U.S. patent application number 10/559307 was filed with the patent office on 2006-06-15 for thiamin production by fermentation.
Invention is credited to Markus G. Goese, John B. Perkins, Ghislain Schyns.
Application Number | 20060127993 10/559307 |
Document ID | / |
Family ID | 33490751 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060127993 |
Kind Code |
A1 |
Goese; Markus G. ; et
al. |
June 15, 2006 |
Thiamin production by fermentation
Abstract
The present invention provides a method for producing thiamin
products using a microorganism containing a mutation that causes it
to overproduce and release thiamin products into the medium.
Biologically pure cultures of the microorganisms and isolated
polynucleotides containing the mutations are also provided. In
addition, methods for detecting a pathogenic microorganism in a
clinical sample, assays for identifying an antibiotic, as well as,
antibiotics identified by such assays are provided.
Inventors: |
Goese; Markus G.; (Basel,
CH) ; Perkins; John B.; (Reinach, CH) ;
Schyns; Ghislain; (Aesch, CH) |
Correspondence
Address: |
Stephen M Haracz;Bryan Cave
1290 Avenue of the Americas
New York
NY
10104
US
|
Family ID: |
33490751 |
Appl. No.: |
10/559307 |
Filed: |
May 27, 2004 |
PCT Filed: |
May 27, 2004 |
PCT NO: |
PCT/CH04/00321 |
371 Date: |
December 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60475323 |
Jun 2, 2003 |
|
|
|
Current U.S.
Class: |
435/122 ;
435/252.31; 435/252.9 |
Current CPC
Class: |
C12R 2001/125 20210501;
C12Q 1/04 20130101; C12P 17/167 20130101; C12Q 1/18 20130101; C12N
15/52 20130101; C12N 1/205 20210501 |
Class at
Publication: |
435/122 ;
435/252.31; 435/252.9 |
International
Class: |
C12P 17/12 20060101
C12P017/12; C12N 1/20 20060101 C12N001/20; C12N 1/21 20060101
C12N001/21 |
Claims
1. A microorganism selected from the group consisting of
Bacillaceae, Lactobacillaceae, Streptococcaceae, Corynebacteriaceae
and Brevibacteriaceae, the microorganism containing a mutation that
deregulates thiamin production and causes thiamin products to be
released into a culture media.
2. The microorganism of claim 1 wherein the mutation is selected
from the group consisting of .DELTA.thiL, txl, tx26 and
combinations thereof.
3. The microorganism of claim 1 which is selected from the group
consisting of Bacillus, Lactobacillus, Lactococcus, Corynebacterium
and Brevibacterium.
4. The microorganism of claim 3 which is a Bacillus subtilis
cell.
5. The microorganism of claim 4 which is B. subtilis TH95 (ATCC
PTA-5221).
6. The microorganism according to claim 1 further comprising a DNA
cassette containing a polynucleotide sequence being selected from
the group consisting of: (a) a polynucleotide sequence that encodes
a thiA gene product, wherein one or more copies of said
polynucleotide sequence are contained in said DNA cassette; (b) a
polynucleotide sequence that encodes gene products from a thiKC
operon, wherein one or more copies of said polynucleotide sequence
are contained in said DNA cassette; and (c) a polynucleotide
sequence that encodes gene products of a tenAl-thiOSGFD operon;
which polynucleotide sequence is operatively controlled by a strong
constitutive promoter.
7. The microorganism according to claim 1 further comprising (a) a
DNA cassette containing a polynucleotide sequence that encodes gene
products of a tenAlthiOSGFD operon and (b) a DNA cassette
containing at least one copy of a polynucleotide sequence that
encodes a thiA gene product, which polynucleotide sequences are
operatively controlled by a strong constitutive promoter.
8. The microorganism according to claim 6 which is selected from
the group consisting of B. subtilis TH116 (ATCC PTA-5224), TH115
(ATCC PTA-5223), TH404 (DSM 16333) and TH405 (DSM 16334).
9. The microorganism according to claim 1 further comprising a
first mutation that deregulates expression of a purine operon of B.
subtilis and a second mutation that blocks conversion of
5-aminoimidazole ribotide (AIR) to carboxyaminoimidazole ribotide
(CAIR).
10. The microorganism according to claim 9 wherein the first
mutation comprises a mutation within the leader region of the pur
operon and the second mutation comprises a mutation within the purE
gene encoding phosphoribosylaminoimidazole carboxylase 1.
11. The microorganism according to claim 10 which is B. subtilis
TH101 (ATCC PTA-5222).
12. A process for producing thiamin products comprising: (a)
culturing, in a suitable medium, a microorganism according to claim
1 that overproduces thiamin products into the medium; and (b)
recovering the thiamin products.
13. The process according to claim 12 further comprising culturing
the microorganism in the presence of a component which is selected
from the group consisting of: (a) thiamin precursors, (b) a thiamin
precursor and a purine source, (c) a precursor of a HET pathway,
(d) a precursor of a HMP pathway, and (e) a derivative of HMP.
14. The process according to claim 13 wherein the thiamin
precursors are selected from the group consisting of
4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP),
5-(2-hydroxyethyl)-4-methylthiazole (HET) and a combination
thereof.
15. The process according to claim 13 wherein the thiamin precursor
is HET and the purine source is xanthine.
16. The process according to claim 13 wherein the precursor of the
HET pathway is selected from the group consisting of glycine,
cysteine, isoleucine, threonine and combinations thereof.
17. The process according to claim 13 wherein the derivative of HMP
is 4-amino-2-methyl-5-pyrimidinemethaneamine (Grewe Diamine).
18. An isolated polynucleotide sequence comprising a txl
mutation.
19. The isolated polynucleotide sequence according to claim 18
wherein the mutation results in a leucine to phenylalanine
substitution at amino acid residue 116.
20. The isolated polynucleotide sequence according to claim 18
wherein the sequence is SEQ ID NO: 31 or a polynucleotide sequence
that hybridizes to SEQ ID NO: 31 under stringent conditions and,
when present in a microorganism, causes a deregulation of thiamin
production.
21. An isolated polynucleotide sequence comprising a first mutation
with 70% linkage to .DELTA.yufR::Tn917 (tx26-1) and a second
mutation with 59% linkage to .OMEGA.motA::Tn917 (tx26-2), wherein
the presence of both of the mutations in a thiamin-producing
microorganism causes a deregulation of thiamin production.
22. The isolated polynucleotide sequence according to claim 21
wherein the tx26-1 mutation is encoded by a polynucleotide sequence
which is SEQ ID NO: 33 or a polynucleotide sequence that hybridizes
to SEQ ID NO: 33 under stringent conditions and wherein the tx26-2
mutation is encoded by a polynucleotide sequence which is SEQ ID
NO: 36 or a polynucleotide sequence that hybridizes to SEQ ID NO:
36 under stringent conditions and, when present in a microorganism,
causes a deregulation of thiamin production.
23. A DNA cassette comprising one or more polynucleotides according
to claim 18.
24. A microorganism containing the DNA cassette of claim 23.
25. A method for detecting a pathogenic microorganism in a clinical
sample from a patient comprising: (a) determining whether a
Gram.sup.+ microorganism is present in the sample, (b) determining
whether the microorganism contains a yloS ortholog, and (c)
determining whether the microorganism contains a thiL ortholog,
wherein the presence of a yloS ortholog and the absence of a thiL
ortholog in a Gram.sup.+ microorganism indicates that the
microorganism is pathogenic.
26. The method according to claim 25 wherein the microorganism is
selected from the group of group consisting of Listeria,
Staphylococcus, Clostridium, Enterococcus, and Streptococcus.
27. The method according to claim 26 wherein the microorganism is
selected from the group consisting of Listeria monocytogenes,
Staphylococcus aureus, Staphylococcus epidermidis, Clostridium
tetani, Clostridium perfringens, Enterococcus sp., Streptococcus
agalactiae, Streptococcus pyogenes, and Streptococcus
pneumoniae.
28. An assay for identifying an antibiotic comprising: (a)
contacting an assay composition comprising a YloS protein with a
test compound, and (b) determining whether the test compound
inhibits YloS protein activity, wherein the compound is identified
as an antibiotic based on the compound's ability to inhibit the
activity of the YloS protein activity.
29. The assay according to claim 28 wherein the YloS protein
comprised in the assay composition is selected from the group
consisting of a purified YloS protein, a partially purified YloS
protein, and a crude cell extract from a cell producing YloS
protein.
30. The assay according to claim 28 wherein the YloS protein is
encoded by a polynucleotide derived from a pathogenic microorganism
selected from the group consisting of Listeria, Staphylococcus,
Clostridium, Enterococcus, and Streptococcus.
31. The assay according to claim 30 wherein the microorganism is
selected from the group consisting of Listeria monocytogenes,
Staphylococcus aureus, Staphylococcus epidermidis, Clostridium
tetani, Clostridium perfringens, Enterococcus sp., Streptococcus
agalactiae, Streptococcus pyogenes, and Streptococcus pneumoniae.
Description
[0001] The present invention relates to methods for producing
thiamin products. More particularly, the present invention relates
to methods for producing thiamin products using a microorganism
containing a mutation that causes it to overproduce and release
thiamin products into the medium. Biologically pure cultures of the
microorganisms and isolated polynucleotides containing the
mutations are also provided. In addition, methods for detecting a
pathogenic microorganism in a clinical sample, assays for
identifying an antibiotic, as well as, antibiotics identified using
such assays are provided.
[0002] Thiamin, also known as vitamin B1, is a member of the
water-soluble B-complex of vitamins and is a nutritional
requirement for mammals. The pyrophosphate form of thiamin acts in
vivo as the coenzyme in many carbohydrate and amino acid metabolic
pathways, like for example those catabolized by pyruvate
dehydrogenase, pyruvate oxidase or transketolase. It is important
to note that unlike other vitamin biosynthetic pathways (e.g.
riboflavin and biotin), thiamin is not part of the de novo pathway,
but is actually part of the salvage pathway.
[0003] Most enzymatic steps and intermediates in thiamin
biosynthesis have been studied in E. coli and to a lesser extent in
Salmonella typhimurium and Rhizobium (for reviews, see Brown and
Williamson (1987) pp. 528-532, In F. C. Neidhardt et al. (ed.)
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, vol. 1. American Society for Microbiology, Washington,
D.C.; White and Spenser (1996) pp. 680-686, In P. C. Neidhardt et
al. (ed.) Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, vol. 2. American Society for Microbiology,
Washington, D.C.; Begley et al. (1999) Arch. Microbiol. 171:
293-300). The E. coli genes encoding the steps in the thiamin
pathway are located at four distinct sites on the chromosome: a
thiCEFSGH operon at 90''; a thiMD operon at 46'', individual thiJ
and thiL genes are clustered in the 9.5'' vicinity and thiK at
25''. All of these genes have been cloned and sequenced and many of
the enzymes encoded by these genes have been overproduced in E.
coli and their enzymatic activities determined.
[0004] The pyrimidine moiety,
4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), is
derived from 5-aminoimidazole ribotide (AIR), an intermediate in
the de novo purine biosynthetic pathway. In Gram-negative bacteria,
conversion of AIR to HMP-P is catalyzed by the thiC gene product.
HMP-P is then phosphorylated to HMP-PP by ThiD kinase prior to
coupling with the thiazole unit.
[0005] The thiazole moiety, 5-(2-hydroxyethyl)-4-methylthiazole
phosphate (HET-P), is derived from L-tyrosine and
1-deoxy-D-xylulose phosphate (DXP); the sulfur atom is most likely
derived from L-cysteine. This reaction requires expression of at
least five genes thiF, thiS, thiG, thiH and thiI.
[0006] Coupling of HMP-PP and HET-P is catalyzed by thiamin
phosphate pyrophosphorylase encoded by thiE, resulting in thiamin
monophosphate (TMP). TMP is then phosphorylated to form thiamin
pyrophosphate (TPP) by the action of thiamin monophosphate kinase,
encoded by thiL. Because thiamin is not part of the de novo
pathway, E. coli requires a salvage enzyme, thiamin kinase, encoded
by thiK to convert exogenous thiamin into TMP.
[0007] Synthesis of thiamin in B. subtilis appears to utilize the
same enzymes and intermediates as found in E. coli (see, e.g.,
Perkins and Pero (2001) pp. 271-286, In Sonenshein et al, (ed.)
Bacillus subtilis and its relatives: from genes to cells, American
Society for Microbiology, Washington, D.C.). However there are
important differences. The traditional gene names are different in
E. coli and B. subtilis. First, the HMP biosynthesis enzyme ThiC,
thiamin-phosphate pyrophosphate ThiE, and hydroxyethylthiazole
kinase ThiM from E. coli have their counterparts named ThiA, ThiC,
and ThiK, respectively. Second, the known B. subtilis thiamin
biosynthetic genes are organized differently, as three clusters:
the thiA locus consisting of only the thiA gene, the thiB locus
consisting of genes thiOSFGD1, and the thiC locus consisting of
thiK and thiC genes. Third, at least one enzymatic step in thiazole
biosynthesis is different. The B. subtilis genome does not contain
a thiH ortholog. Instead thiO (yjbR in the thiB locus) is predicted
to encode an oxidase activity involved in thiazole biosynthesis.
This gene is not present in the E. coli genome, nor does it show
amino acid homology to ThiH. It is homologous to one of the genes
(thiO) associated with thi genes from Rhizobium etli. Fourth, two
orthologs of E. coli thiD have been found in B. subtilis, yjbV
(thiD1) and ywdB (thiD2), which could encode the biosynthetic and
salvage HMP kinases. Finally, the thiC locus contains an unknown
gene, ywbI that displays strong similarity to the lysR family of
transcriptional regulators.
[0008] The present invention provides a microorganism selected from
the group consisting of Bacillaceae, Lactobacillaceae,
Streptococcaceae, Corynebacteriaceae and Brevibacteriaceae, wherein
the microorganism contains a mutation that deregulates thiamin
production and causes thiamin products to be released from the
cell.
[0009] "Thiamin products" means thiamin, thiamin monophosphate
(TMP) and/or thiamin pyrophosphate (TPP), either alone or in any
combination.
[0010] It is understood that a microorganism as used for the
present invention means a "biologically pure culture" of said
microorganism, i.e., a microorganism that is separated from
constituents, cellular and otherwise, in which the microorganism is
normally associated with in nature.
[0011] The following materials have been deposited with the
American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,
Va. 20108 USA on May 12, 2003, and with the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1B,
D-38124 Braunschweig, Germany on Apr. 5, 2004, respectively, with
the corresponding accession numbers as indicated below, in
accordance with the stipulations of the Budapest Treaty: Bacillus
subtilis TH95 (ATCC PTA-5221), Bacillus subtilis TH101 (ATCC
PTA-5222), Bacillus subtilis TH115 (ATCC PTA-5223), Bacillus
subtilis TH116 (ATCC PTA-5224), Bacillus subtilis TH404 (DSM
16333), and Bacillus subtilis TH405 (DSM 16334).
