polynucleotide comprising the coding region. The promoter sequence
contains transcriptional control sequences, which mediate the
expression of the polypeptide. The promoter may be any nucleic acid
sequence which shows transcriptional activity in the host cell of
choice including mutant, truncated, and hybrid promoters, and may
be obtained from genes encoding extracellular or intracellular
polypeptides, either homologous or heterologous to the host
cell.
[0123] For bacterial host cells, suitable promoters for directing
the transcription of the nucleic acid constructs of the present
disclosure, include the promoters obtained from the E. coli lac
operon, Streptomyces coelicolor agarase gene (dagA), Bacillus
subtilis levansucrase gene (sacB), Bacillus licheniformis
alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic
amylase gene (amyM), Bacillus amyloliquefaciens alphaamylase gene
(amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus
subtilis xylA and xylB genes, and prokaryotic .beta.-lactamase gene
(VIIIa-Kamaroff et al., 1978, Proc Natl Acad Sci USA 75:
3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc
Natl Acad Sci USA 80:21-25). Additional promoters are described in
"Useful proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; and in Sambrook et al., 1989, supra. For
filamentous fungal host cells, suitable promoters for directing the
transcription of the polynucleotide constructs of the present
disclosure include, among others, promoters obtained from the genes
for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic
proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus
niger acid stable alpha-amylase, Aspergillus niger or Aspergillus
awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus
oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum
trypsin-like protease (WO 96/00787), as well as the NA2-tpi
promoter (a hybrid of the promoters from the genes for Aspergillus
niger neutral alpha-amylase and Aspergillus olyzae triose phosphate
isomerase), and mutant, truncated, and hybrid promoters
thereof.
[0124] In a yeast host, useful promoters can be from the genes for
Porphyromonas gingivalis enolase (ENO-I), Porphyromonas gingivalis
galactokinase (GAL1), Porphyromonas gingivalis alcohol
dehydrogenaselglyceraldehyde-3-phosphate dehydrogenase (ADH21GAP),
and Porphyromonas gingivalis 3-phosphoglycerate kinase. Other
useful promoters for yeast host cells are described by Romanos et
al., 1992, Yeast 8:423-488.
[0125] In some embodiments, the control sequence may also be a
suitable transcription terminator sequence, a sequence recognized
by a host cell to terminate transcription. The terminator sequence
is typically operably linked to the 3' terminus of the nucleic acid
sequence encoding the polypeptide. Any terminator, which is
functional in the host cell of choice, may be used in the present
disclosure.
[0126] Terminators for filamentous fungal host cells can be from
the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger
glucoamylase, Aspergillus nidulans anthranilate synthase,
Aspergillus niger alpha-glucosidase, and Fusarium oxysporum
trypsin-like protease.
[0127] Terminators for yeast host cells can be from the genes for
Porphyromonas gingivalis enolase, Porphyromonas gingivalis
cytochrome C (CYC1), and Porphyromonas gingivalis
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., supra.
[0128] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA, which is important for
translation by the host cell. The leader sequence is typically
operably linked to the 5' terminus of the nucleic acid sequence
encoding the polypeptide. Any leader sequence that is functional in
the host cell of choice may be used in the present disclosure.
Leader sequences for filamentous fungal host cells can be from the
genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans
triose phosphate isomerase. Suitable leaders for yeast host cells
can be from the genes for Porphyromonas gingivalis enolase (ENO-I),
Porphyromonas gingivalis 3-phosphoglycerate kinase, Sacchuromyces
cerevisiae alpha-factor, and Sacchuromyces cerevisiae alcohol
dehydrogenaselglyceraldehyde-3-phosphate dehydrogenase
(ADHYGAP).
[0129] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleic acid
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence that is functional in the host cell of
choice may be used in the present disclosure. In some embodiments,
polyadenylation sequences for filamentous fungal host cells can be
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger glucoamylase, Aspergillus nidulans anthranilate synthase,
Fusarium oxysporum trypsin-like protease, and Aspergillus niger
alpha-glucosidase. Useful polyadenylation sequences for yeast host
cells are described by Guo and Sherman, 1995, Mol Cell Biol
5:5983-5990.
[0130] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleic acid sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region that encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region that is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region.
[0131] Alternatively, the foreign signal peptide coding region may
simply replace the natural signal peptide coding region in order to
enhance secretion of the polypeptide. However, any signal peptide
coding region that directs the expressed polypeptide into the
secretory pathway of a host cell of choice may be used in the
present disclosure.
[0132] Effective signal peptide coding regions for bacterial host
cells can be the signal peptide coding regions from the genes for
Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus
alpha-amylase, Bacillus licheniformis subtilisin, Bacillus
licheniformis .beta.-lactamase, Bacillus stearothermophilus neutral
proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further
signal peptides are described by Simonen and Palva, 1993,
Microbiol. Rev 57:109-137, incorporated herein by reference.
[0133] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and
Humicola lanuginosa lipase.
[0134] Useful signal peptides for yeast host cells are obtained
from the genes for Porphyromonas gingivalis alpha-factor and
Porphyromonas gingivalis invertase. Other Useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
[0135] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Porphyromonas gingivalis alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila lactase (WO
95133836).
[0136] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino-terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0137] It may also be desirable to add regulatory sequences, which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. In prokaryotic host cells,
suitable regulatory sequences include the lac, tac, and trp
operator systems. In yeast host cells, suitable regulatory systems
include the ADH2 system or GAL1 system. In filamentous fungi,
suitable regulatory sequences include the TAKA alpha-amylase
promoter, Aspergillus niger glucoamylase promoter, and Aspergillus
oryzae glucoamylase promoter.
[0138] Other examples of regulatory sequences are those, which
allow for gene amplification. In prokaryotes, these include
replication mutants that result in high copy number of plasmids
(e.g., runaway replication plasmids; see, e.g., Yasuda and Takagi,
1983, J Bacteriol. 154(3):1153-1161). In eukaryotic systems, these
include the dihydrofolate reductase gene, which is amplified in the
presence of methotrexate, and the metallothionein genes, which are
amplified with heavy metals. In these cases, the nucleic acid
sequence encoding the AAM polypeptide of the present disclosure
would be operably linked with the regulatory sequence.