[0012] "Mutation" is used interchangeably herein with modification
to mean a change in the wild-type DNA sequence of a microorganism,
such as a bacterium, that conveys a phenotypic change to the
microorganism compared to the wild type microorganism, e.g. that
allows an increase or decrease of thiamin or a thiamin product
either in the cell or out of the cell by any mechanism. The
mutation may be caused in a variety of ways including one or more
frame shifts, substitutions, insertions and/or deletions, including
nonsense mutations (amber (UAG), ocher (T/UAA) and opal (T/UGA)).
The deletion may be of a single nucleotide or more, including
deletion of the entire gene.
[0013] "Amino acid substitution" means a one-for-one amino acid
replacement. Such substitutions are conservative in nature when the
substituted amino acid has similar structural and/or chemical
properties. Examples of conservative replacements include
substitution of a leucine with an isoleucine or valine, an
aspartate with a glutamate, or a threonine with a serine.
Non-conservative substitutions within the scope of the present
invention include replacement of amino acids having aliphatic side
chains with those that have aromatic side chains, such as
replacement of leucine with phenylalanine.
[0014] Amino acid "insertions" or "deletions" mean changes to or
within an amino acid sequence. They typically fall in the range of
about 1 to 5 amino acids. The variation allowed in a particular
amino acid sequence may be experimentally determined by producing
the peptide synthetically or by systematically making insertions,
deletions, or substitutions of nucleotides in the sequence using
recombinant DNA techniques.
[0015] "Deregulates" or "deregulation" means an alteration or
modification of the expression of a gene that encodes an
enzyme/protein in a biosynthetic pathway, such that the level or
activity of said enzyme/protein is altered or modified, which
results in, but is not limited to, an increase in the production of
a thiamin product or the release of thiamin products out of the
cell, by e.g., secretion, efflux, and the like. Alterations or
modifications of gene expression can occur by changes in the DNA
sequence of the gene itself or regions outside of the gene,
including non-protein encoding DNA regions. "Deregulates" or
"deregulation" can also mean any perturbation of the intracellular
levels of a metabolite that alters the expression of a biosynthetic
gene of the cell, such that an increase in the production or the
release of thiamin products occurs.
[0016] In one embodiment, the mutation that deregulates thiamin
production in a microorganism as defined above is selected from the
group consisting of .DELTA.thiL, txl, tx26 and combinations
thereof. Such a mutation includes .DELTA.thiL combined with txl,
.DELTA.thiL combined with tx26, txl combined with tx26, and
.DELTA.thiL combined with both txl and tx26. Preferred is a
microorganism comprising all three mutations .DELTA.thiL, txl and
tx26.
[0017] In a preferred embodiment, the microorganism is selected
from the group consisting of Bacillus, Lactobacillus, Lactococcus,
Gorynebacterium, and Brevibacterium. More preferably, the
microorganism is selected from the genus Bacillus, most preferably
it is a B. subtilis cell.
[0018] In one embodiment, the microorganism containing the mutation
as defined above is B. subtilis TH95.
[0019] In one embodiment, the present invention provides a
microorganism as defined above containing a mutation which is
selected from the group consisting of .DELTA.thiL, txl, tx26 and
combinations thereof further comprising a DNA cassette containing
at least one copy of a polynucleotide sequence that encodes a thiA
gene product, which polynucleotide sequence is operatively
controlled by a strong constitutive promoter. A preferred
microorganism is B. subtilis TH116.
[0020] In a further embodiment, the present invention provides a
microorganism as defined above containing a mutation which is
selected from the group consisting of .DELTA.thiL, txl, tx26 and
combinations thereof further comprising a DNA cassette containing
at least one copy of a polynucleotide sequence that encodes gene
products from a thiKC operon, which polynucleotide sequence is
operatively controlled by a strong constitutive promoter. A
preferred microorganism is B. subtilis TH115.
[0021] In a further embodiment, the present invention provides a
microorganism as defined above containing a mutation which is
selected from the group consisting of .DELTA.thiL, txl, tx26 and
combinations thereof further comprising a DNA cassette containing a
polynucleotide sequence that encodes gene products of a
tenAl-thiOSGFD operon, which polynucleotide sequence is operatively
controlled by a strong constitutive promoter. A preferred
microorganism is B. subtilis TH404.
[0022] In a further embodiment, the present invention provides a
microorganism as defined above containing a mutation which is
selected from the group consisting of .DELTA.thiL, txl, tx26 and
combinations thereof further comprising (a) a DNA cassette
containing a polynucleotide sequence that encodes gene products of
a tenAl-thiOSGFD operon and (b) a DNA cassette containing at least
one copy of a polynucleotide sequence that encodes a thiA gene
product, which polynucleotide sequences are operatively controlled
by a strong constitutive promoter. A preferred microorganism is B.
subtilis TH405.
[0023] In the present invention, "DNA cassette" means a DNA coding
sequence or segment of DNA that codes for an expression product
that can be inserted into a vector at defined restriction sites.
The cassette restriction sites are designed to ensure insertion of
the cassette in the proper reading frame. Generally, foreign DNA is
inserted at one or more restriction sites of the vector DNA, and
then is carried by the vector into a host cell along with the
transmissible vector DNA. DNA fragments can also be inserted into
the vector DNA without restriction enzymes by the use of
topoisomerase bound at the ends of linearized vector DNA; this is
especially useful for direct cloning of PCR-prepared DNA fragments.
A segment or sequence of DNA having inserted or added DNA, such as
an expression vector, can also be called a "DNA construct". A
common type of vector is a "plasmid", which generally is a
self-contained molecule of double-stranded DNA, usually of
bacterial origin, that can readily accept additional (foreign) DNA
and which can be readily introduced into a suitable host cell. A
plasmid often contains coding DNA and promoter DNA and has one or
more restriction sites suitable for inserting foreign DNA. "Coding
DNA" is a DNA sequence that encodes a particular amino acid
sequence for a particular protein or enzyme. The DNA cassette may,
in addition to the specific nucleotide sequence, contain additional
transcription control elements including enhancers and promoters
for controlling transcription of the specific nucleotide sequence.
"Promoter DNA" is a DNA sequence which initiates, regulates, or
otherwise mediates or controls the expression of the coding DNA.
Promoter DNA and coding DNA may be from the same gene or from
different genes, and may be from the same or different organisms. A
large number of vectors, including plasmid and fungal vectors, have
been described for replication and/or expression in a variety of
eukaryotic and prokaryotic hosts. Non-limiting examples include
those specifically described in the Examples, as well as, pKK
plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc.,
Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego,
Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.),
pCR2.1Topo (Invitrogen, San Diego, Calif.), pXLTopo (Invitrogen,
San Diego, Calif.), and many appropriate host cells, using methods
disclosed or cited herein or otherwise known to those skilled in
the relevant art. Recombinant cloning vectors will often include
one or more replication systems for cloning or expression, one or
more markers for selection in the host, e.g. antibiotic resistance,
and one or more expression cassettes.
[0024] The DNA cassette may contain one or more copies of the
coding DNA such as for example from 1-50 copies of the sequence,
preferably from 1-25 copies, such as from 1-5,1-10, 1-15 and 1-20
copies. The sequences may be arranged in any order, including for
example, tandemly, i.e., in a head-to-tail arrangement.
[0025] "Operatively controlled" means that the transcription of the
coding DNA is controlled or mediated by e.g., a promoter or
transcription enhancer. Such promoters or transcription enhancers
may be adjacent to the coding DNA or may be located upstream or
downstream from the coding DNA.
[0026] A "strong constitutive promoter" is one which causes mRNAs
to be initiated at high frequency compared to a native host cell.
Strong constitutive promoters are well known and an appropriate one
may be selected according to the specific sequence to be controlled
in the host cell. Examples of such strong constitutive promoters
from Gram-positive microorganisms include, but are not limited to,
SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), and
amyE. Examples of promoters from Gram-negative microorganisms
include, but are not limited to, tac, tet, trp-tet, lpp, lac,
lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, .lamda.-P.sub.R,
and .lamda.-P.sub.L.
[0027] In another aspect, the present invention provides a
microorganism as defined above containing a mutation which is
selected from the group consisting of .DELTA.thiL, txl, tx26 and
combinations thereof further comprising (a) a first mutation that
deregulates expression of a purine operon of B. subtilis and (b) a
second mutation that blocks conversion of 5-aminoimidazole ribotide
(AIR) to carboxyaminoimidazole ribotide (CAIR). In a preferred
embodiment, the first mutation comprises a mutation within the
leader region of the pur operon and the second mutation comprises a
mutation within the pure gene encoding phosphoribosylaminoimidazole
carboxylase I. More preferably, the microorganism is B. subtilis
TH101.
[0028] The term "blocks conversion" means that the mutation
prevents the cellular machinery from converting AIR to CAIR. In the
present invention, the conversion of AIR to CAIR is preferably
completely blocked; but blockage of the conversion of greater than
75%, such as greater than 85-90% is also acceptable.
[0029] In one embodiment, the present invention is a method or a
process for producing thiamin products. This method comprises
culturing, in a suitable medium, a microorganism as defined above
containing a mutation which is selected from the group consisting
of .DELTA.thiL, txl, tx26 and combinations thereof that causes it
to overproduce thiamin products and to release them into the
medium. The thiamin products are then recovered from the
medium.
[0030] "Overproduce" means that the microorganism(s) of the present
invention or the microorganism(s) used in the methods of the
present invention is/are engineered to produce one or more thiamin
products in excess of what the native microorganism would produce
as measured by any of the methods set forth in the examples. A
substantial amount of such thiamin products are released into the
culture media, by e.g., secretion or efflux. As used herein, a
"substantial amount" means more than 75%, preferably more than 85%,
such as between 90-95% of the thiamin products produced by the cell
are released into the media.
[0031] "Recovering" when used in conjunction with "thiamin
products" means separating the thiamin products from the medium
and/or isolating the recovered thiamin products into pure or
semi-pure form. Any conventional method for recovering thiamin
products from, e.g., a fermentation broth may be used in the
present invention. Recovering can also mean isolation of thiamin
products by use of HPLC.
[0032] In one aspect, the method as defined above further comprises
culturing said microorganism in the presence of thiamin precursors.
Preferred precursors are selected from the group consisting of
4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP),
5-(2-hydroxyethyl)-4-methylthiazole (HET) and a combination
thereof.
[0033] If in the method for producing thiamin products as defined
above said microorganism further comprises a mutation that
deregulates the expression of a purine operon of B. subtilis as
described above, it is another aspect of the present invention to
provide a method wherein said microorganism is cultured in the
presence of a thiamin precursor and a purine source. In a preferred
embodiment, the thiamin precursor is HET and the purine source is
xanthine.
[0034] In another aspect, the method as defined above further
comprises culturing said microorganism in the presence of a
precursor of a HET pathway. A "precursor of a HET pathway" means a
carbon-containing compound that is utilized to make
5-(2-hydroxyethyl)-4-methylthiazole (HET). Such precursors are
preferably selected from the group consisting of glycine, cysteine,
isoleucine, threonine, and combinations thereof.
[0035] In another aspect, the method as defined above further
comprises culturing said microorganism in the presence of a
precursor of a HMP pathway or derivative of HMP. A "precursor of a
HMP pathway" means a carbon-containing compound that is utilized to
make 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Non-limiting
examples of such a precursor include 5-aminoimidazole ribotide
(AIR). A "derivative of HMP" means any chemically modified variant
of HMP that functions in the same manner as
4-amino-2-methyl-5-pyrimidinemethaneamine (Grewe Diamine).
[0036] Thus, the present invention is directed to a method for
producing thiamin products comprising (a) culturing, in a suitable
medium, a microorganism selected from the group consisting of
Bacillaceae, Lactobacillaceae, Streptococcaceae, Corynebacteriaceae
and Brevibacteriaceae, the microorganism containing a mutation that
causes it to overproduce thiamin products into the medium; and (b)
recovering the thiamin products.
[0037] In one embodiment, the invention is directed to a method as
defined above wherein the microorganism contains a mutation, said
mutation comprising .DELTA.thiL, txl, and tx26.
[0038] In one embodiment, the invention is directed to a method as
defined above wherein the microorganism further comprises a DNA
cassette containing at least one copy of a polynucleotide sequence
that encodes a thiA gene product, which polynucleotide sequence is
operatively controlled by a strong constitutive promoter.
[0039] In one embodiment, the invention is directed to a method as
defined above wherein the microorganism further comprises a
mutation that deregulates expression of a purine operon of B.
subtilis and a mutation that blocks conversion of 5-aminoimidazole
ribotide (AIR) to carboxyaminoimidazole ribotide (CAIR).
[0040] In one embodiment, the invention is directed to a method as
defined above wherein the microorganism further comprises a DNA
cassette containing at least one copy of a polynucleotide sequence
that encodes gene products from a thiKC operon, which
polynucleotide sequence is operatively controlled by a strong
constitutive promoter.
[0041] In one embodiment, the invention is directed to a method as
defined above wherein the microorganism further comprises a DNA
cassette containing at least one copy of a polynucleotide sequence
that encodes gene products of a tenAl-thiOSGFD operon, which
polynucleotide sequence is operatively controlled by a strong
constitutive promoter.
[0042] In one embodiment, the invention is directed to a method as
defined above wherein the microorganism further comprises (a) a DNA
cassette containing at least one copy of a polynucleotide sequence
that encodes gene products of a tenAl-thiOSGFD operon and (b) a DNA
cassette containing at least one copy of a polynucleotide sequence
that encodes a thiA gene product, which polynucleotide sequence is
operatively controlled by a strong constitutive promoter.
[0043] In another embodiment, the invention is an isolated
polynucleotide sequence comprising a txl mutation. Such mutation is
useful for the construction of a recombinant microorganism wherein
the production of thiamin is increased, e.g. a microorganism
selected from the group consisting of Bacillaceae,
Lactobacillaceae, Streptococcaceae, Corynebacteriaceae and
Brevibacteriaceae, containing a mutation that deregulates thiamin
production and causes thiamin products to be released into the
culture medium. Preferred is a mutation which results in a leucine
to phenylalanine substitution at amino acid residue 116 (see SEQ ID
NO: 31 for a copy of the amino acid sequence having the Leu to Phe
substitution on position 116 in comparison to the wild type YloS
sequence ID NO: 32).
[0044] As used herein, an "isolated" polynucleotide (e.g., an RNA,
DNA or a mixed polymer) or polypeptide means substantially
separated from components that accompany it in its natural state.
In the case of polynucleotides, "isolated" means separated from
other cellular components which naturally accompany a native
sequence, e.g., ribosomes, polymerases, many other genome sequences
and proteins. The term embraces a polynucleotide that has been
removed from its naturally occurring environment, and includes
recombinant or cloned DNA isolates and chemically synthesized
analogs or analogs biologically synthesized by heterologous
systems. With respect to polypeptides, the term "isolated" means a
protein or a polypeptide that has been separated from components
that accompany it in its natural state. A monomeric protein is
isolated when at least about 60 to 75% of a sample exhibits a
single polypeptide sequence. An isolated protein will typically
comprise about 60 to 90% w/w of a protein sample, more usually
about 95%, and preferably will be over about 99% pure. Protein
purity or homogeneity may be indicated by a number of means well
known in the art, such as polyacrylamide gel electrophoresis of a
protein sample, followed by visualizing a single polypeptide band
upon staining the gel. For certain purposes, using HPLC or other
means well known in the art may provide higher resolution for
purification.
[0045] In one embodiment, the isolated polynucleotide sequence as
defined above is SEQ ID NO: 30 or a polynucleotide sequence that
hybridizes to SEQ ID NO: 30 under stringent conditions and, when
present in a microorganism, causes deregulation of thiamin
production.
[0046] Nucleic acids which hybridize under "stringent conditions"
to the polynucleotide sequences identified herein and that retain
the same function, i.e., when introduced into an appropriate cell
cause a deregulation of thiamin production, are within the scope of
the present invention. "Stringent conditions" are known in the art;
see for example Maniatis et al., Molecular Cloning: A Laboratory
Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology,
ed. Ausubel, et al., both of which are hereby incorporated by
reference. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, "Overview of principles of hybridization and the strategy
of nucleic acid assays" (1993). Generally, stringent conditions are
selected to be about 5-10.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
pH. The Tm is the temperature (under defined ionic strength, pH and
nucleic acid concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g. greater than 50 nucleotides). Stringent conditions may
also be achieved with the addition of destabilizing agents such as
formamide. For the purposes of this disclosure, suitable "stringent
conditions" for such hybridizations are those which include
hybridization in a buffer of 40% formamide, 1M NaCl, 1% sodium
dodecyl sulfate (SDS) at 37.degree. C., and at least one wash in
0.2.times.SSC at a temperature of at least about 50.degree. C.,
usually about 55.degree. C. to about 60.degree. C., for 20 minutes,
or equivalent conditions. A positive hybridization is at least
twice above the level of background. Those of ordinary skill will
readily recognize that alternative hybridization and wash
conditions can be utilized to provide conditions of similar
stringency.
[0047] The phrase "nucleic acid sequence" means a single or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. It includes chromosomal DNA,
self-replicating plasmids, infectious polymers of DNA or RNA and
DNA or RNA that performs a primarily structural role. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); Cassol et al., 1992; Rossolini et al.,
Mol. Cell. Probes 8:91-98 (1994)).
[0048] In another embodiment, the invention is two unlinked
mutations: an isolated polynucleotide sequence comprising a first
mutation tx26-1 and a second mutation tx26-2 wherein the presence
of both mutations in a thiamin-producing microorganism causes a
deregulation of thiamin production. The first mutation, tx26-1,
exhibits a 70% linkage to .DELTA.yufR::Tn917 (BGSC# 1A642) and the
second mutation, tx26-2, exhibits a 59% linkage to
.OMEGA.motA::Tn917 (BGSC# 1A631). Thus, the present invention is
directed to an isolated polynucleotide sequence comprising a first
mutation with 70% linkage to .DELTA.yufR::Tn917 (tx26-1) and a
second mutation with 59% linkage to .OMEGA.motA::Tn917 (tx26-2)
wherein the presence of both of the mutations in a
thiamin-producing microorganism causes a deregulation of thiamin
production.
[0049] Preferably, the tx26-1 mutation is encoded by a
polynucleotide sequence which is SEQ ID NO: 33 or a polynucleotide
sequence that hybridizes to SEQ ID NO: 33 under stringent
conditions and, when present in a microorganism in combination with
a tx26-2 mutation, causes a deregulation of thiamin production.
[0050] Preferably, the tx26-2 mutation is encoded by a
polynucleotide sequence which is SEQ ID NO: 36 or a polynucleotide
sequence that hybridizes to SEQ ID NO: 36 under stringent
conditions and, when present in a microorganism in combination with
a tx26-1 mutation, causes a deregulation of thiamin production.
[0051] Furthermore, a DNA cassette comprising one or more
polynucleotides as defined above, i.e., (a) an isolated
polynucleotide sequence comprising a first mutation tx26-1 and a
second mutation tx26-2 or (b) an isolated polynucleotide sequence
comprising a txl mutation, as well as a microorganism containing
such DNA cassette is provided by the present invention.
[0052] A further embodiment is a method for detecting a pathogenic
microorganism in a clinical sample from a patient. This method
comprises determining whether a Gram-positive (Gram.sup.+)
microorganism is present in the sample, determining whether the
microorganism contains a yloS ortholog, and determining whether the
microorganism contains a thiL ortholog, wherein the presence of a
yloS ortholog and the absence of a thiL ortholog in a Gram.sup.+
microorganism indicates that the microorganism is pathogenic.
[0053] A "clinical sample" means any assayable specimen taken from
a patient, which is a mammal, preferably a human or a feed animal.
An assayable specimen may be selected from blood, urine, fecal,
sputum, tissue or other biological sources from which
microorganisms, if present, may be identified and characterized as
disclosed in the Examples.
[0054] The microorganisms that are detected in a clinical sample
from a patient as defined above are preferably selected from the
group consisting of Listeria, Staphylococcus, Clostridium,
Enterococcus, and Streptococcus, most preferably from Listeria
monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis,
Clostridium tetani, Clostridium perfringens, Enterococcus sp.,
Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus
pneumoniae.
[0055] The YloS protein is a valuable target for identifying
bacteriocidal compounds because many Gram.sup.+ bacteria that
contain only a yloS ortholog and not a thiL ortholog are known
pathogens. Accordingly, the present invention also provides a
screening assay (or method) for identifying modulators, i.e.,
candidate or test compounds or agents (e.g., peptides,
peptidomimetics, small molecules or other drugs) that bind to YloS,
or have a stimulatory or inhibitory effect on, for example, yloS
expression or YloS activity.
[0056] In one embodiment, an assay is provided for identifying an
antibiotic comprising (a) contacting an assay composition
comprising a YloS protein with a test compound and (b) determining
whether the test compound inhibits YloS protein activity, wherein
the compound is identified as an antibiotic based on the compound's
ability to inhibit the activity of the YloS protein activity.
Preferably, the assay comprises a purified YloS protein, a
partially purified YloS protein, a crude cell extract from a cell
producing YloS protein, or the YloS protein is encoded by a
polynucleotide derived from a pathogenic microorganism selected
from the group consisting of Listeria, Staphylococcus, Clostridium,
Enterococcus, and Streptococcus. Such pathogenic microorganisms
include, but are not limited to, Listeria monocytogenes,
Staphylococcus aureus, Staphylococcus epidermidis, Clostridium
tetani, Clostridium perfringens, Enterococcus sp., Streptococcus
agalactiae, Streptococcus pyogenes, and Streptococcus
pneumoniae.
[0057] An "assay composition" in reference to the assays for
identifying an antibiotic means the components in combination that
are required to conduct such an assay. Such an assay composition
requires at a minimum the YloS protein or biologically active
portion thereof and the test compound, i.e., the peptide,
peptidomimetic, small molecule or other drug to be tested.
[0058] As used herein, "YloS activity" means any detectable or
measurable activity of the YloS protein, i.e., the protein encoded
by the yloS gene. In the present invention, YloS activity is at
least one of the following: (1) modulation of at least one step in
the YloS biosynthetic pathway; (2) promotion of YloS biosynthesis;
or (3) complementation of a YloS mutant. In reference to the assays
for identifying an antibiotic, a test compound. "inhibits YloS
protein activity" if it causes a decrease in YloS protein
translation, yloS transcription or loss of YloS activity.
[0059] The test compounds of the present invention may be obtained
using any of the numerous approaches in chemical compound library
methods known in the art, including: natural compound libraries,
biological libraries, spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[0060] Examples of methods for the synthesis of molecular libraries
may be found in the art, for example in: De Witt et al. (1993) PNAS
90:6909; Erb et al. (1994) PNAS 91:11422; Zuckermann et al. (1994)
J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell
et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.
(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al.
(1994) J. Med. Chem. 37:1233. Libraries of compounds may be
presented in solution, on beads, on chips, in bacteria, in spores
(U.S. Pat. No. 5,223,409), on plasmids or on phage.
[0061] In one embodiment, the assay is a microorganism-based assay
in which a recombinant microorganism that expresses a YloS protein
or biologically active portion thereof is contacted with a test
compound and the ability of the test compound to modulate YloS
activity is determined. Determining the ability of a test compound
to modulate YloS activity may be accomplished by monitoring, for
example, growth, intracellular YloS concentrations or secreted YloS
concentrations (as compounds that inhibit YloS will result in a
buildup of YloS protein in the test microorganism). YloS substrate
may be labeled with a radioisotope, enzymatic label or other
soluble or insoluble signal generating moiety such that modulation
of YloS activity may be determined by, e.g. detecting a conversion
of labeled substrate to intermediate or product. For example, YloS
substrates may be labeled with .sup.32P, .sup.14C or .sup.3H,
either directly or indirectly, and the radioisotope detected by
direct counting of radioemission, by scintillation counting.
Alternatively, the YloS substrates may be labeled directly or
indirectly with a soluble or insoluble signal generating moiety and
the signal detected by a calorimetric, enzymatic or fluorometric
assay. Determining the ability of a compound to modulate YloS
activity may alternatively be determined by detecting the induction
of a reporter gene (comprising a yloS-responsive regulatory element
operatively linked to a nucleic acid encoding a detectable marker,
e.g., luciferase) or detecting a CoA-regulated cellular
response.
[0062] In another embodiment of the invention, the screening assay
of the present invention is a cell-free assay in which the YloS
protein or a biologically active portion thereof is contacted with
a test compound in vitro and the ability of the test compound to
bind to or modulate the activity of the YloS protein or
biologically active portion thereof is determined. In one such
embodiment, the assay includes contacting the. YloS protein or
biologically active portion thereof with known substrates to form
an assay mixture, contacting the assay mixture with a test
compound, and determining the ability of the test compound to
modulate enzymatic activity of the YloS protein on its substrate.
In one embodiment, the known substrate is the YloS protein. In
another embodiment, the known substrate is a YloS analog. The
phrase "YloS analog" means compounds similar in structure to the
YloS protein that functions in the same or a similar manner as
YloS. Exemplary analogs include labeled YloS protein and/or other
detectable YloS protein derivatives. The term YloS analog also
includes compounds closely related to or derived from the YloS
protein, for example, structurally related compounds capable of
acting as YloS substrate.
[0063] Screening assays may be accomplished in any vessel suitable
for containing the microorganisms, proteins, and/or reactants.
Examples of such vessels include microtiter plates, test tubes and
micro-centrifuge tubes. In more than one embodiment of the assay
methods of the present invention, it may be desirable to immobilize
either the YloS protein, YloS substrate, substrate analogs or a
recombinant microorganism expressing the YloS protein to facilitate
separation of products, ligands, and/or substrates, as well as to
accommodate automation of the assay. For example,
glutathione-S-transferase/YloS fusion proteins may be adsorbed onto
glutathione sepharose beads (Sigma Chemical Co., St. Louis, Mich.)
or glutathione derivatized microtiter plates. Other techniques for
immobilizing proteins on matrices (e.g., biotin-conjugation and
streptavidin immobilization or antibody conjugation) may also be
used in the screening assays of the invention.
[0064] This invention also includes novel agents identified by the
above-described screening assays. Accordingly, it is within the
scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, a
YloS modulating agent identified as described herein (e.g., an
anti-bactericidal compound) may be used in an infectious animal
model to determine the efficacy, toxicity or side effects of
treatment with such an agent and/or to treat a specific disease
state caused or induced by a pathogenic microorganism. In a
preferred embodiment, said novel agent is an antibiotic.
[0065] YloS modulators may further be designed based on the crystal
structure of any one of the YloS proteins of the present invention.
In particular, based, at least in part, on the discovery of YloS in
many Gram.sup.+ pathogenic bacteria, one may produce significant
quantities of the YloS protein, for example using the recombinant
methodologies as described herein, purify and crystallize the
protein, subject the protein to X-ray crystallographic procedures
and, based on the determined crystal structure, design modulators
(e.g., active site modulators, for example, competitor molecules,
active site inhibitors, and the like), and test the designed
modulators according to any one of the assays described herein.
[0066] Some of the most important results of the present invention
are summarized in the following figures:
[0067] FIG. 1 depicts the structure of the P.sub.26 thiA expression
cassette contained in plasmid pTH43 with a
chloramphenicol-resistance gene (A) and plasmid pTH47 with a
tetracycline resistance gene (B).
[0068] FIG. 2 depicts the structure of the P.sub.26 thiKC
expression cassette contained in plasmid pTH48 with a tetracycline
resistance gene.
[0069] FIG. 3 describes the two-step procedure used to construct
strain TH404, that overexpresses the B. subtilis thiB operon. In
the first step, a thiamin-auxotroph strain was build by
substitution of the thiB promoter region with a chloramphenicol
acetyltransferase (cat) cassette. Restoration of prototrophy was
then used to select for strains that have integrated the
bacteriophage strong constitutive P26 promoter in front of the thiB
operon.
[0070] The following examples are illustrative only and are not
intended to limit the scope of the invention in anyway.
EXAMPLE 1
General Methodology
Strains
[0071] Bacillus subtilis strains of the present invention are
derived from strain PY79 (prototroph SP.beta..sup.c; Cat. # 1A747,
Bacillus Genetic Stock Center (BGSC), The Ohio State University,
Columbus, Ohio 43210 USA) and 1012 (leuA8 metB5; Saito et al.
(1979) Mol. Gen. Genet. 170:117-122). The neomycin-resistance gene
(neo) cassette and tetracycline-resistance gene (tet) cassette were
obtained from plasmid pBEST501 (Cat. # ECE47, BGSC) and pDG1514
(Cat # ECE100, BGSC), respectively.
Media
[0072] Standard minimal medium (MM) for B. subtilis contains
1.times.Spizizen salts, 0.04% sodium glutamate, and 0.5% glucose.
Standard solid complete medium is Tryptone Blood Agar Broth (TBAB,
Difco). Standard liquid complete medium is Veal Infusion-Yeast
Extract broth (VY). For testing thiamin production in liquid test
tube cultures, a thiamin-free medium is used (Difco). For fed-batch
fermentations, VF medium is used. The compositions of these media
are described below or are standard formula described previously
(Harwood and Archibald (1990) pp. 1-26 and 545-552 (Appendix 1), In
Cutting and Harwood (ed.) Molecular biological methods for
Bacillus. John Wiley and Sons, New York).
[0073] TBAB medium: 33 g Difco Tryptone Blood Agar Broth, qsp 1 L
water. Autoclave.
[0074] VY medium: 25 g Difco Veal Infusion Broth, 5 g Difco Yeast
Extract, qsp 1 L water. Autoclave.
[0075] Minimum medium (MM): 100 ml 10.times.Spizizen salts; 10 ml
50% glucose; 1 ml 40% sodium glutamate, qsp 1 L water.
[0076] 10.times.Spizizen salts: 140 g K.sub.2HPO.sub.4; 20 g
(NH.sub.4).sub.2SO.sub.4; 60 g KH.sub.2PO.sub.4; 10 g
Na.sub.3(citrate).2H.sub.2O; MgSO.sub.40.7H.sub.2O; qsp 1 L
water.
[0077] Thiamin assay medium: 85 g Difco thiamin assay medium, qsp 1
L water. Autoclave (Difco Manual (1998) pp. 499-501, Difco
Laboratories, Maryland, USA).
[0078] Trace elements solution: 1.4 g MnSO.sub.4.H.sub.2O; 0.4 g
CoCl.sub.2.6H.sub.2O; 0.15 g
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O; 0.1 g
AlCl.sub.3.6H.sub.2O; 0.075 g CuCl.sub.2.2H.sub.2O; qsp 200 ml
water. Filter-sterilize.