7.4 Expression Vectors
[0139] As discussed above, the present disclosure is also directed
to a recombinant expression vector comprising a polynucleotide
encoding an engineered AMM polypeptide, and one or more expression
regulating regions. An expression regulating region includes a
promoter, a terminator, a replication origin, etc., depending on
the type of hosts into which they are to be introduced. The various
nucleic acid and control sequences described above may be joined
together to produce a recombinant expression vector which may
include one or more convenient restriction sites to allow for
insertion or substitution of the nucleic acid sequence encoding the
polypeptide at such sites. Alternatively, the nucleic acid sequence
of the present disclosure may be expressed by inserting the nucleic
acid sequence or a nucleic acid construct comprising the sequence
into an appropriate vector for expression. In creating the
expression vector, the coding sequence is located in the vectors
that the coding sequence is operably linked with the appropriate
control sequences for expression.
[0140] The recombinant expression vector may be any vector (e.g., a
plasmid or virus), which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the polynucleotide sequence. The choice of the vector will
typically depend on the compatibility of the vector with the host
cell into which the vector is to be introduced. The vectors may be
linear or closed circular plasmids.
[0141] The expression vector may be an autonomously replicating
vector, i.e., a vector that exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0142] In various embodiments, the expression vector can have one
or more selectable markers, which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like. Examples of
bacterial selectable markers include among others, the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers, which
confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol (Example 1) or tetracycline resistance. Suitable
markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,
TRP1, and URA3.
[0143] Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Embodiments for use in an
Aspergillus cell include the amdS and pyrG genes of Aspergillus
nidulans or Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
[0144] In some embodiments, the expression vectors can contain an
element(s) that permits integration of the vector into the host
cell's genome or autonomous replication of the vector in the cell
independent of the genome. For integration into the host cell
genome, the vector may rely on the nucleic acid sequence encoding
the polypeptide or any other element of the vector for integration
of the vector into the genome by homologous or nonhomologous
recombination.
[0145] Alternatively, the vector may contain additional nucleic
acid sequences for directing integration by homologous
recombination into the genome of the host cell. The additional
nucleic acid sequences enable the vector to be integrated into the
host cell genome at a precise location(s) in the chromosome(s). To
increase the likelihood of integration at a precise location, the
integrational elements should preferably contain a sufficient
number of nucleic acids, such as 100 to 10,000 base pairs,
preferably 400 to 10,000 base pairs, and most preferably 800 to
10,000 base pairs, which are highly homologous with the
corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding nucleic acid sequences. On the other hand,
the vector may be integrated into the genome of the host cell by
nonhomologous recombination.
[0146] For autonomous replication, the vector can further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of bacterial
origins of replication are P15A, pSC101, pMB1 and Co1E1. Origins of
replication of plasmids pBR322 (which has a pMB1 origin of
replication) pUC19 (which has a Co1E1 origin of replication),
pACYC177 and pACYC184 (which have a P15A origin of replication),
permit replication in E. coli; origins of replication for plasmids
pUB110, pE194, pTA1060, or pAM f31 permit replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are
the 2 micron origin of replication, ARS 1, ARS4, the combination of
ARS 1 and CEN3, and the combination of ARS4 and CEN6. The origin of
replication may be one having a mutation which conditional for
replication (e.g., temperature-sensitive) in the host cell (see,
e.g., Ehrlich et al., 1978, Proc Natl Acad Sci USA 75:1433).
[0147] More than one copy of a nucleic acid sequence of the present
disclosure may be inserted into the host cell to increase
production of the gene product. An increase in the copy number of
the nucleic acid sequence can be obtained by integrating at least
one additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the nucleic
acid sequence where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
nucleic acid sequence, can be selected for by cultivating the cells
in the presence of the appropriate selectable agent.
[0148] Many of the expression vectors for use in the present
disclosure are commercially available. Suitable commercial
expression vectors include p3xFLAGTM.TM. expression vectors from
Sigma-Aldrich Chemicals, St. Louis Mo., which includes a CMV
promoter and hGH polyadenylation site for expression in mammalian
host cells and a pBR322 origin of replication and ampicillin
resistance markers for amplification in E. coli. Other suitable
expression vectors include, among others, pBluescriptII SK(-) and
pBK-CMV, which are commercially available from Stratagene, LaJolla
Calif., and plasmids which are derived from pBR322 (Gibco BRL), pUC
(Gibco BRL), pREP4, pCEP4 (Invitrogen) and pPoly (Lathe et al.,
1987, Gene 57:193-201).
7.5 Host Cells for Expression of AAM
[0149] In another aspect, the present disclosure provides a host
cell comprising a polynucleotide encoding an improved AAM
polypeptide of the present disclosure, the polynucleotide being
operatively linked to one or more control sequences for expression
of the AAM enzyme in the host cell. Host cells for use in
expressing the AAM polypeptides encoded by the expression vectors
are well known in the art and include but are not limited to,
bacterial cells, such as E. coli, Streptomyces and Salmonella
typhimurium cells; fungal cells, such as yeast cells (e.g.,
Porphyromonas gingivalis or Pichia pastoris (ATCC Accession No.
201178)); insect cells such as Drosophila S2 and Spodoptera Sf9
cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma
cells; and plant cells. Appropriate culture mediums and growth
conditions for the above-described host cells will be apparent to
the skilled artisan based on the choice of the host cell.
[0150] By way of example, a mutant E. coli BW25113 .DELTA.panD
strain (Datsenko et al., 2000, Proc Natl Acad. Sci. USA
97:6640-6645) can be transformed by an expression vector for
expressing the AAM polynucleotides of the present disclosure. The
expression vector is created by operably linking a polynucleotide
of the present disclosure to the lac promoter under control of the
lad repressor gene. The expression vector also contains the P15A
origin of replication and the chloramphenicol resistance gene. The
transformed E. coli BW25113 .DELTA.panD can be readily cultured
under appropriate culture medium containing chloramphenicol such
that only transformed E. coli cells that expressed the expression
vector survives. See, e.g., Example 1.
7.6 Method of Generating and Using Engineered AAM Polypeptides
[0151] To make the improved AAM polynucleotides and polypeptides of
the present disclosure, the polynucleotide encoding the
naturally-occurring or engineered AMM enzymes can be subjected to
mutagenesis and/or directed evolution methods, as discussed above.
An exemplary directed evolution technique is mutagenesis and/or DNA
shuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA
91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230;
WO 00/42651; WO 01/75767; and U.S. Pat. No. 6,537,746. Other
directed evolution procedures that can be used include, among
others, staggered extension process (StEP), in vitro recombination
(Zhao et al., 1998, Nat Biotechnol. 16:258-261), mutagenic PCR
(Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140), cassette
mutagenesis (Black et al., 1996, Proc Natl Acad Sci USA
93:3525-3529), and other techniques referenced previously. Any of
these methods can be applied to a reference polynucleotide encoding
an AAM polypeptide, including any of the polynucleotides listed in
the Sequence Listing beginning from SEQ ID NO:5, to generate
variants of those AAM polynucleotides. To maximize any diversity,
several of the above-described techniques can be used sequentially.
Typically, a library of mutant polynucleotides, such as variants
polynucleotides of the AMM encoding polynucleotides disclosed
herein, is created by one mutagenic or evolutionary technique, and
their expression products are screened to find the polypeptide
having the highest AAM activity. Then a second mutagenic or
evolutionary technique is applied to polynucleotides encoding the
most active polypeptides to create a second library, which in turn
is screened for AAM activity by the same technique. The process of
mutating and screening can be repeated as many times as needed,
including the insertion of point mutations, to arrive at a
polynucleotide that encodes a polypeptide with the desired improved
property, such as activity, thermostabiliy, or cofactor
preference.
[0152] Exemplary methods are described in further detail in the
Examples. In the present disclosure, as discussed above, a series
of directed evolution techniques was applied to an initial parent
molecule SEQ ID NO:3, which encodes the polynucleotide of SEQ ID
NO:4. International patent publication WO 03/062173 reports that
SEQ ID NO: 3 was obtained by modifying the DNA of SEQ ID NO:1 (P.
gingivalis KAM). P. gingivalis KAM (SEQ ID NO: 2), which is thus
the "grandparent" molecule of the disclosure molecules described
herein and is also reported to have no detectable AAM activity.
Parental polypeptide (SEQ ID NO: 4) has detectable, but low alanine
2,3-aminomutase activity in the assay of Example 4.
[0153] AAM polypeptides, including variants of AAM polypeptides in
the Sequence Listing, can be readily screened to ascertain whether
they have AAM activity. For example, after each round of
mutagenesis or directed evolution is performed, the resulting
libraries of polypeptides can be screened. In some embodiments,
screening for transformed cells that express a polypeptide having
AAM activity can be, in general, a two-step process. First, the
cells are physically separated and then screened to identify which
cells do and do not possess a desired property. Selection can be a
form of screening in which identification and physical separation
are achieved simultaneously by expression of a selection marker,
which, in some genetic circumstances, allows cells expressing the
marker to survive while other cells die (or vice versa). Exemplary
screening markers include luciferase, .beta.-galactosidase, and
green fluorescent protein. Selection markers include drug and toxin
resistance genes, such as resistance to chloramphenicol,
ampicillin, kanamycin, tetracyclin, and the like. Although
spontaneous selection can and does occur in the course of natural
evolution, in the present methods selection is performed by man.
The cells can then be screened for production of .beta.-alanine
using any assay sufficiently sensitive for measuring
.beta.-alanine, including the assays described in the Examples.
Thus, in some embodiments, the assay comprises a LC/MS/MS assay of
Example 4.
[0154] In some embodiments, the screens for the improved AAM
polypeptides can be based on measuring the ultimate products of the
biochemical pathway involving AAM enzymes (see FIG. 2). Thus in
some embodiments, the assay can measure the production of 3-HP. In
such embodiments, the expression of enzymes involved in the pathway
can be increased in a single cell to enhance the production of the
substrate .alpha.-alanine as well as conversion of the product
.beta.-alanine to 3-HP. The genes encoding the other enzymes in the
3-HP pathway can be cloned into a synthetic operon, thereby
allowing co-expression of the enzymes. The polynucleotide encoding
the AAM polypeptide to be tested can be part of the synthetic
operon or introduced in a separate expression vector. Enhancements
in AAM enzyme activity can be detected by the increase in the
formation of 3-HP. An exemplary assay of this type is described in
Example 5, which describes the construction of the synthetic operon
that includes the polynucleotide encoding the AAM polypeptide. Any
standard assay for detecting 3-HP can be used. An exemplary
LC/MS/MS assay is provided in Example 6. Additional descriptions of
use of synthetic operons for expressing the enzymes of the 3-HP
pathway are given in Jessen et al., U.S. application Ser. No.
60/824,031, entitled "Beta-Alanine/Alpha-Ketoglutarate
Aminotransferase for 3-Hydroxypropionic Acid Production," filed
Aug. 30, 2006, incorporated herein by reference.
[0155] If the sequence of the engineered polypeptide is known, the
polynucleotides encoding the AAM enzyme can be prepared by standard
solid-phase methods, according to known synthetic methods. In some
embodiments, fragments of up to about 100 bases can be individually
synthesized, then joined (e.g., by enzymatic or chemical ligation
methods, or polymerase mediated methods) to form any desired
continuous sequence. For example, polynucleotides and
oligonucleotides of the present disclosure can be prepared by
chemical synthesis using, e.g., the classical phosphoramidite
method described by Beaucage et al., 1981, Tet Lett 22:1859-69, or
the method described by Matthes et al., 1984, EMBO J. 3:801-05,
e.g., as it is typically practiced in automated synthetic methods.