[0079] Fe-solution: 0.21 g FeSO.sub.4.7H.sub.2O; qsp 10 ml water.
Filter-sterilize.
[0080] CaCl.sub.2-solution: 15.6 g CaCl.sub.2.2H.sub.2O; qsp 500 ml
water. Filter-sterilize.
[0081] Mg/Zn-solution: 100 g MgSO.sub.4.7H.sub.2O; 0.4 g
ZnSO.sub.4.7H.sub.2O; qsp 200 ml water. Filter-sterilize.
[0082] VF fermentation medium: 0.75 g sodium glutamate; 4.71 g
KH.sub.2PO.sub.4; 4.71 g K.sub.2HPO.sub.4; 8.23 g
Na.sub.2HPO.sub.4.12H.sub.2O; 0.23 g NH.sub.4Cl; 1.41 g
(NH.sub.4).sub.2SO.sub.4; 11.77 g Yeast extract (Merck); 0.2 ml
Basildon antifoam; qsp 1 L water. Sterilize in place. [0083] Added
separately to the fermentor: glucose.H.sub.2O to 27.3 g/L final
concentration. [0084] Added separately to the fermentor (final
concentrations): 2 ml/L trace elements solution; 2 ml/L
CaCl.sub.2-solution; 2 ml/L Mg/Zn-solution; 2 ml/L Fe-solution.
[0085] Modifications of the batch for feeding studies will be
presented specifically in the following examples. Glucose was fed
as needed. Feed solutions can contain minerals, defined or food
nutrients, as reported in the following compositions:
[0086] Fermentation feeding solution for fed-batch process with NB
(Nutrient Broth): Final concentrations (after autoclavation): 660
g/L glucose H.sub.2O; 2 g/L MgSO.sub.4.7H.sub.2O; 14.6 mg/L
MnSO.sub.4.H.sub.2O; 4 mg/L ZnSO.sub.4.H.sub.2O; 47.8 g/L Nutrient
Broth (Difco, autoclave separately in 1 g/ml solution).
[0087] Fermentation feeding solution for fed-batch process with
HMP: Final concentrations (after autoclavation): 660 g/L
glucose.H.sub.2O; 2 g/L MgSO.sub.4.7H.sub.2O; 14.6 mg/L
MnSO.sub.4.H.sub.2O; 4 mg/L ZnSO.sub.4.H.sub.2O. Add HMP to 0.54
g/L or 2.7 g/L (dissolve HMP in water/HCl conc.;
filter-sterilize).
[0088] Fermentation feeding solution for fed-batch process with
HET: Final concentrations (after autoclavation): 660 g/L
glucose.H.sub.2O; 2 g/L MgSO.sub.4.7H.sub.2O; 14.6 mg/L
MnSO.sub.4.H.sub.2O; 4 mg/L ZnSO.sub.4.H.sub.2O. Add HET to 0.54
g/L or 2.7 g/L (dissolve HET in water; filter-sterilize).
[0089] Fermentation feeding solution for fed-batch process with HMP
and HET: Final concentrations (after autoclavation): 660 g/L
glucose.H.sub.2O; 2 g/L MgSO.sub.4.7H.sub.2O; 14.6 mg/L
MnSO.sub.4.H.sub.2O; 4 mg/L ZnSO.sub.4.H.sub.2O. Add HMP to 0.54
g/L or 2.7 g/L (dissolve HMP in water/HCl conc.; filter-sterilize).
Add HET to 0.54 g/L or 2.7 g/L (dissolve HET in water;
filter-sterilize).
Thiamin Assays
[0090] Biological assays: Total thiamin compounds were assayed
using indicators derived from Salmonella typhimurium using known
methods (Difco Manual (1998) pp. 499-501, Difco Laboratories,
Maryland, USA). Strain DM456 (thiD906::MudJ) responds to thiamin,
TMP and TPP in minimal medium, whereas strain DM1864
(thiL934::Tn10d) responds to only TPP (Webb and Downs (1997) J.
Biol. Chem. 272:15702-15707; Peterson and Downs (1997) J.
Bacteriol. 179:4894-4900). The response of DM456 to known amounts
of thiamin, TMP, and TPP was similar, ranging from 0.0256 to 100
.mu.g/liter. In addition, DM456 was found to be more sensitive to
TPP than DM1854. To assay B. subtilis cultures, supernatants were
filter-sterilized before preparation of dilutions. Intracellular
thiamin levels were measured from dilutions of filter-sterilized
cellular extracts that were obtained by French press-breaking of
the cells and centrifugation at 10,000 g for 10 min. Indicator
strains were grown overnight at 37.degree. C. in thiamin assay
medium (TAM). Turbidity readings were made at 600 nm and compared
to a range of standard solutions.
[0091] HPLC/Thiochrome: Individual thiamin compounds, thiamin, TMP,
and TPP were measured using a modified thiochrome-HPLC assay
procedure described previously (Chie et al. (1999) Biochemistry
38:6460-6470). Briefly, 100 .mu.l of culture supernatant or
intracellular extracts are added to 200 .mu.l of 4M potassium
acetate. The sample is then oxidized by the addition of 100 .mu.l
fresh 3.8 mM potassium ferricyanide in 7 M NaOH. The mixture is
vigorously mixed and then quenched by addition of 100 .mu.l fresh
0.06% H.sub.2O.sub.2 in saturated KH.sub.2PO.sub.4. Samples are
transferred to HPLC vials and injected onto a Supelcosil LC-18-T
column (15 cm.times.4.6 mm, 3 .mu.m) (Supelco-Ref. No 58970-U).
Elution is made by a 10%-35% methanol (H.sub.2O 50%-25%) gradient
in the presence of 40% 0.1 M K.sub.2HPO.sub.4 (pH 6.6) and 4 mM
tetrabutyl ammonium hydrogen sulfate. Fluorescence is measured at
444 nm after excitation at 365 nm. The chronological order of
elution from the column is thiamin, TMP, and TPP. This procedure
was utilized to monitor both internal and external thiamin
production during fermentation.
[0092] HPLC/DAD: To directly measure thiamin and the intermediates
HMP and HET in the fermentation broth, chromatography of samples
was performed on a Phenomenex LUNA C18 column, using an Agilent
1100 HPLC system equipped with a thermostatted autosampler and a
diode array detector (DAD). The column dimensions are 150.times.4.6
mm, particle size 5 micron. The column temperature was kept
constant at 20.degree. C. The mobile phase is a mixture of 0.4 g
pentane sulfonate in water, pH 2 (A) and methanol (B). Gradient
elution is applied, ranging from 2% A (3 min) to 20% A in 20
minutes. The flow rate is 1 ml/min. The detection method is UV
absorption at 254 nm. The selectivity of the method was verified by
injecting 10 .mu.l standard solutions of the relevant reference
compounds, thiamin, HMP, and HET, each at 100 .mu.g/ml. The target
compounds were completely separated without special sample
preparation.
Molecular and Genetic Techniques
[0093] Standard genetic and molecular biology techniques are
generally know in the art and have been previously described
(Maniatis et al. (1982) Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller
(1972) Experiments in molecular genetics. Cold Spring Harbor
Laboratory, Cold Spring Harbor). DNA transformation, PBS1
generalized transduction, and other standard B. subtilis genetic
techniques are also generally know in the art and have been
described previously (Cutting and Horn (1990) pp. 27-74, In Cutting
and Harwood (ed.) Molecular biological methods for Bacillus. John
Wiley and Sons, New York).
Fermentations
[0094] Thiamin producing strains were grown in stirred tank
fermentors, for example, in BIOPLO 4500 New Brunswick 20 liter
vessels with 6-12 liter working volume. Computer control and data
collection was done by NBS Biocommand 32 commercial software (New
Brunswick Scientific Co., Inc., Edison, N.J., USA).
[0095] The inoculum size was usually 5% of the initial media volume
in the vessel. A pH of 6.8 was kept constantly in the reactor by
the automatic addition of ammonium hydroxide solution (28% in
water). The fermentation temperature was 39.degree. C. and a
constant airflow of 6 liter/min was provided. Antifoam (Basildon)
was added manually as needed and a constant pressure of 2 psi was
kept in the vessel. A minimum concentration of 15%-dissolved oxygen
(pO.sub.2) was achieved by automatic cascading of the stirrer. The
minimum stirrer speed was set to 400 rpm.
[0096] Fermentations can be batch processes but are preferably,
carbohydrate-limited, fed-batch processes. Therefore a defined feed
solution (s. above) was provided to the reactor after consumption
of the initial glucose which was usually the case after 6-8 hours
process time. At that time the pressure was increased to 8 psi and
the addition of the feed solution was initiated at a rate of 70 g
h.sup.-1 increasing linearly to 102.5 g h.sup.-1 in a period of 8
hours and then kept constant at 102.5 g h.sup.-1.
EXAMPLE 2
Culturing Mutant Microorganisms Producing Thiamin and Thiamin
Compounds de novo
[0097] This example describes the isolation of thiamin biosynthesis
and deregulation mutations .DELTA.thiL, txl, tx26 mutations and
their combination to produce a B. subtilis strain that overproduces
thiamin compounds.
[0098] Intra- and extracellular levels of thiamin products from
wild type and engineered B. subtilis strains were determined from
cells grown in 30 ml minimal medium shake flasks cultures at
37.degree. C. for 24 hours. As a positive control, thiamin
deregulated E. coli PT-R1 mutant was also tested. Bioassay results
indicated that thiamin products were readily detected from extracts
of sonicated cells, but little or none were detected from the
culture medium (Table 1). The intracellular level of thiamin
products in logarithmic or stationary phase wild-type B. subtilis
was calculated to be approximately 100-200 .mu.g/L. As reported for
E. coli, intracellular thiamin products in B. subtilis are likely
to be in the form of TPP. Intracellular levels of thiamin products
were significantly higher in the thiamin deregulated E. coli PT-R1
strain, reaching approximately 1.6 mg/L in stationary phase cells.
TABLE-US-00001 TABLE 1 Thiamin production in various E. coli and B.
subtilis strains. Extracellular.sup.a Intracellular.sup.b Predicted
Strain (.mu.g/L) (.mu.g/L) Extracellular.sup.c E. coli K-12 <0.1
80 3 E. coli PT-R1 6 1600-2800 50-90 B. subtilis prototroph <0.1
100-200 3-6 .sup.aThiamin concentration in minimal medium (30 ml)
after 24 hours growth at 37.degree. C. .sup.bThiamin concentration
of 1 ml supernatant of sonicated cells collected from a 30 ml
minimal medium culture after 24 hours growth at 37.degree. C.
.sup.cAssumes all intracellular thiamin products are excreted into
the culture medium. Calculation: (Intracellular thiamin
concentration (.mu.g/L) .times. 0.001 L) .times. 1000 ml/L/30
ml.
[0099] Comparison of the E. coli ThiL protein sequence to the
protein database of Subtilist detected significant similarity to
only one protein sequence: YdiA (P(N)=8.1e.sup.-15). The gene
encoding this protein, ydiA, is 975 base pairs in length and is the
first gene of a five-gene operon located at 55.degree. on the B.
subtilis chromosome. Non-polar insertional vectors pMUTIN2 and 4,
which contain an IPTG-inducible P.sub.spac promoter that controls
transcription of genes downstream from the site of insertion, were
used to generate thiL disruption mutants (Vagner et al. (1998)
Microbiology 144:3097-3104). Using two oligonucleotide primers,
BsuydiA1 (SEQ ID NO: 7) and BsuydiA2 (SEQ ID NO: 8), corresponding
to the YdiA sequence between nucleotides 264 and 612, a 348 bp DNA
fragment was prepared by standard PCR methods. This fragment was
cloned between the HindIII and BamHI sites of pMUTIN2 vector,
generating the E. coli plasmid pTH1. This plasmid was then inserted
into the ydiA (thiL) gene of B. subtilis PY79 by DNA transformation
selecting for colonies resistant to 5 .mu.g/ml erythromycin. One
Erm.sup.r colony was recovered and named TH5 (.OMEGA.thiL::pMUTIN).
By comparing bacterial growth on TBAB medium with erythromycin in
the presence or absence of IPTG, it was determined that expression
of one or more of the genes downstream of ydiA was required for
cell growth. Similar results were also obtained when additional
ydiA (thiL) disruptions were generated by inserting a
chloramphenicol acetyltransferase cassette containing (cat.sub.2)
or lacking (cat.sub.4) the endogenous rho-independent transcription
termination site between nucleotide 267 and 272 of ydiA (thiL). PCR
primer pairs cat#1 (SEQ ID NO: 9)-cat#2 (SEQ ID NO: 10) and
cat#1-cat#4 (SEQ ID NO: 11) were used to generate DNA cassettes
cat.sub.2 or cat.sub.4 gene, respectively, which were ligated to
ydiA (thiL) PCR DNA fragments generated using primers
ydiA/atp/for/bam (SEQ ID NO: 12)-ydiA/atp/rev/sma (SEQ ID NO: 13)
and ydiA/ctp/for/sma/2 (SEQ ID NO: 14)-ydiA/ctp/rev/ecorI/2 (SEQ ID
NO: 15). The .DELTA.thiL::cat cassettes were then inserted directly
into the chromosomal ydiA (thiL) gene of strain PY79 by DNA
transformation selecting for colonies resistant to 5 .mu.g/ml
chloramphenicol. Only Cm.sup.r colonies containing
.DELTA.thiL::cat.sub.4 (TH12) grew normally on TBAB medium;
Cm.sup.r colonies containing .DELTA.thiL::cat.sub.2 grew as tiny,
pinpoint colonies. Strains TH11 and TH12 containing
.DELTA.thiL::cat.sub.2 and .DELTA.ydiA::cat.sub.4 were saved,
respectively. Surprisingly, strains TH5 and TH12 were not thiamin
or TPP auxotrophs. Instead both strains were thiamin bradytroph: on
minimal medium the colony size of TH5 and TH12 was half the
diameter of PY79 control colonies. Since thiL null mutants of
Salmonella and E. coli are strict thiamin auxotrophs, B. subtilis
appeared to contain a second kinase activity or an alternate route
that could convert TMP to TPP. Interestingly, ydiA (thiL) mutants
were able to cross-feed B. subtilis thiF or thiG mutants (strains
called respectively TH3 and TH4) on minimal medium after one day of
incubation at 37.degree. C., whereas PY79 took three or more days.
This suggested that ydiA mutants are partially deregulated for
thiamin biosynthesis and release more diffusible thiamin products
than the wild-type strain. Bioassay results (Table 2) showed that
the total intracellular thiamin production (thiamin+TMP+TPP) level
was slightly higher (2- to 3-fold) in TH5 and TH12 than PY79.