According to the phosphoramidite method, oligonucleotides are
synthesized, e.g., in an automatic DNA synthesizer, purified,
annealed, ligated and cloned in appropriate vectors. In addition,
essentially any nucleic acid can be obtained from any of a variety
of commercial sources, such as The Midland Certified Reagent
Company, Midland, Tex., The Great American Gene Company, Ramona,
Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc.,
Alameda, Calif., and many others.
[0156] In some embodiments, the polynucleotides can also be
synthesized by well known techniques as described in the technical
literature. See, e.g., Caruthers et al., 1992, Cold Spring Harbor
Symp. Quant. Biol. 47:411-418. Double stranded DNA can then be
obtained either by synthesizing the complementary strand and
annealing the strands together under appropriate conditions, or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0157] In some embodiments, the AAM polypeptides can be synthesized
by standard peptide chemistry techniques. Polypeptides may be
prepared using conventional step-wise solution or solid phase
synthesis (see, e.g., Chemical Approaches to the Synthesis of
Peptides and Proteins, Williams et al., Eds., 1997, CRC Press, Boca
Raton Fla., and references cited therein; FMOC Solid Phase Peptide
Synthesis: A Practical Approach, Chan & White, Eds., 2000, IRL
Press, Oxford, England, and references cited therein). In other
embodiments, the polypeptides may be prepared by way of segment
condensation, as described, for example, in Liu et al., 1996,
Tetrahedron Lett. 37(7):933-936; Baca et al., 1995, J Am Chem. Soc.
117:1881-1887; Tam et al., 1995, Int J Peptide Protein Res.
45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and
Tam, 1994, J Am Chem Soc. 116(10):4149-4153; Liu and Tam, 1994,
Proc Natl Acad Sci USA 91:6584-6588; Yamashiro and Li, 1988, Int.
J. Peptide Protein Res. 31:322-334.
[0158] Manipulation of the polynucleotides and polypeptide can
follow general molecular biological techniques, such as those
described in Berger and Kimmel, "Guide to Molecular Cloning
Techniques," Meth. Enzy., Volume 152, Academic Press, Inc., San
Diego, Calif.; Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual, 2.sup.nd Ed., Volumes 1-3, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; and Current Protocols
in Molecular Biology, F. M. Ausubel et al. eds., Green Publishing
Associates and John Wiley & Sons, updates to 2000. Examples of
techniques sufficient to direct persons of skill for in vitro
amplification methods, including the polymerase chain reaction,
ligase chain reaction, Q.beta.-replicase amplification, and other
RNA polymer mediated techniques (e.g., NASBA) can be found in the
references cited above (e.g., Sambrook, supra) as well as U.S. Pat.
No. 4,683,202; PCR Protocols: A Guide to Methods and Applications,
Innis et al. eds., Academic Press, Inc., San Diego, Calif. (1990),
Arnheim and Levinson, 1990, Chemical and Engineering News 36-47;
Kwoh et al., 1989, Proc Natl Acad Sci USA 86:1173; Guatelli et al.,
1990, Proc Natl Acad Sci USA 87:1874; Lomell et al., 1989, J Clin
Chem. 35:1826; Landegren et al., 1988, Science 241:1077-1080; Van
Burnt, 1990, Biotechnology 8:291-294; Wu and Wallace, 1989, Gene
4:560; Barringer et al., 1990, Gene 89:117; and Sookman and Malek,
1995, Biotechnology 13:563-564. Improved methods of cloning in
vitro amplified nucleic acid are described in U.S. Pat. No.
5,426,039. Improved methods of amplifying large nucleic acids by
PCR are summarized in Cheng et al., 1994, Nature 369:684-685, and
the references cited therein.
[0159] Engineered AAM enzymes expressed in a host cell can be
recovered from the cells and or the culture medium using any one or
more of the well known techniques for protein purification,
including, among others, lysozyme treatment, sonication,
filtration, salting-out, ultra-centrifugation, and chromatography.
Suitable solutions for lysing and the high efficiency extraction of
proteins from bacteria, such as E. coli, as commercially available
under the trade name CelLytic B.TM. from Sigma-Aldrich of St. Louis
Mo.
[0160] Chromatographic techniques for isolation of the AAM
polypeptide include, among others, reverse phase chromatography
high performance liquid chromatography, ion exchange
chromatography, gel electrophoresis, and affinity chromatography.
Conditions for purifying a particular enzyme will depend, in part,
on factors such as net charge, hydrophobicity, hydrophilicity,
molecular weight, molecular shape, etc., and will be apparent to
those having skill in the art (see, e.g., Scopes, 2006, Protein
Purification, 3rd Ed., Springer; Protein Purification Techniques: A
Practical Approach, 2.sup.nd Ed. (Roe, S. ed.) Oxford University
Press (2006).
[0161] In some embodiments, affinity techniques may be used to
isolate the improved AAM enzymes. For affinity chromatography
purification, any antibody which specifically binds the AAM
polypeptide may be used. For the production of antibodies, various
host animals, including but not limited to rabbits, mice, rats,
etc., may be immunized by injection with an AAM polypeptide or a
fragment of the AAM polypeptide. The AAM polypeptide or its
fragments may be attached to a suitable carrier, such as BSA, by
means of a side chain functional group or linkers attached to a
side chain functional group. Various adjuvants may be used to
increase the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacilli Calmette
Guerin) and Corynebacterium parvum.
[0162] The engineered AAM polypeptides of the present disclosure
can be used in the biocatalytic route to produce 3-hydroxypropionic
acid (3-HP) from .beta.-alanine in accordance with the methods
described in International patent publication WO 03/062173, which
is incorporated herein by reference in its entirety.
[0163] Typically, the AAM polypeptides can be used in the form of
whole cell catalysts or in as a crude preparations of the enzyme.
In some embodiments, the AAM polypeptides may be isolated and
further purified under anoxic conditions. For example, once the AAM
polypeptides are expressed by the AAM polynucleotides in E. coli,
the polypeptides can be extracted and purified from the cells and
or the culture medium using any one or more of the well known
techniques for protein purification under anoxic conditions. An
exemplary process for purifying AAM polypeptides sufficiently from
a cell lysate under anaerobic conditions for applications in a
chemical process is disclosed in Petrovich et al., 1991, J. Biol.