Slightly higher total thiamin levels were also detected in the
culture medium relative to PY79, but well below the intracellular
levels. Interestingly, the increase in thiamin production was not
sufficient to provide resistance to the thiamin analog
pyrithiamine. TABLE-US-00002 TABLE 2 Thiamin production of B.
subtilis thiL insertional mutants. Extracellular.sup.a
Intracellular.sup.b Strain thiL mutation (.mu.g/L) (.mu.g/L) B.
subtilis PY79 -- <0.1 190 B. subtilis TH5 .OMEGA.thiL::pMUTIN2
0.7 550 B. subtilis TH12 .DELTA.thiL::cat.sub.4 0.9 530
.sup.aThiamin concentration in minimal medium (30 ml) after 24
hours growth at 37.degree. C. .sup.bThiamin concentration of 1 ml
supernatant of sonicated cells collected from a 30 ml minimal
medium culture after 24 hours growth at 37.degree. C.
[0100] The strategy to isolate thiamin deregulated mutants of B.
subtilis was to mutagenize bacteria that contained a thiA-lacZ
fusion and then screen for colonies on XGAL-containing medium that
were Lac.sup.+ (i.e. blue colonies) in the presence of TPP or
thiamin. A 732 bp-long DNA fragment containing 417 bp of the 5'
promoter region of thiA was prepared by PCR using standard methods
and cloned unidirectionally in front of the promoterless lacZ gene
of the pDG1728 vector (Guerout-Fleury et al. (1996) Gene
180:57-61)) resulting in plasmid pTH12. This vector is designed to
introduce ectopic transcriptional lacZ fusions into the
non-essential amyE locus of B. subtilis. Plasmid pTH12 was
linearized by restriction enzyme digestion and transformed into B.
subtilis PY79, selecting for colonies that were resistant to 100
.mu.g/ml spectinomycin. One resulting colony, designated TH21
(.OMEGA.amyE::thiA-lacZ), showed unambiguous thiamin regulation
when tested under different nutrient growth conditions. When grown
to early logarithmic phase (OD600=0.8-0.9) in shake-flask cultures
containing 1 .mu.M thiamin, the expression of thiA-lacZ was
repressed approximately 80-fold compared to cells grown in minimal
medium without thiamin. HMP (1 .mu.M) also repressed expression of
the fusion, but to a lesser extend (6- to 7-fold). Both thiazole
and adenosine (1 .mu.M each) showed repressing activity. In a time
course experiment, expression of thiA-lacZ was highest (200 Miller
Units) when cells were at early logarithmic phase (OD600=0.8-0.9).
Expression gradually decreased (to 50 Miller Units) when cells
enter stationary phase (OD600.gtoreq.1).
[0101] The regulation of thiA-lacZ fusion was also assessed in
several mutants, In a sporulation-deficient mutant strain
(.DELTA.spo0A::erm), expression of the fusion was regulated,
however, the level of repression by thiamin was less than in the
wild type. In strains containing a deletion of ydiA/thiL (TH22
(.DELTA.thiL::cat.sub.4, .OMEGA.amyE::thiA-lacZ)), the fusion was
partially deregulated: LacZ activity was 2- to 3-fold higher under
both repressing and derepressing growth conditions.
[0102] Based on these results, thiA-lacZ reporter strain TH22
(.DELTA.thiL::cat.sub.4) was used to screen for deregulated mutants
under repressing growth conditions. Two methods were used to
isolate such mutants. In the first method, MM agar plates were
prepared that contain 1 .mu.M thiamin and 251 .mu.g/ml XGAL. After
applying a uniform dilution of logarithmic growth phase TH22 cells,
a paper disk containing 3 drops of ethylmethane sulfonate (EMS,
d=1.17 g/ml solution) was placed in the center of the plate.
Lac.sup.+ colonies appeared over a period of 7 days incubation at
37.degree. C. Deregulated mutants Tx1-Tx10 were recovered. In the
second method banks of EMS-mutagenized cells were prepared and
screened. Accordingly, logarithmic stage TH22-cells were treated
with 9.4 mM EMS for 90 minutes and aliquots frozen in 10% glycerol
at -90.degree. C. Cells from the frozen stocks were diluted in VY
medium, incubated at room temperature for 30 minutes and then
plated onto MM medium containing 1 .mu.M thiamin and 25 .mu.g/ml.
XGAL. Screening of Lac.sup.+ colonies led to mutants Tx11 to Tx26.
These mutants could be grouped into three classes based on the
intensity and timing of the appearance of blue color under thiamin-
and TPP-repressing conditions (Table 3), and based on additional
phenotypes. One mutant (Tx1) was found to be a strong thiamin
bradytroph suggesting that this mutation either inactivated (1) a
residual TMP kinase activity, or (2) a gene involved in a second
TMP to TPP route, via TMP. Another mutant, Tx26, was resistant to
10 .mu.M pyrithiamine. In terms of synthesis of thiamin products,
mutant Tx7 (class 2) excreted 2- to 3-times more total thiamin
products relative to the parental strain, TH22
(.DELTA.thiL::cat.sub.4, .OMEGA.amyE::thiA-lacZ), although the
intracellular levels of thiamin products were similar (Table 4).
Mutant Tx26 (class 1), excreted 10- to 15-times more total
extracellular thiamin products into the culture medium than the
TH22 control strain (Table 4). Little over 50% of the excreted
thiamin products were in the form of TPP. Class 3 mutants,
represented by Tx1 and Tx23, appear to be affected in the
thiamin-TMP-TPP pathway based on the differential Lac expression in
the presence of thiamin or TPP. TABLE-US-00003 TABLE 3 Phenotype of
B. subtilis thiamin-deregulated mutants. Class Phenotype Mutant 1
Thiamin prototroph with strong Lac activity Tx2; Tx4; Tx6; Tx9;
after 1 day growth on minimal medium Tx11; Tx12; Tx13; containing
either thiamin or TPP Tx14; Tx15; Tx16; Tx17; Tx21; Tx22; Tx24;
Tx26 2 Thiamin prototroph with weak Lac activity Tx3; Tx5; Tx7;
Tx8; after >3 days growth on minimal medium Tx10; Tx25
containing thiamin, and weak or no Lac activity in the presence of
TPP 3 Thiamin auxotroph (or strong bradytroph) Tx1 and Tx23 with
strong Lac activity after 1 day growth on minimal medium containing
thiamin or TMP, but little or no Lac activity in the presence of
TPP
[0103] TABLE-US-00004 TABLE 4 Thiamin production of B. subtilis
thiamin-deregulated mutants. Extracellular.sup.a
Intracellular.sup.b Strain (.mu.g/L) (.mu.g/L) B. subtilis PY79
<0.1 150 B. subtilis TH22 1 500 B. subtilis Tx7 5 500 B.
subtilis Tx26 15 500 .sup.aThiamin concentration in minimal medium
(30 ml) after 24 hours growth at 37.degree. C. .sup.bThiamin
concentration of 1 ml supernatant of sonicated cells collected from
a 30 ml minimal medium culture after 24 hours growth at 37.degree.
C.
[0104] In exogenous precursor feeding studies, conversion of HMP
and HET to total thiamin products also differed between Tx7 and
Tx26 (Table 5). Each strain was grown in minimal medium cultures
containing the indicated amounts of HET and HMP for 18 hours at
37.degree. C. Culture media and cell extracts were analyzed for
thiamin production. (Thiamin+TMP+TPP) and (TPP) were measured by a
biological assay using S. typhimurium indicators DM456
(.OMEGA.thiD906::MudJ) and DM1856 (.OMEGA.thiL934::Tn10),
respectively. Thiamin products were not detected in the medium. In
Tx7, most of the thiamin products were found within the cells,
predominantly in the form of TPP. In contrast, 90% of the total
thiamin products in Tx26 was found in the culture medium mostly as
thiamin+TMP. Extracellular accumulation was approximately 40-fold
higher than Tx26 grown without added HMP+HET. TABLE-US-00005 TABLE
5 HMP and HET feeding studies of B. subtilis thiamin-deregulated
mutants Tx7 and Tx26. Extracellular Intracellular (.mu.g/L)
(.mu.g/L) Thiamin + Thiamin + HMP HET TMP + TMP + Strain (10 .mu.M)
(10 .mu.M) OD.sub.600 TPP TPP TPP TPP Tx7 - - 1.3 0.2 0 750 360 Tx7
+ - 1.1 0.3 0 210 390 Tx7 - + 1.5 0.3 0 890 1400 Tx7 + + 1.2 4 8
1800 2000 Tx26 - - 1.2 2 0.8 500 280 Tx26 + - 1.7 40 18 610 640
Tx26 - + 1.1 2 0.6 420 240 Tx26 + + 1.2 80 10 610 650
[0105] A B. subtilis strain was built that contains a combination
of mutations tx26, thiL, and tx1. This strain could serve as a host
for integrated and amplified engineered thiamin biosynthetic genes.
As a first step, the mutations in Tx26 were transferred into TH12
(.DELTA.thiL::cat.sub.4) by DNA transformation and selecting
colonies that were resistant to 10 .mu.M pyrithiamine. One
Pyr.sup.r colony that was also Lac.sup.+ in the presence of thiamin
was recovered and designated TH48. Each strain was grown in minimal
medium supplemented with micronutrients and 2.5% Difco nutrient
broth (NB) for 18 hours at 37.degree. C. Supernatants were analyzed
for thiamin production by bioassays using indicators Salmonella
DM456 (thiD906::MudJ) for (Thiamin+TMP+TPP) and Salmonella DM1856
(thiL934::Tn10) for (TPP). When grown in minimal medium shake-flask
cultures, TH48 produced similar levels of thiamin products compared
to the Tx26 parent (Table 6). The cat-interrupted thiL gene was
next replaced by an in-frame deletion. Using standard PCR methods,
an in-frame deletion of thiL (removing amino acid residues Gly79
and Gly202) was first constructed and inserted between the BamHI
and EcoRI sites of the E. coli plasmid vector pEpUC.DELTA.1
creating pTH30. pEpUC.DELTA.1 (S. Seror, Universite Paris-Sud,
91405 Orsay, France) contains a selectable erythromycin-resistance
(erm) cassette and a temperature-sensitive origin of replication
that does not function over 51.degree. C. TH48 cells were
transformed at 51.degree. C. with pTH30 selecting for erythromycin
resistance. One Erm.sup.r colony that was also Cm.sup.r was
recovered and was grown overnight at 28.degree. C. in the absence
of antibiotic selection for 72 hours. Bacteria were then plated
onto TBAB agar plates, and the plates incubated overnight at
37.degree. C. Approximately 25% of the colonies were found to be
sensitive to both erythromycin and chloramphenicol antibiotics. PCR
analysis of chromosomal DNA from several Erm.sup.S Cm.sup.S
colonies confirmed the presence of the in-frame .DELTA.thiL
mutation and the absence of the .DELTA.thiL::cat.sub.4 mutation.
This resulted in strain TH83. The txl mutation was next introduced
by transduction into TH83 by PBS1 transduction by standard
procedures using linkage to a silent Tn917 insertion,
.OMEGA.yloA::Tn917 (60% linkage to tx1; strain 1A633 of the
Bacillus Genetic Stock Center, also called CU4153 or
.OMEGA.zdi-82::Tn917). The resulting strain was called TH95.
TABLE-US-00006 TABLE 6 Thiamin production of B. subtilis
thiamin-deregulated strains containing .DELTA.thiL and tx26
mutations. Extracellular (.mu.g/L) Thiamin + TMP + Strain Genotype
OD.sub.600 TPP TPP Tx26 .DELTA.thiL::cat.sub.4 thiA-lacZ tx26 16.5
320 460 TH48 .DELTA.thiL::cat.sub.4 tx26 18.7 280 310 TH49
.DELTA.thiL::cat.sub.4 thiA-lacZ tx26 16.5 300 470 TH12
.DELTA.thiL::cat.sub.4 18.4 2.6 2.2 TH22 .DELTA.thiL::cat.sub.4
thiA-lacZ 18.4 3.2 2.4
[0106] In standard fed-batch fermentations using 20-liter lab scale
fermentors, NB was found to enhance thiamin production in TH95.
Results showed that extracellular thiamin product levels were
approximately 2- to 3-fold higher using feed medium containing 4%
NB compared to feed without NB (Table 7). Production reached a
maximum level of 6-7 mg/liter between 30-48 hours of growth. More
importantly, as judged by thiochrome/HPLC assay, at least 65% of
extracellular thiamin products were in the form of thiamin, whilst
in the fermentation without NB in the feed, most of the product was
TMP and TPP. Simultaneously, increasing the amount of NB in the
seed (10%) and removing NB from the feed, delayed production of
thiamin-related products to 24 hours cultivation time. Moreover,
excreted thiamin products were decreased and mainly in the form of
TMP. Addition of NB (4%) to the batch also led to a decrease in
total thiamin production and a change in the excretion profile, in
which all the thiamin forms (THI, TMP, and TPP) were in almost
equimolar quantities. TABLE-US-00007 TABLE 7 Thiamin production of
TH95 in 6-liter fermentation with the addition of nutrient broth
(NB). Thiamin products (mg/L) Feed OD.sub.600 Thiamin TMP TPP
Strain 60% Glucose plus 48 hr 48 hr 48 hr 48 hr TH95 -- 120 0.7 1.4
1.0 TH95 4% NB 140 4.5 1.0 1.2
EXAMPLE 3
Producing Thiamin Compounds Using HMP and HET
[0107] This example describes a method to produce thiamin compounds
growing thiamin-deregulated strains in the presence of thiamin
precursors HMP and HET.
[0108] Fermentation of strain TH95 (tx26 txl .DELTA.thiL
yloA::Tn917) with thiamin precursor co-feed was performed in 6- and
1-liter scale under fed-batch conditions. Feed solutions containing
0.54 g/liter hydroxyethylthiazole (HET) and 0.54 g/liter
hydroxymethylpyrimidine (HMP) led to a significant accumulation of
thiamin in the culture medium. Thiamin titers reached 120 mg/L
(Table 8) after 48 hours, which represents a molar yield of 25%
based on the concentration of either precursor. Conversely, TMP and
TPP titers were very low (4 and 2 mg/liter, respectively)
accounting for less than 3% of the total amount of thiamin-related
excreted products. Feeding of either HMP or HET alone led to a very
low titer of all thiamin products. Increasing the concentration of
HMP and HET to 2.7 g/liter each or in combination did not result in
a significant increase in thiamin production levels. Extending the
fermentation of TH95 in the presence of 0.54 g/liter HET and 0.54
g/liter HMP led to a preferential increase in thiamin titers. After
70 hours, thiamin (THI) titers reached 250 mg/liter whereas TMP and
TPP levels (7 and 2 mg/liter) were similar to those at the 48-hour
time point (Table 9). TABLE-US-00008 TABLE 8 Thiamin production of
TH95 in 6-liter fermentation with the addition of HMP or HET.
Thiamin products (mg/L) OD.sub.600 Thiamin TMP TPP Strain Feed: 60%
Glucose plus 48 hr 48 hr 48 hr 48 hr TH95 0.54 g/L HMP 80 6 1 0.8
TH95 0.54 g/L HMP, 0.54 g/L HET 90 120 4 2 TH95 0.54 g/L HET 70 1
0.5 0.3 TH95 2.7 g/L HMP 110 7 1 1 TH95 2.7 g/L HMP, 2.7 g/L HET 80
125 12 4 TH95 2.7 g/L HET 90 3 1 1
[0109] TABLE-US-00009 TABLE 9 Thiamin production of TH95 in 1-liter
fermentation with the addition of HMP and HET. Feed Thiamin
products (mg/L) 60% Glucose OD.sub.600 OD.sub.600 THI TMP TPP THI
TMP TPP Strain plus 48 hr 70 hr 48 hr 48 hr 48 hr 70 hr 70 hr 70 hr
TH95 0.54 g/L HMP, 75 85 110 4 2 250 7 2 0.54 g/L HET
EXAMPLE 4
Thiamin Producing Strains with Increased ThiA Synthesis
[0110] This example describes the construction of a DNA cassette
containing the B. subtilis thiA gene which can be used to
overexpress said gene resulting in overproduction and excretion of
thiamin products.