Chem. 266:7656-7660, which is incorporated herein by reference in
its entirety.
[0164] The AMM polypeptide in various forms can be used to produce
.beta.-alanine. In some embodiments, the method comprises
cultivating a host cell expressing an AAM polypeptide disclosed
herein under conditions suitable for production of the
.beta.-alanine. Various conditions may be used for cultivating the
host cell. In some embodiments, the method further comprises
culturing the cell in presence of glucose under conditions suitable
for the production of .beta.-alanine. In some embodiments, the
method for producing .beta.-alanine comprises contacting a
preparation of AAM polypeptide, such as a crude extract or isolated
preparation of the AAM as described herein, with .beta.-alanine
under conditions suitable for conversion to .beta.-alanine.
[0165] The methods further provide optionally recovering the
.beta.-alanine and/or using the .beta.-alanine as a substrate in a
biocatalytic pathway as described, for example in International
patent publication WO 03/062173, which is incorporated by reference
herein in its entirety. Conditions suitable for the production of
.beta.-alanine are described herein in Example 4, as well as in
International patent publication WO 03/062173 and in Examples 5 and
7 of U.S. application Ser. No. 60/726,925, entitled "Increasing the
Activity of Radical S-Adenosyl Methionine (SAM) Enzymes," filed
Oct. 14, 2005, both of which examples are incorporated herein by
reference for the disclosed procedures and conditions.
8. EXAMPLES
8.1 Example 1
Transformation Protocol for aam Libraries into .DELTA.panD KIfldA
Strain
[0166] A mutant E. coli strain .DELTA.panD derived from BW25113,
which is described in Datsenko et al., 2000, Proc Natl Acad Sci USA
97:6640-6645, was used as the host strain for screening of the aam
(alanine 2,3-aminomutase) gene libraries. The protocol used to make
the deletion is detailed in Example 4 of International patent
publication WO 03/062173. Optimally, a strain additionally having
an increased expression of the flavodoxin (fldA) gene was used as
the host strain for screening of the aam gene libraries since
increased flavodoxin enhances aminomutase activity when produced in
E. coli. Description for production of .beta.-alanine from cells
that express AAM and overexpress flavodoxin are given in Examples
1-4 of U.S. application Ser. No. 60/726,925, by Liao et al., filed
Oct. 14, 2005, entitled "Increasing the Activity of Radical
S-Adenosyl Methionine (SAM) Enzymes," incorporated herein by
reference. This same application (i.e., Liao, et al.) describes in
Example 4 the construction of a strain of E. coli in which an
artificial Plac/ara hybrid promoter was placed immediately upstream
of the fldA gene. Strains carrying the artificial promoter before
the fldA gene are designated KIfldA, where KI refers to
"knock-in").
[0167] Competent cells of E. coli .DELTA.panD KIfldA were prepared
either chemically or electrochemically using standard protocols.
Competent E. coli .DELTA.panD KIfldA was removed from -80.degree.
C. frozen storage and thawed. Thereafter, it was kept on ice until
used. An aliquot (100 .mu.l per transformation) was transferred
into a sterile 1.5 ml centrifuge tube. A KCM (5.times.) salt
solution was added until the concentration in the aliquot was
1.times.. KCM consists of 700 mM KCl; 10 mM
morpholinopropanesulphonic acid (MOPS) adjusted to pH 5.8. A 1-5
.mu.l of the ligation mixture was added to the cells. The cells
containing the ligation mixture were first incubated on ice for 30
minutes. The cells were heat shocked at 42.degree. C. for 1 min,
and subsequently incubated on ice for 2 minutes. 500 .mu.l of SOC
(Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, 1st
Ed., pp. A.2 and A.3, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.) was added to the cells, and the cells were incubated
at 37.degree. C. for 1 hour with agitation. The cells were then
centrifuged at 5000 rpm for 3 minutes, and the SOC was removed.
Pellets were subsequently resuspended in a medium appropriate for
either the complementation assay (Example 3) or the
biotransformation assay (Example 4).
8.2 Example 2
Cloning of aam Genes into pCK110900-I-Bla Series Vectors
[0168] The strategy employed for cloning the alanine aminomutase
genes into an inducible expression system involved the isolation of
the aam gene by PCR and cloning of the PCR fragment into the SfI
restriction sites downstream from a mutant lac promoter/operator
system. Initially, PCR primers were designed to contain a
nucleotide sequence that is specific to the 5' and 3' ends of the
aam gene, as well as the Shine-Delgarno sequence of the
ribosome-binding site, and the unique SfiI restriction sites. The
fragment was then amplified from a template, purified and digested
with the restriction endonuclease SfiI. The restriction digested
PCR fragment was purified using the QIAquick PCR purification kit
(Qiagen) and cloned into the S'I sites of the expression vector
pCK110900-I Bla under the control of a lac promoter and lad
repressor gene. The expression vector also contained the P15A
origin of replication and the chloramphenicol resistance gene.
Shuffled aam gene libraries were cloned by the same method. Several
clones were found that expressed an active alanine 2,3-aminomutase
(as per the method of Example 4) and the synthetic genes were
sequenced. A polynucleotide of SEQ ID NO: 3 was used as a starting
material for mutation and shuffling. SEQ ID NO: 3 has 99%
nucleotide identity with the wild-type Porphyromonas gingivalis
lysine aminomutase (SEQ ID NO: 1) (GenBank Accession No.
AE017175).
8.3 Example 3
Growth of Aminomutase Variants
[0169] A. Complementation assay for colonies possessing alanine
aminomutase activity. To identify genes encoding polypeptides that
can perform the alanine 2,3-aminomutase reaction, an efficient
screen or selection for the desired activity is needed. Therefore,
a selection method was developed by recognizing that E. coli uses
.beta.-alanine for the synthesis of pantothenic acid, which in turn
is a component of coenzyme A (CoA) and of acyl carrier protein
(ACP). CoA and ACP are the predominant acyl group carriers in
living organisms, and are essential for growth.
[0170] In E. coli, the primary route to .beta.-alanine is from
aspartate in a reaction catalyzed by aspartate decarboxylase (E.C.
4.1.1.11), which is encoded by the panD gene. A functional deletion
mutation of panD (i.e., .DELTA.panD) results in .beta.-alanine
auxotrophy and growth inhibition, which can be alleviated by the
exogenous addition of pantothenate or .beta.-alanine, or by the
production of .beta.-alanine from another source. This method is
described in International Patent Publication WO 03/062173 (see
Example 4), which is incorporated herein by reference.
[0171] Transformed cell pellets (Example 1) were re-suspended in
500 .mu.l of M9 selection medium ((Maniatis et al, supra) and
incubated at 30.degree. C. for 2-4 hours with agitation. The cells
were then plated onto M9 minimal agar medium supplemented with 1%
mannose, 20 .mu.M iron citrate, 5.0 g/l .alpha.-alanine, 0.1 mM
isopropyl-.beta.-D-thiogalactoside (IPTG) (Sigma Chemical Corp.,
St. Louis, Mo.), 50 mM MOPS, 25 mM bicarbonate, and 30 .mu.g/ml
chloramphenicol. The plated cells were incubated at 30.degree. C.
until colonies were of sufficient size to be picked using the
Q-BOT.TM. robot colony picker (Genetix USA, Inc, Boston Mass.).
[0172] B. Non-selective growth of aminomutase variants. In some
cases, non-selective growth of aminomutase variants was a desirable
alternative to the complementation assay. To accomplish this,
transformed cell pellets (Example 1) were resuspended in 500 .mu.l
rich medium using glucose repression conditions (2.times.YT media
(Yeast Extract Tryptone Medium) containing 1% glucose and 30
.mu.g/ml chloramphenicol). Cells were then plated onto 2.times.YT
agar medium supplemented with 30 .mu.g/ml chloramphenicol and 1%
glucose. The plated cells were incubated overnight at 30.degree.
C., and the resultant colonies were picked using the Q-BOT.TM.
robot colony picker (Genetix USA, Inc, Boston Mass.).
8.4 Example 4
Assay for .beta.-alanine Production
[0173] A. Sample Preparation. This assay for .beta.-alanine
production can be accomplished using either colonies from the
complementation assay (Example 3A), or from colonies grown under
non-selective conditions (Example 3B). Library colonies were picked
with a Q BOT.TM.-robot colony picker (Genetix USA, Inc. Boston
Mass.) and grown overnight at 30.degree. C. in liquid 2.times.YT
media (Yeast Extract Tryptone Medium) containing 1% glucose and 30
.mu.g/ml chloramphenicol. This culture was then diluted 10-fold
into fresh 2.times.YT containing 30 .mu.g/ml chloramphenicol, 20
.mu.M ferric ammonium citrate and 10 .mu.M pyridoxine and after 2
hours of growth at 30.degree. C., the cultures were induced for
aminomutase expression with the addition of 1 mM IPTG (isopropyl
thiogalactoside). The cultures were then allowed to grow for
another 5 hrs at 30.degree. C. before pelleting by
centrifugation.
[0174] The cell pellets were suspended into buffered minimal media,
BMM (12.8 g/L KH.sub.2PO.sub.4; 3 g/L Na.sub.2HPO.sub.4.7H.sub.2O;
0.5 g/L NaCl; 1 g/L NH.sub.4C1; 2 mM MgSO.sub.4; 0.04 mM
CaCl.sub.2; 2% mannose; 1 mM IPTG; 20 .mu.M ferric ammonium
citrate; 10 .mu.M pyridoxine; 30 .mu.g/ml chloramphenicol; 50 mM
MOPS pH 7; 25 mM sodium bicarbonate pH 9, and 200 mM
.alpha.-alanine) incubated at 30.degree. C. for 17 hours under
anaerobic conditions. Those having ordinary skill in the art will
appreciate that the incubation time may be varied to allow for the
generation of more .beta.-alanine product. Cell growth was
monitored by measuring the OD at 600 nm. The cell load is kept
constant between samples to facilitate comparison of activity.
Those having ordinary skill in the art will appreciate that the
cell load may be varied to allow for the generation of more
.beta.-alanine product. An aliquot of the cell suspension was
diluted 1:10 fold with cold 50:50 water:methanol mixture. The final
solution was subsequently filtered before subjecting to the
LC/MS/MS for analysis.
[0175] This assay was used to monitor .beta.-alanine production
over time by withdrawing samples at time intervals.
[0176] B. LC/MS/MS Analysis. The quantity of .beta.-alanine was
determined using a combination of liquid chromatography and mass
spectrometry.
[0177] The liquid chromatography (LC) phase was performed using an
ASTEC CHIROBIOTIC.TM. T 4.6 cm.times.50 mm chiral LC column
(Advanced Separation Technologies, Inc., Whippany, N.J., USA). The
mobile phase consisted of two solutions: A: 0.25% aqueous acetic
acid; and B: 0.25% (v/v) acetic acid in methanol. The elution was
isocratic @ 0.6 ml/minute.
[0178] The mass spectrometer (MS) analysis was performed on a
Micromass Ultima Triple Quad mass spectrometer, using the following
tune parameters:
[0179] Capillary: 3.5 kV; cone: 20 V; hex 1: 15 V; aperture: 1.0V;
source temp: 100.degree. C.; desolvation temp: 350.degree. C.; cone
gas: 40 L/hr; desolvation gas: 500 L/h; low mass resolution (Q1):
12; high mass resolution (Q1): 12; ion energy (Q1): 0.1; collision
cell entrance: -5; collision energy: 14; exit: 1; low mass
resolution (42): 15 high mass resolution (Q2): 15; ion energy (42):
3.0; multiplier: 650 V.
[0180] MS Method
[0181] Alanine Transitions
TABLE-US-00012 Analyte Parent Ion (m/z) Daughter Ion (m/z) Dwell
Time (sec) .alpha.-alanine 90 44.7 0.1 .beta.-alanine 90 30.2 0.1
The inter-channel delay was 0.1 seconds.