[0111] In order to increase expression, an amplifiable engineered
thiA cassette was constructed in which the native promoter region
was replaced by a strong constitutive SP01-26 promoter derived from
the B. subtilis bacteriophage SPO1 (designated P.sub.26). First a
1770 bp-long DNA fragment containing the entire thiA gene was
amplified by PCR using standard methods and synthetic oligo DNA
primers thiA/for/pXI22/NdeI (SEQ ID NO: 1) and thiA/rev/pXI22/Bam
(SEQ ID NO: 2). After digestion with NdeI and BamHI, the PCR
product was inserted between the NdeI and BamHI sites of pXI22mod
and transformed into competent E. coli cells using standard
methods. pXI22mod is a 7.2 kb E. coli plasmid that contains the
P.sub.26 promoter, a synthetic Bacillus RBS, cryT terminator,
selectable ampicillin (bla) resistance and chloramphenicol (cat)
resistance genes, and NdeI/BamHI cloning sites located between the
RBS and cryT terminator (the NdeI site generates the ATG start
site). The P.sub.26 promoter is inactivated due to the placement of
the E. coli ColE1 replicon and the bla gene between the -35 and -10
consensus regions of the promoter. In addition, a NdeI site within
pXI22 was modified by standard methods to remove an undesired NdeI
restriction site located within the replicon region. This resulted
in plasmid pTH43 (FIG. 1). Another thiA expression cassette was
also prepared by replacing the cat cassette with a selectable
tetracycline-resistance (tet) gene. To do this, a 2042 bp DNA
fragment containing the tet gene from plasmid pDG1514 (BGSC, Cat #
ECE100) gene was amplified by PCR using standard methods and
synthetic oligo DNA primers tet/for/PmeI (SEQ ID NO: 3) and
tet/rev/NotI (SEQ ID NO: 4). This fragment was then cloned between
the PmeI and NotI sites of pTH43 to give pTH47 (FIG. 1).
[0112] The next task was to integrate the P.sub.26thiA-cat cassette
in thiamin deregulated strains. First, plasmid pTH43 was digested
by BsaI and a 3352 bp long fragment was purified from the agarose
gel using standard methods. The purified fragment was ligated to
itself at high DNA concentration and transformed into TH95
competent cells using standard methods. Transformants were selected
on TBAB medium containing 5 .mu.g/ml chloramphenicol. One Cm.sup.r
colony, designated TH116, was saved for further studies. The
expression of thiA was increased by obtaining colonies that were
resistant to successively higher levels of chloramphenicol.
Specifically, a strain of TH116 resistant to 60 .mu.g/ml
chloramphenicol could be obtained. SDS-PAGE analysis of crude cell
extracts of THI 16 strains resistant to 60 .mu.g/ml chloramphenicol
showed significantly higher levels of ThiA protein than TH 116
strains resistant to only 5 .mu.g/ml chloramphenicol. Thiamin
production with the TH 116 engineered strain was tested in 20-liter
lab scale fermentors using standard fed-batch conditions with HET
co-feeding (0.54 g/liter, w/w). Strain TH 116 resistant to 60
.mu.g/ml chloramphenicol produced between 18-21 mg/liter thiamin
(Table 10), which is a 3-fold increase in thiamin production
compared to TH95 fermentation (Table 8). Thiamin production,
however, was significantly lower than observed in feeding studies
(see Example 3). This result indicates that formation of HMP is
rate limiting, which could be caused by insufficient quantities of
an additional enzymatic activity or low levels of AIR pools.
TABLE-US-00010 TABLE 10 Thiamin production of TH116 in 6-liter
fermentation. Thiamin products (mg/L) OD.sub.600 Thiamin TMP TPP
Thiamin TMP TPP Strain 48 hr 24 hr 24 hr 24 hr 48 hr 48 hr 48 hr
TH116 65 18 4 6 21 3 5
EXAMPLE 5
Methods for Producing Thiamin Compounds Using Microorganisms With
Increased AIR Formation
[0113] This example describes experiments that increase thiamin
production by altering the purine pathway to increase
aminoimidazole ribotide (AIR) formation. This was achieved by
simultaneously deregulating the expression of the B. subtilis
purine operon using a mutation within the leader region of the
purine operon (purO) and blocking conversion of AIR to
carboxyaminoimidazole ribotide (CAIR) through introduction of a
mutation within the purE gene encoding phosphoribosylaminoimidazole
carboxylase I.
[0114] To construct a purO mutation, the upstream region of the
operon promoter was amplified by PCR using primers YebF+1 (SEQ ID
NO: 18) and YebG-1 (SEQ ID NO: 19) to generate a 667-bp product.
Genomic DNA prepared from wild-type B. subtilis 1012 was used as a
template and the PCR reaction conditions consisted of 30 cycles of
denaturation at 95.degree. C. for 1 min., annealing at 55.degree.
C. for 1 min. and extension at 72.degree. C. for 1 min. The PCR
product was purified using the Wizard PCR purification kit
(Promega) and double-digested with EcoRI-BamHI. The PCR product was
cloned into EcoRI-BamHI-digested pUC19 to give plasmid pNMR72.
[0115] The purE promoter was amplified by PCR using primers PurE+3
(SEQ ID NO: 20) and PurE-1 (SEQ ID NO: 21) to give a 768-bp
product. The PCR product was purified using the Wizard PCR
purification kit (Promega) and double-digested with BamHI-PstI and
cloned into BamHI-PstI-digested pNMR72 generating plasmid
pNMR76.
[0116] Plasmid pNMR76 was linearized with BamHI and ligated to a
BclI-digested neomycin-resistance (neo) gene cassette from pBEST50
to give plasmids pNMR79, with the neo cassette in the same
orientation as pur transcription, and pNMR80, with the neo cassette
in the opposite orientation. Both plasmids were linearized with
ScaI and transformed into competent B. subtilis 1012 cells.
Transformants were selected on TBAB plates containing neomycin to a
final concentration of 2.5 .mu.g ml.sup.-1. 67 colonies were
observed for the pNMR79 transformation, and 18 colonies for the
pNMR80 transformation. 6 colonies from each transformation
experiment were picked and analyzed by PCR. Two clones were
identified as containing the truncated pur operons integrated as
double crossovers for each transformation. These clones were
renamed BS1566 and BS1567 for the pNMR79 transformation, and BS1568
and BS11569 for the pNMR80 transformation.
[0117] To combine the purO deletion mutation with the purE6
mutation, B. subtilis strain 1A320 (purE6 trpC2; Bacillus Genetic
Stock Center, The Ohio State University, Columbus, Ohio 43210 USA)
was transformed with chromosomal DNA from strain BS1567, resulting
in strain TH94. Since purO::neo purE6 are closely linked, both
mutations were simultaneously transferred to thiamin deregulated
strain TH95 by PBS 1 generalized transduction under standard
conditions, resulting in strain TH101 (tx26 txl .DELTA.thiL
yloA::Tn917 .DELTA.purO::neo purE). To determine thiamin
production, strain TH101 was grown under standard 1-L fed-batch
conditions with a HET (0.54 g/liter) co-feeding. Xanthine was also
added to the batch medium and feed solution at 0.01% (w/w) and 1%
(w/w, dissolved in 7.35 N NaOH), respectively, to satisfy the
purine requirement. Xanthine does not feedback inhibit any of the
purine de novo enzymes, nor is it toxic at high concentrations. In
addition, the feed also contained NH.sub.4Cl (9.6% w/w) and the pH
was controlled using H.sub.2SO.sub.4 (10%) and NaOH (7.35 M).
Results indicated that TH101 produced approximately 6-times more
thiamin in the broth than the control TH95 fermentation under the
same experimental conditions (Table 11). TABLE-US-00011 TABLE 11
Thiamin production of TH95 in 1-liter fermentation containing the
purOE mutations. purOE- OD.sub.600 Thiamin products mg/L Strain
deletion 48 hr Thiamin TMP TPP TH95 - 90 0.5 0.5 BD TH101 + 52 2.7
0.6 0.3 BD, below detection
EXAMPLE 6
Methods for Increasing Thiamin Compounds Production by Enhancing
the Thiamin Coupling Gene
[0118] This example describes the construction of a DNA cassette
containing the B. subtilis thiC gene which can be used to
overexpress said gene resulting in overproduction and excretion of
thiamin products. This gene is located in an operon containing
thiK, a gene that encodes the salvage enzyme, HET kinase.
[0119] In order to increase expression of thiC, an amplifiable
cassette was constructed in which the native promoter region was
replaced by a strong constitutive SP01-26 promoter derived from the
B. subtilis bacteriophage SP01 (designated P.sub.26). To do this,
the thiA cassette was removed from pTH47 and replaced by a DNA
fragment containing thiKC. First a 1555 bp fragment containing
thiKC structural gene was amplified by PCR using standard methods
and synthetic oligo DNA primers thiKC/for3/pXI22/NdeI (SEQ ID NO:
5) and thiCop/rev2/pXI22/SmaI (SEQ ID NO: 6). After digestion with
NdeI and BamHI, the PCR product was inserted between the NdeI and
BamHI sites of pTH43, resulting in plasmid pTH48 (FIG. 2).
[0120] This P.sub.26 thiKC-tet cassette was introduced into TH95 by
first digesting pTH48 with BsaI and purifying a 4078 bp fragment
from the agarose gel using standard methods. The purified fragment
was ligated to itself at high DNA concentration and transformed
into TH95 competent cells using standard methods. Transformants
were selected on TBAB medium containing 20 .mu.g/ml tetracycline.
One Tet.sup.r colony, designated TH115, was saved for further
study. The expression of thiKC was increased by obtaining colonies
that were resistant to successively higher levels of tetracycline.
Specifically, a strain of TH115 resistant to 45 .mu.g/ml
tetracycline could be obtained. SDS-PAGE analysis of crude cell
extracts showed significantly higher levels of ThiK and ThiC
protein than TH115 strains resistant to only 20 .mu.g/ml
tetracycline.
[0121] In the presence of HMP and HET co-feeding (0.54 g/liter,
each), two fed-batch fermentations of TH115 resulted in an increase
in thiamin production (210 mg/L at 48 hours, and 300 mg/L after 78
hours). The molar yields on substrates HMP or HET were 45% each at
both 48 and 78 hours. Two other fermentations led to a slight
decrease in thiamin production. Interestingly, higher thiamin
excretion coincided with a severe growth-limiting event that
occurred during the first hours of the fermentation, and which
could be overcome by the addition of 25 g of nutrient broth (NB)
and 1.6 mg of TPP to the 6-L cultivation. HPLC/DAD confirmed the
presence of thiamin in the fermentation broth, and could be used to
purify thiamin from the other UV-detectable compounds.
Interestingly, neither HMP nor HET was detected by HPLC/DAD,
indicating complete uptake of the co-feed applied at 0.54 g/liter.
In any event, these results indicate that TH115 has a large
capacity to synthesize TMP from exogenously-added precursors,
dephosphorylate TMP to thiamin, and excrete thiamin into the
culture medium. In addition, the ThiC coupling activity is
apparently not rate limiting in this process.
EXAMPLE 7
Methods for Increasing Thiamin Compounds Production by Enhancing
the Expression Level of Thiazole Biosynthetic Enzymes
[0122] This example describes the construction of a DNA cassette
containing the B. subtilis thiB operon containing genes
tenAI-thiOSGFD, which can be used to overexpress said genes
resulting in overproduction and excretion of thiamin products.
[0123] First, a thiazole auxotroph strain, deleted for the native
promoter region in front of the tenAI-thiOSGFD operon, was
generated. To do that, two DNA fragments (downstream TenA and
upstream YjbQ fragments), located on each side of the promoter
region, were first amplified from B. subtilis PY79 chromosome using
primer pairs YjbQ+_BamHI (SEQ ID NO: 22)/YjbQ-_MluI (SEQ ID NO: 23)
and TenA+_KpnI (SEQ ID NO: 24)/TenA-_XhoI (SEQ ID NO: 25). A third
fragment, containing a chloramphenicol acetyltransferase cassette,
was amplified from TH11 chromosomal DNA (PY79
.DELTA.thiL::cat.sub.2) using primers TenA-cat+_KpnI (SEQ ID NO:
26) and TenA-cat-_MluI (SEQ ID NO: 27). Assembling of these three
fragments was then performed in pUC19 to generate plasmid pTH401,
which contains the
`yjbQ.sub.3'-MluI-.sub.3'cat.sub.5'-KpnI-.sub.5'tenA` DNA
construction inserted between the PstI and EcoRI restriction sites.
Transformation of plasmid pTH401 in TH95 and selection on Cm 5
.mu.g/ml, yielded thiamin-, and thiazole-auxotroph TH403.
[0124] To construct a P.sub.26 tenAI-thiOSGFD cassette, the cat
cassette in pTH401 was excised by KpnI and MluI, and, then, was
substituted by a PCR fragment containing the P.sub.26 strong
constitutive promoter, derived form the bacteriophage SPO1. This
fragment was amplified from plasmid pUCSPO1-26 using primers
P26+_MluI (SEQ ID NO: 28) and P26-_KpnI (SEQ ID NO: 29).
Introduction of the thiB overexpressing cassette was then made by
selecting for restoration of prototrophy in TH403 background. After
transformation of the ligation mix creating the P.sub.26 tenA'
in-frame fusion into TH403, prototrophic transformants were
selected for their ability to grow on minimal medium plates. Their
chloramphenicol sensitivity and the presence of the P.sub.26
promoter in front of the tenAI-thiOSGFD operon were confirmed. The
resulting strain was named TH404 (FIG. 3).
[0125] Thiamin production with TH404 engineered strain was tested
in 20-liter lab scale fermentors using standard fed-batch
conditions (start volume: 6 liter) with HMP co-feeding (0.54
g/liter, w/w). Strain TH404 produced up to 315 mg/liter thiamin
(Table 12). That number is significantly higher than the number
obtained after 48 hours for strain TH95 (110 mg/liter, Table 9).
TABLE-US-00012 TABLE 12 Thiamin production of TH404 in 6-liter
fermentation. Thiamin products mg/L OD.sub.600 Thiamin TMP TPP
Strain 48 hr 48 hr 48 hr 48 hr TH404 85 315 31 5
EXAMPLE 8
Methods for Increasing Thiamin Compounds Production by Enhancing
the Expression Levels of both ThiA and Thiazole Biosynthetic
Enzymes
[0126] This example describes the combination of DNA cassettes
containing the B. subtilis thiA gene and the B. subtilis thiB
operon containing genes tenAI-thiOSGFD, which can be used to
overexpress said gene resulting in overproduction and excretion of
thiamin products.