[0182] .beta.-Alanine was quantified in rate units of .mu.M
.beta.-alanine produced per hour. It is useful to assess relative
performance of a particular AAM polypeptide to a reference
polypeptide, e.g., as fold improvement. The polypeptides are
typically utilized in the form of a whole cell biocatalyst (i.e., a
host cell transformed with the corresponding AAM polynucleotide).
Exemplary reference polypeptides are the polypeptide of SEQ ID NO:
4 and the polypeptide of SEQ ID NO: 36.
8.5 Example 5
Generation and Use of Synthetic Operons for Screening for Improved
Aminomutase Activity
[0183] A. Transformation and growth protocol for Pgaam Libraries/E.
coli 11303.DELTA.panD-panC .DELTA.ldhA KIfldA. A mutant E. coli
11303 .DELTA.panD-C .DELTA.ldhA KIfldA was used as the host strain
for screening of the aam gene libraries cloned into the context of
the 3-HP operon. The protocol used for the construction of the host
strain is detailed in Example 4 of the International patent
publication WO 03/062173. Electroporation-competent cells of E.
coli 11303 .DELTA.panD-C .DELTA.ldhA KIfldA, an E. coli B strain,
were prepared by growing 200 ml of cells in LB in 1 L flask to
OD.sub.600.about.0.5. The cells were cooled in ice-water mixture
for 10 min before centrifugation at 4,000 g for 10 min at 4.degree.
C. The cells were washed successively with 100, 50 and 20 ml of
ice-cold 10% glycerol solution. The cells were then resuspended
into a final volume of 0.8 ml 10% glycerol solution and saved at
-80.degree. C. in 100 aliquots until used for electroporation.
[0184] ECM 630 Electro Cell Manipulator (BTX A Division of
Genetronics, Inc.) was used to electroporate cells.
Electroporation-competent E. coli 11303 .DELTA.panD-C .DELTA.ldhA
KIfldA were removed from -80.degree. C. storage and thawed on ice.
Thereafter, they were kept on ice until used. Electroporation
cuvettes (BTX) with a 1 mm gap were also placed on ice before use.
An aliquot of cells (100 .mu.l per transformation) was transferred
into a sterile 1.5 ml centrifuge tube and 1-5 .mu.l of ligation
mixture was added to the cells. Cells were electroporated using the
following parameters: Voltage: 1700V; Resistor: 200 Ohm; Capacitor:
25 .mu.F. One milliliter of SOC medium (Maniatis et al., supra) was
added to the cells, and the cells were incubated at 37.degree. C.
for 1 hour with agitation.
[0185] The cells were then plated onto LB agar medium supplemented
with 30 .mu.g/ml chloramphenicol and 1% glucose. The plated cells
were incubated overnight at 37.degree. C., and the resultant
colonies were picked using the Q-BOT.TM. robot colony picker
(Genetix USA, Inc, Boston Mass.).
[0186] B. Cloning of Pgaam genes into pCEK-Pgaam-Baat-PpmmsB-Spaat
vector. A synthetic operon expression vector,
pCEK-Pgaam-Baat-PpmmsB-Spaat, is described in Jessen et al., U.S.
application Ser. No. 60/824,031, entitled
"Beta-Alanine/Alpha-Ketoglutarate Aminotransferase for
3-Hydroxypropionic Acid Production," filed Aug. 30, 2006,
incorporated herein by reference. The expression vector comprises
the genes Pgaam (P. gingivalis alanine aminomutase), Baat
(.beta.-alanine amino transferase), PpmmsB (Pseudomomas putida
methyl-malonyl semialdehyde dehydrogenase) and Spaat
(Schizosaccharomyces pombe glutamate-pyruvate aminotransferase) in
a single operon under the control of a lac promoter.
[0187] The strategy employed for cloning alanine aminomutase gene
into an inducible expression system involved PCR amplification and
cloning of the PCR fragment into the NdeI-HindIII restriction sites
downstream from a hybrid lac-ara promoter/operator system (or a
mutant lac promoter which possesses an additional lac operator
sequences and the recognition site for AraC). The PCR primers were
designed to contain a nucleotide sequence that is specific to the
5' and 3' ends of the aam gene and the unique NdeI and HindIII
restriction sites. The fragment was then amplified from a template,
purified and digested with the restriction endonucleases NdeI and
HindIII. The digested PCR fragment was purified using the QIAquick
PCR purification kit (Qiagen), and cloned into the NdeI-HindIII
sites of the expression vector pCEK-Pgaam-Baat-PpmmsB-Spaat under
the control of a lac promoter. The expression vector also contained
the ColE1 origin of replication and the kanamycin resistance
gene.
[0188] C. Cloning 3-HP operon into pCK110900 I-Bla vector. For
better control of expression of the 3-HP pathway genes, the four
pathway genes were cloned into the inducible pCK110900-I-Bla vector
(see FIG. 4). The genes were assembled on a polycistronic message
in the 5' to 3' order Pgaam-Baat-PpmmsB-Spaat with regions between
the genes encoding ribosome binding sites (RBS). The operon was
cloned downstream from the mutant lac promoter-operator system. The
pCK110900-I-Bla vector contains a P15A origin of replication (low
copy), lad repressor gene, chloramphenicol resistance gene and two
terminators located 3' to the pathway genes (see, e.g., WO
03/062173).
[0189] Several restriction sites were modified in order to clone
the 3-HP pathway genes into the described vector. An internal SfiI
site in PpmmsB was destroyed by changing K33 (AAG) to K33 (AAA), a
HindIII site between Pgaam and Baat was replaced with AvrII, an
XbaI between PpmmsB and Spaat was replaced with SalI, an XbaI site
at 3'-end of Spaat was replaced with SphI, and SfiI sites were
added to both ends of the 3-HP operon. PCR primers containing
corresponding restriction site sequences were used to amplify Baat
and Spaat genes from the pCEK-Pgaam-Baat-PpmmsB-Spaat vector. The
Pgaam variants were amplified from pCK-Pgaam using appropriate
primers. The SfiI site within mmsB was destroyed by the SOEing
method (splicing by overlap extension) (Ho et al., 1999, Gene,
77:51-59), while at the same time new restriction sites were added
to the 5' and 3' end of the gene. PCR products were cloned using
TOPO TA Cloning Kit (Invitrogen Corp., Carlsbad, Calif.). Several
clones of each construct were sequenced and clones with the correct
sequence were taken into further cloning steps. TOPO-mmsB plasmid
DNA was digested with BamHI/SalI and mmsB was ligated with the
TOPO-Spaat vector digested with the same enzymes to yield
TOPO-mmsB-Spaat construct. The presence of the insert was confirmed
by restriction analysis. TOPO-mmsB-Spaat plasmid was digested with
BamHI/SfiI and a fragment containing two genes was gel purified.
Subsequently, all the genes were assembled into the operon in a
four-way ligation
(SfiI-Pgaam-AvrII+AvrII-Baat-BamHI+BamHI-mmsB-Spaat-SfiI+the
pCK110900-I-Bla/SfiI vector) using T4 DNA ligase (New England
Biolabs, Beverly, Mass.). The ligation mixture was incubated
overnight at 16.degree. C., transformed into E. coli XL1-Blue
(Stratagene, La Jolla, Calif.), and colonies grown on agar plates
were screened by colony PCR for the presence of the full size
operon. DNA was isolated from the clones exhibiting the correct
sized band by PCR, and sequenced. The correct sequence was
identified and the corresponding plasmids isolated. Shuffled Pgaam
libraries were cloned into NdeI-AvrII sites in place of a stuffer
fragment (gfp) into the context of the operon. These plasmids were
subsequently transformed into E. coli 11303 .DELTA.ldhA
.DELTA.panD-C KIfldA strain.
8.6 Example 6
Assay for 3-HP Production in Screens Using Synthetic Operons
[0190] A. Sample Preparation. Library colonies were picked with a
QBOT.TM. robot colony picker (Genetix USA, Inc. Boston Mass.) and
grown overnight at 30.degree. C. in liquid LB media (Yeast Extract
Tryptone Medium) containing 30 .mu.g/ml chloramphenicol. This
culture was then diluted 10-fold into fresh 2.times.YT containing
30 .mu.g/ml chloramphenicol, 20 .mu.M ferric ammonium citrate, 100
.mu.M pyridoxine HCl and 10 .mu.M pantothenate. After 2 hours of
growth at 30.degree. C., the cultures were induced for 3-HP pathway
enzymes expression with the addition of 1 mM IPTG (isopropyl
thiogalactoside). The cultures were then allowed to grow for
another 6 hrs at 30.degree. C. before pelleting the cells by
centrifugation.
[0191] The cell pellets were suspended into buffered minimal media,
BMM (12.8 g/L KH.sub.2PO.sub.4; 3 g/L Na.sub.2HPO.sub.4-7H.sub.2O;
0.5 g/L NaCl; 20 .mu.M ferric ammonium citrate; 100 .mu.M
pyridoxine; 5 .mu.M pantothenate, 100 mM MOPS pH 8; 1% glucose)
incubated at 30.degree. C. for 14 hours under anaerobic conditions.
Those having ordinary skill in the art will appreciate that the
incubation time may be varied to allow for the generation of more
3-HP product. Cell growth was monitored by measuring the OD at 600
nm. The cell load is kept constant between samples to facilitate
comparison of activity. Those having ordinary skill in the art will
appreciate that the cell load may be varied to allow for the
generation of more 3-HP product. An aliquot of the cell suspension
was diluted 1:10 fold with 90:10 water:acetonitrile mixture. The
final solution was subsequently filtered before subjecting the
sample to analysis by LC/MS/MS.
[0192] This assay was used to monitor 3-HP production over time by
withdrawing samples at timed intervals.
[0193] B. LC/MS/MS Analysis. The quantity of 3-HP was determined
using a combination of liquid chromatography and mass spectrometry
in the ESI negative ion mode. The ms/ms transitions from 89>59
represents (3-HP-H).sup.->(CH.sub.3CO.sub.2).sup.-.
[0194] The liquid chromatography (LC) phase was performed using a
Phenomenex Aqua 2.0.times.250 mm 5 u 125 A C18 column (Phenomenex
Inc, Torrance, Calif., USA). The mobile phase consisted of two
solutions: A: 98.5% of 0.1% formic acid in water and B: 1.5%
acetonitrile. The elution was isocratic @ 0.4 ml/minute.
[0195] The mass spectrometer (MS) analysis was performed on a
Waters, Micromass Ultima Triple Quad mass spectrometer, using the
following tune parameters:
[0196] Capillary: 3.3 kV; cone: 22 V; hex 1: 15 V; aperture: 1.0V;
source temp: 100.degree. C.; desolvation temp: 350.degree. C.; cone
gas: 47 L/hr; desolvation gas: 702 L/h; low mass resolution (Q1):
11.5; high mass resolution (Q1): 12; ion energy (Q1): 0.7;
collision cell entrance: -1; collision energy: 9; exit: 1; low mass
resolution (Q2): 11.5 high mass resolution (Q2): 12; ion energy
(Q2): 1.1; multiplier: 650 V.
[0197] MS Method
[0198] 3-HP Transitions
TABLE-US-00013 Analyte Parent Ion (m/z) Daughter Ion (m/z) Dwell
Time (sec) 3-HP 88.7 58.7 0.5 The inter-channel delay was 0.02
seconds.
[0199] 3-HP was quantified as the total 3-HP produced (.mu.M 3-HP
produced after 14 hrs of anaerobic biotransformation). It is useful
to assess relative performance of a particular AAM polypeptide to a
reference polypeptide, e.g., as fold improvement. The polypeptides
are typically utilized in the form of a whole cell biocatalyst
(i.e., a host cell transformed with the corresponding AAM
polynucleotide).
[0200] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
[0201] While various specific embodiments have been illustrated and
described, it will be appreciated that various changes can be made
without departing from the spirit and scope of the disclosure(s).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120040437A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120040437A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References