[0127] In order to combine the B. subtilis P.sub.26 tenAI-thiOSGFD
cassette (described in Example 7) and the amplifiable P.sub.26
thiA-cat cassette (described in Example 4), competent cells of
strain TH404 were transformed with non-congressional concentration
of chromosomal DNA extracted from strain TH116 using standard
methods. Transformants were selected for resistance to 5 .mu.g/ml
chloramphenicol. PCR analysis confirmed that they contain the
P.sub.26 tenAI-thiOSGFD and P.sub.26 thiA-cat cassettes. One
Cm.sup.r colony, designated TH405, was saved for further studies.
The expression of thiA in TH405 was increased by obtaining colonies
that were resistant to successively higher levels of
chloramphenicol. Specifically, a strain of TH405 resistant to 60
.mu.g/ml chloramphenicol could be obtained. SDS-PAGE analysis of
crude extracts of strain TH405 resistant to 60 .mu.g/ml
chloramphenicol showed a significantly higher level of ThiA protein
than strain TH405 resistant to only 5 .mu.g/ml, which is identical
to the level obtained after amplification of the P.sub.26 thiA-cat
cassette in strain TH116 (Example 4).
[0128] Thiamin production with the TH405 engineered strain was
tested in 20-liter lab scale fermentors using standard fed-batch
conditions (start volume: 6 liter). Strain TH405 resistant to 60
.mu.g/ml chloramphenicol produced between 34-37 mg/liter thiamin in
48 h (Table 13), which is a 6-fold increase in thiamin production
compared to TH95 fermentation with 0.54 g/liter co-feed of HMP
(Table 8). Thiamin production, however, was significantly lower
than observed in co-feeding studies or with a strain overexpressing
the thiB operon only (Table 12, Example 7). This result confirms
our observation from Example 4, i.e. formation of HMP is
rate-limiting, and that the putative missing gene that needs to be
overexpressed in addition to thiA and the thiB operon, is not part
of the thiB operon. TABLE-US-00013 TABLE 13 Thiamin production of
TH404 in 6-liter fermentation. Thiamin products (mg/L) OD.sub.600
OD.sub.600 Thiamin TMP Thiamin Strain 24 hr 48 hr 24 hr 24 hr TPP
24 hr 48 hr TMP 48 hr TPP 48 hr TH405 78 20 34 2 1 37 2 1
EXAMPLE 9
Methods for Increasing Thiamin Compounds Production by Increasing
Glycine or Cysteine Availability
[0129] This example describes experiments that increase thiamin
production by growing thiamin-deregulated strains in the presence
of glycine or cysteine, which are both precursors in the HET
pathway.
[0130] Thiamin production with B. subtilis TH95 (tx26 txl
.DELTA.thiL) was tested in 6-liter lab scale fermentation using
standard fed-batch conditions with HMP (0.54 g/liter) and glycine
co-feeding (2 g/liter). Thiamin, TMP, and TPP production reached 14
mg/liter, 3 mg/liter, and 0.5 mg/liter, respectively, in 48 hours
(Table 14), which is substantially higher than thiamin products
levels in TH95 grown with just a HMP feed (Table 8).
[0131] Next, thiamin production with B. subtilis TH95 (tx26 txl
.DELTA.thiL) was tested in 6-liter lab scale fermentation using
standard fed-batch conditions with HMP (0.54 g/liter) and cysteine
co-feeding (0.5 g/liter); threonine (0.4 g/liter) and isoleucine
(0.2 g/liter) were also added to facilitate cysteine assimilation.
Thiamin production reached 8 mg/liter in 48 hours (Table 14), which
is substantially higher than thiamin titers in TH95 grown with just
a HMP feed (Table 8).
[0132] Increasing thiamin production could also be achieved by
addition of all four amino acids, glycine, cysteine, isoleucine,
and threonine to the fermentation of TH95 (Table 14). Moreover
increasing the expression of biosynthetic genes involved in the
synthesis of these amino acids, or introducing mutations in
regulatory genes or cis-acting regulatory sites that lead to
increased expression of the said amino acid biosynthetic genes or
by introducing mutations that lead to increased activity of said
biosynthetic enzymes could increase thiamin productivity.
TABLE-US-00014 TABLE 14 Thiamin production of TH95 in 6-liter
fermentation with the addition of glycine,
cysteine-isoleucine-threonine, or glycine
cysteine-isoleucine-threonine. Feed OD.sub.600 Thiamin products
(mg/L) Strain 60% Glucose plus 48 hr Thiamin TMP TPP TH95 0.54 g/L
HMP, 65 14 3 0.5 2 g/L glycine TH95 0.54 g/L HMP, 60 8 2 0.5 0.54
g/L L-cysteine, 0.4 g/L D,L-threonine, 0.2 g/L L-isoleucine TH95
0.54 g/L HMP, 75 11 2 0.4 2 g/L glycine, 0.2 g/L L-cysteine, 0.4
g/L D,L-threonine, 0.2 g/L L-isoleucine
EXAMPLE 10
Methods for Producing Thiamin Compounds Using Grewe Diamine as a
Precursor
[0133] This example describes experiments demonstrating the
production of thiamin by growing thiamin-deregulated strains in the
presence of a derivative of HMP,
4-amino-2-methyl-5-pyrimidinemethaneamine (Grewe Diamine).
[0134] Grewe Diamine is a derivative of HMP in which the C-5
hydroxymethyl group is substituted by an aminomethyl group. Thiamin
production with B. subtilis TH95 (tx26 txl .DELTA.thiL) was tested
in 6-liter lab scale fermentation using standard fed-batch
conditions with Grewe Diamine and HET co-feeding (0.54 g/liter
each). Thiamin production reached 53 mg/liter in 45.5 hours (Table
15). Increasing the level of Grewe Diamine in the feed solution to
2.7 g/liter increased thiamin production to 120 mg/liter in 46.5
hours (Table 15). These results demonstrate the feasibility of
developing a fermentation process to produce thiamin from Grewe
Diamine and HET. Moreover, these results demonstrate that Bacillus
subtilis encodes one or more enzyme activities that can convert
Grewe Diamine to HMP or a structurally similar compound that can
then be used to produce thiamin. TABLE-US-00015 TABLE 15 Thiamin
production of TH95 in 6-liter fermentation with a Grewe Diamine and
HET co-feed. Feed OD.sub.600 Thiamin products (mg/L) Strain 60%
Glucose plus 48 hr Thiamin TMP TPP TH95 0.54 g/L HET, 44 53 7 1
0.54 g/L Grewe Diamine TH95 2.7 g/L HET, 60 118 8 1 2.7 g/L Grewe
Diamine
EXAMPLE 11
Isolated Genes and Mutations
yloS/tx1
[0135] Strain Tx1 carries the txl mutation (see SEQ ID NO: 30 for a
copy of the polynucleotide sequence having the mutation), which was
isolated by screening thiA-lacZ fusion-containing bacteria for the
Lac.sup.+ phenotype in the presence of TPP. This strain
(.DELTA.thiL::cat4 amyE::thiA-lacZ tx1) was shown to be a strong
thiamin bradytroph indicating that this mutation appeared to
inactivate a residual TMP kinase activity or an unknown gene
product involved in a secondary route that leads to the formation
of TPP from thiamin. Reconstitution studies indicated that the
Lac.sup.+ phenotype of Tx1 was not caused by a general defect in
the thiazole or HMP pathways as judged by analysis of TH22
derivatives containing a block in either the HET
(.OMEGA.thiF::pMUTIN) or HMP-P (.OMEGA.thiA::Tn917) pathways.
[0136] Strains with txl were confirmed to be bradytrophs based on
their ability to grow in minimal medium supplemented with 2.5%
nutrient broth, which does not contain any significant amounts of
thiamin products. In these cultures, Tx1 produced approximately
10-fold more extracellular thiamin products than the control strain
TH22 (.OMEGA.thiL::cat.sub.4 .OMEGA.amyE::thiA-lacZ). Interestingly
all the detectable thiamin produced was either in the form of
thiamin or TPP; TMP was not detected in the culture medium. Genetic
studies indicated that the thiamin bradytroph phenotype of Tx1
required .DELTA.thiL::cat.sub.4.
[0137] Genetic mapping studies using PBS1 generalized transduction
under standard conditions showed that the txl mutation was not
linked to either .DELTA.thiL::cat.sub.4 or .OMEGA.amyE::thiA-lacZ.
However, txl showed 90% transduction linkage to zdi-82::Tn917
(BGSC# 1A633) located at 140'. The transformation linkage to the
same marker was 8%. Several Tn917-linked mutations located between
121.degree. and 140.degree. also showed significant linkage to tx1.
One mutation, urc83::Tn917 (strain 1A611), showed high transduction
linkage (>60%) and significant transformation linkage (10%).
This insertion, which causes an auxotrophy for uracil+cysteine or
uracil+methionine, is at the junction between the pyr operon and
the cys operon (139.degree.). A second mutation, yloA::Tn917 showed
a similar linkage to tx1. Much higher transduction and DNA
transformation linkage of txl to .OMEGA.spoVM::Tn917 mutation was
observed suggesting that txl was allelic to yloS, which is adjacent
to spoVM. The yloS gene shows weak amino acid similarity (P=0.23)
to the TNR3 gene protein of Schizosaccharomyces pombe, which has
been previously shown to have thiamin pyrophosphokinase activity.
To determine if txl is allelic to yloS, a stable 448 bp long
deletion mutation, starting at base 124 of the yloS gene
(.DELTA.yloS::cat.sub.4) was constructed by PCR using standard
methods and introduced into PY79. After introduction of
.OMEGA.thiL::pMUTIN, the resulting double mutant was phenotypically
similar to the original Tx1 mutant. Moreover, DNA sequencing of
yloS in the Tx1 mutant revealed a single base mutation that
resulted in a leucine-to-phenylalanine substitution at amino acid
residue 116 (L116>F116; see SEQ ID NO: 31).
[0138] Based on these results, B. subtilis contains two
biosynthetic routes to synthesize TPP from TMP: (1) direct
enzymatic transformation of TMP to TPP by the product of thiL; and
(2) enzymatic transformation of TMP to THI by an unknown
phosphatase, followed by the pyrophosphorylation of THI to TPP by
the product of yloS.
[0139] Route (1) has been shown to be present in Gram-negative
organisms (e.g. Salmonella typhimurium and E. coli). Route (2) is
present only in several Gram.sup.+ bacteria and some other
eukaryotic microorganisms, including yeast (Llorente et al. (1999)
Mol. Microbiol. 32:1140-1152). In addition, B. subtilis must
contain a kinase activity that converts thiamin to TMP. This
conclusion is based on genetic studies that showed that strain
TH109 containing mutations .DELTA.yloS::cat.sub.4 and
.OMEGA.thiA84::Tn917 could grow on minimal medium containing
thiamin.
[0140] Protein database searches indicated that at least 13
bacterial genera contain one or more genes that encode a protein
with significant homology to YloS: Oceanobacillus, Listeria,
Staphylococcus, Enterococcus, Streptococcus, Clostridium,
Fusobacterium, Tropheryma, Mesorhizobium, Brucella, Thermotoga,
Agrobacterium, and Helicobacter. Most of these microorganisms are
Gram.sup.+ bacteria. Weaker homology to genes from non-bacterial
organisms (e.g. yeast, Drosophila melanogaster, Mus musculus, and
Treponema pallidum) was also detected. Interestingly, most of the
yloS-containing bacterial species do not contain a thiL ortholog
gene, and conversely most of the thiL-containing bacterial species
do not contain a yloS ortholog (Table 16). This latter group
consisted mostly of Gram-negative genera. Like B. subtilis,
Oceanobacillus iheyensis contains both genes. These results
indicate that eubacteria can be classified into two groups
depending on the ability to form TPP by pyrophosphorylation of
thiamin or by phosphorylation of TMP. Moreover, many of the
Gram.sup.+ bacteria that contain only a yloS ortholog and not a
thiL ortholog are known pathogens, suggesting the yloS gene could
be used as a target for developing anti-bacterial agents.
TABLE-US-00016 TABLE 16 Presence or absence of yloS and thiL
orthologs in various bacterial genera. Microorganism yloS ortholog
thiL ortholog Bacillus subtilis 168 Yes Yes Oceanobacillus
iheyensis HTE831 Yes Yes Bacillus stearothermophilus Yes No
Listeria monocytogenes EGD-e Yes No Staphylococcus aureus Yes No
Enterococcus faecalis V583 Yes No Enterococcus faecium Yes No
Streptococcus pneumoniae TIGR4 and R6 Yes No Streptococcus pyogenes
Yes No Clostridium acetobutylicum ATCC-824D Yes No Clostridium
tetani E88 Yes No Clostridium perfringens str.13 Yes No Listeria
monocytogenes EGD-e Yes No Escherichia coli K12 No Yes Escherichia
coli 0157: H7 EDL933 No Yes Shigella flexneri 2a str. 301 No Yes
Haemophilus influenzae Rd No Yes Salmonella typhimurium LT2 No
Yes
yuaJ/tx26-1
[0141] PBS1 generalized transduction experiments, using standard
conditions, showed that the Tx26 mutant contained two mutations
located at different regions of the chromosome. In these
experiments, phage lysates were first prepared on standard
wild-type B. subtilis strains containing a phenotypically-silent
Tn917 insertion located around the chromosome (Bacillus Genetic
Stock Center). Two of these lysates, one carrying
.OMEGA.yujR::Tn917 (BGSC# 1A642) at map position 277.degree. and
the other carrying .OMEGA.motA::Tn917 (BGSC# 1A631) at map position
122.5.degree., were able to revert the Tx26 phenotype to wild type
(reversion of Lac.sup.+ to Lac.sup.- on minimal medium containing 1
.mu.M TPP and reversion of pyrithiamine-resistance to
pyrithiamine-sensitivity using 0.1 .mu.M pyrithiamine). These
unexpected results indicated that both mutations are required for
the thiamin-deregulation phenotype exhibited by Tx26. One mutation,
designated tx26-1, showed 70% linkage to .OMEGA.yufR::Tn917, and
the other mutation, designated tx26-2, showed 59% linkage to
.OMEGA.motA::Tn917. Moreover, in back-cross experiments, the
pyrithiamine-resistance marker of Tx26 could be transferred into
sensitive B. subtilis strains by congression DNA transformation.
These pyrithiamine-resistant transformants were also
thiamin-deregulated (Lac.sup.+ on minimal medium containing 1 .mu.M
TPP and resistant to 0.1 .mu.M pyrithiamine).
[0142] Three-factor cross experiments using donor strains
containing different combinations of antibiotic insertions in
yufR/maeN (277.1.degree.), yuiGH (281.degree.), yurI
(285.6.degree.), gerAB and yvaC (294.degree.) further mapped the
tx26-1 mutation close to yuaJ, a thiamin-regulated gene, which was
identified using microarray analysis (see below). To determine if
tx26-1 is allelic to yuaJ, a deletion mutation of yuaJ was first
constructed using standard PCR methods. To achieve this, a 324
bp-long internal fragment of yuaJ starting at position 353 was
PCR-amplified and inserted between the BamHI and HindIII sites of
pMUTIN2 using primers BsyuaJ/for/Hind3 (SEQ ID NO: 16) and
BsyuaJ/rev/Bam (SEQ ID NO: 17), creating plasmid pTH31. As
expected, introduction of .OMEGA.yuaJ::pMUTIN into wild type
strains, (e.g. PY79) or thiA mutants were without phenotype.
However, in several genetic crosses, the .OMEGA.yuaJ::pMUTIN2
disruption showed very high transduction and transformation
linkages (100%) to tx26-1. These linkage results placed tx26-1
within or near yuaJ. Moreover, transduction and transformation of
.OMEGA.yuaJ::pMUTIN into strain TH112 (tx26-1.sup.+ tx26-2
.DELTA.thiL), resulted in Erm.sup.r colonies that were resistant to
0.1 .mu.M pyrithiamine. Finally, DNA sequence analysis of yuaJ from
Tx26 confirmed that tx26-1 (see SEQ ID NO: 33 for a copy of the
polynucleotide sequence containing the mutation) was an allele of
yuaJ. Comparison of DNA sequences from four independent PCR
fragments from Tx26 and two from the wild type parent strain (PY79)
detected a single base mutation that resulted in the change of a
glutamine residue at amino acid position 35 to an Ocher stop codon
(Q35 (CAA)>Stop (TAA); see SEQ ID NO: 34 in comparison to the
amino acid sequence ID NO: 35 of the wild type YuaJ). Protein
database searches indicated that yuaJ encodes a thiamin permease or
a regulator of surface antigen protein genes. Hydrophobicity
analysis indicated that YuaJ contains six membrane-spanning
domains. Introduction of the tx26-1 mutation is predicted to
produce a truncated 35 amino acid protein, which is likely to
undergo proteolysis. These results, together with the genetic data
presented above, suggest that loss-of-function of yuaJ is
responsible for the thiamin-deregulation phenotype. Moreover,
microarray data (see below) indicated that expression of yuaJ is
regulated by TPP and inspection of the predicted 5' leader region
revealed a DNA sequence with strong homology to the consensus THI
box regulatory sequence.
tx26-2
[0143] Generalized transduction mapping studies (two-factor
crosses), using a collection of PBS1 phage lysates prepared on
strains containing Tn917 insertions, showed 60% linkage of tx26-2
to .OMEGA.motA::Tn917 (BGSC# 1A631) located at 122.5.degree. on the
B. subtilis genome. This genetic map position corresponded to a
cluster of genes, ykoFEDC, whose transcript levels were shown to
increase in microarray studies of the thiamin deregulated Tx26
strain (see Table 17). Moreover, these genes appeared to be
organized as an operon and contain a THI box regulatory elements in
the promoter region upstream of ykoF. DNA sequencing of this
operon, including a 400 bp region upstream of ykoF, detected a
single base mutation in the ykoD gene that resulted in a Asp.sub.80
(GAC) to Asn.sub.180 (AAC) substitution (see SEQ ID NO: 37 for the
amino acid sequence in comparison to amino acid sequence ID NO: 38
of the wild type YkoD). Protein database searches indicated that
ykoD encodes a HMP transport ATP-binding protein. Two other genes
in this operon, ykoE and C, are predicted to encode HMP transport
permeases. These results indicated that the tx26-2 mutation is an
allele of ykoD and affect cellular transport of thiamin (see SEQ ID
NO: 36 for a copy of the polynucleotide sequence containing the
mutation).
Thiamin-regulated Genes Identified Through Microarray Profiling
[0144] In order to perform microarray profiling, PY79 was grown in
shake-flask cultures that contained 50 ml Spizizen minimal medium
with or without added thiamin pyrophosphate. Overnight cultures
were diluted to Klett=10 units into fresh medium and grown to
exponential growth phase (Klett=100 units). Cells from half of the
culture were collected by centrifugation, and the total RNA was
immediately extracted as previously described (Lee et al. (2001) J.
Bacteriol. 183:7371-7380). The remaining culture was grown to early
stationary phase before RNA extraction. Early stationary phase was
judged to be 30 min after glucose exhaustion; glucose content in
the medium was measured by a glucose analyzer 2 (Beckman,
Fullerton, Calif.) using standard procedures. Preparation of
labeled cDNA targets, microarray hybridization and staining
procedures, and data analysis are described in Lee et al.
above.
[0145] In addition to known thiamin-regulated B. subtilis genes
thiA and tenAI-thiOSGFD, analysis of the results also showed a
3-fold or higher transcript level of several other genes in cells
grown in the absence of TPP. These genes are listed in Table 17.
These results were confirmed by comparison of microarray data of
wild-type and thiamin-deregulated (Tx26) strains grown in minimal
medium in the presence of TPP. Moreover, in several of these genes,
a consensus cis-acting regulatory site (thi box) could be
visualized within the 5' leader region, confirming regulation by
TPP. It can be anticipated that increasing or decreasing the
expression of these genes individually or in concert together, or
in combination with known biosynthetic genes, could also lead to
higher thiamin, HMP and/or thiazole production. TABLE-US-00017
TABLE 17 Change in transcript levels of genes in B. subtilis in
response to TPP.sup.a. wt.sup.-vit/ deg.sup.+vit/ Gene
Enzyme/Function wt.sup.+vit wt.sup.+vit thiA Biosynthesis of
hydroxymethylpyrimidine 62 22 phosphate thiK Hydroxyethylthiazole
kinase n/c 4.7 thiC Thiamin phosphate pyrophosphorylase n/c 2.4
thiO Glycine oxidase 67 19 (goxB) thiS Biosynthesis of
hydroxyethylthiazole 84 33 (yjbS) phosphate thiG Biosynthesis of
hydroxyethylthiazole 82 13 (yjbT) phosphate thiF Biosynthesis of
hydroxyethylthiazole 90 11 (yjbU) phosphate thiD1 Possible
phosphomethylpyrimidine kinase 32 14 (yjbV) thiD2 Possible
phosphomethylpyrimidine kinase n/c.sup.b n/c thiL Unknown/possible
thiL ortholog (TMP n/c n/c (ydiA) kinase) ytbJ Unknown/possible
thiI ortholog (sulfur n/c n/c transferase) dxs 1-deoxy-D-xylulose
synthase n/c n/c (yqiE) ykoC Unknown; similar to unknown proteins
12 4 ykoD Unknown; similar to cation ABC 26 5 transporter ykoE
Unknown 17 32 ykoF Unknown 20 5 yloS Thiamin pyrophosphorylase 1.6
0.3 yuaA Thiamin permease 7.4 5.3 ylmB Unknown; similar to
acetylornithine 5.0 7.6 deacetylase .sup.aTranscript ratios were
calculated by dividing the average difference values (after
normalization) from hybridization experiments of wild-type cells
grown to exponential phase in minimal medium without TPP treatment
by those with TPP treatment (wt.sup.-/wt.sup.+) or from
hybridization experiments
[0146]
Sequence CWU 1
1
38 1 29 DNA Artificial PCR primer 1 atgccatatg caaaacaatt cagtgcagc
29 2 36 DNA Artificial PCR primer 2 gcatggatcc tcattattga
tataaattgc ttcccg 36 3 32 DNA Artificial PCR primer 3 acgtgtttaa
acgcaggttg ttctcaatgt cg 32 4 33 DNA Artificial PCR primer 4
acgtgcggcc gcgatcaatt ttgaactctc tcc 33 5 27 DNA Artificial PCR
primer 5 atgccatatg gatgcacaat cagcagc 27 6 36 DNA Artificial PCR
primer 6 gcatcccggg tcagtctgaa aaccttgatg gacagc 36 7 31 DNA
Artificial PCR primer 7 gggaagcttt gcggtacctt caaaatggac t 31 8 29
DNA Artificial PCR primer 8 gggggatcca atctgccgac gcttactct 29 9 28
DNA Artificial PCR primer 9 gggggtaccg aaaattggat aaagtggg 28 10 29
DNA Artificial PCR primer 10 gggacgcgtt caactaacgg ggcaggtta 29 11
33 DNA Artificial PCR primer 11 gggacgcgta agtacagtcg gcattatctc
ata 33 12 23 DNA Artificial PCR primer 12 atgcggatcc cgtccggacc gcc
23 13 25 DNA Artificial PCR primer 13 cgatcccggg gcctcccatc gcggc
25 14 33 DNA Artificial PCR primer 14 atgccccggg atttgcctaa
gcttcatcct aac 33 15 29 DNA Artificial PCR primer 15 cgatgaattc
agcccttctg caaaacctt 29 16 33 DNA Artificial PCR primer 16
agctaagctt ggcagccgtt attttagaca ttg 33 17 34 DNA Artificial PCR
primer 17 tgcaggatcc ataaaaactg cgctgaccac tgaa 34 18 24 DNA
Artificial PCR primer 18 gagagaattc gctgaaagga cagc 24 19 35 DNA
Artificial PCR primer 19 tctcggatcc ttagatcaat ttcccttcaa atacg 35
20 26 DNA Artificial PCR primer 20 gagaggatcc atcgttgaca ttatcc 26
21 27 DNA Artificial PCR primer 21 ctctctgcag ctttctaaca ctgtctg 27
22 30 DNA Artificial PCR primer 22 atcaggatcc cgctcctgct gcttgcgctg
30 23 53 DNA Artificial PCR primer 23 tatgagataa tgccgactgt
acttacgcgt ccttatttgg tcaagattta tcc 53 24 64 DNA Artificial PCR
primer 24 cccactttat ccaattttcg ggtacctaag gaggtaactc atatgatttg
tgaagttttc 60 agaa 64 25 31 DNA Artificial PCR primer 25 ctgctcgagc
cagccttctt ttcgataggc c 31 26 64 DNA Artificial PCR primer 26
cttctgaaaa cttcacaaat catatgagtt acctccttag gtacccgaaa attggataaa
60 gtgg 64 27 53 DNA Artificial PCR primer 27 ggataaatct tgaccaaata
aggacgcgta agtacagtcg gcattatctc ata 53 28 33 DNA Artificial PCR
primer 28 gggttacgcg tggccgctaa ctacactaac agc 33 29 31 DNA
Artificial PCR primer 29 gggttggtac ctttaattct cgagtgttaa g 31 30
642 DNA Bacillus subtilis mutation (346)..(346) Mutation tx1 in
yloS gene 30 atgaaaacaa ttaatatcgt tgcgggaggc ccgaaaaatc tcattcccga
tctaaccggc 60 tatacggatg aacacacgct ttggatcggt gttgacaaag
gcaccgtcac tctcttagat 120 gccgggatca ttcctgttga agccttcgga
gattttgaca gcataacgga gcaagaacgc 180 cggcgaatag aaaaagccgc
tcccgccctt catgtgtatc aagcagaaaa agatcaaaca 240 gatttagacc
tcgcccttga ttgggcgctg gaaaagcagc cggatattat tcagattttc 300
ggcattacag gcggcagagc tgatcatttt ttaggaaaca ttcagtttct gtataaaggt
360 gtaaaaacga acataaaaat taggctgata gacaaacaaa atcatattca
aatgttccct 420 cctggtgaat atgatattga gaaggatgaa aataagcgat
atatctcctt cataccgttt 480 tccgaagaca tacatgagct gaccctgacc
ggttttaaat atcctctaaa taattgtcat 540 attacgctcg gttcaacact
atgtattagt aacgaactca ttcattcacg aggtactttt 600 tcgtttgcaa
aaggcatatt aataatgata agaagcacgg at 642 31 214 PRT Bacillus
subtilis 31 Met Lys Thr Ile Asn Ile Val Ala Gly Gly Pro Lys Asn Leu
Ile Pro 1 5 10 15 Asp Leu Thr Gly Tyr Thr Asp Glu His Thr Leu Trp
Ile Gly Val Asp 20 25 30 Lys Gly Thr Val Thr Leu Leu Asp Ala Gly
Ile Ile Pro Val Glu Ala 35 40 45 Phe Gly Asp Phe Asp Ser Ile Thr
Glu Gln Glu Arg Arg Arg Ile Glu 50 55 60 Lys Ala Ala Pro Ala Leu
His Val Tyr Gln Ala Glu Lys Asp Gln Thr 65 70 75 80 Asp Leu Asp Leu
Ala Leu Asp Trp Ala Leu Glu Lys Gln Pro Asp Ile 85 90 95 Ile Gln
Ile Phe Gly Ile Thr Gly Gly Arg Ala Asp His Phe Leu Gly 100 105 110
Asn Ile Gln Phe Leu Tyr Lys Gly Val Lys Thr Asn Ile Lys Ile Arg 115
120 125 Leu Ile Asp Lys Gln Asn His Ile Gln Met Phe Pro Pro Gly Glu
Tyr 130 135 140 Asp Ile Glu Lys Asp Glu Asn Lys Arg Tyr Ile Ser Phe
Ile Pro Phe 145 150 155 160 Ser Glu Asp Ile His Glu Leu Thr Leu Thr
Gly Phe Lys Tyr Pro Leu 165 170 175 Asn Asn Cys His Ile Thr Leu Gly
Ser Thr Leu Cys Ile Ser Asn Glu 180 185 190 Leu Ile His Ser Arg Gly
Thr Phe Ser Phe Ala Lys Gly Ile Leu Ile 195 200 205 Met Ile Arg Ser
Thr Asp 210 32 214 PRT Bacillus subtilis 32 Met Lys Thr Ile Asn Ile
Val Ala Gly Gly Pro Lys Asn Leu Ile Pro 1 5 10 15 Asp Leu Thr Gly
Tyr Thr Asp Glu His Thr Leu Trp Ile Gly Val Asp 20 25 30 Lys Gly
Thr Val Thr Leu Leu Asp Ala Gly Ile Ile Pro Val Glu Ala 35 40 45
Phe Gly Asp Phe Asp Ser Ile Thr Glu Gln Glu Arg Arg Arg Ile Glu 50
55 60 Lys Ala Ala Pro Ala Leu His Val Tyr Gln Ala Glu Lys Asp Gln
Thr 65 70 75 80 Asp Leu Asp Leu Ala Leu Asp Trp Ala Leu Glu Lys Gln
Pro Asp Ile 85 90 95 Ile Gln Ile Phe Gly Ile Thr Gly Gly Arg Ala
Asp His Phe Leu Gly 100 105 110 Asn Ile Gln Leu Leu Tyr Lys Gly Val
Lys Thr Asn Ile Lys Ile Arg 115 120 125 Leu Ile Asp Lys Gln Asn His
Ile Gln Met Phe Pro Pro Gly Glu Tyr 130 135 140 Asp Ile Glu Lys Asp
Glu Asn Lys Arg Tyr Ile Ser Phe Ile Pro Phe 145 150 155 160 Ser Glu
Asp Ile His Glu Leu Thr Leu Thr Gly Phe Lys Tyr Pro Leu 165 170 175
Asn Asn Cys His Ile Thr Leu Gly Ser Thr Leu Cys Ile Ser Asn Glu 180
185 190 Leu Ile His Ser Arg Gly Thr Phe Ser Phe Ala Lys Gly Ile Leu
Ile 195 200 205 Met Ile Arg Ser Thr Asp 210 33 576 DNA Bacillus
subtilis mutation (103)..(103) Mutation tx26-1 in yuaJ 33
atgaatcaat ctaagcaact ggttcgcctt attgaaattg ccattatgac agcggcagcc
60 gttattttag acattgtctc aggaatgttt cttagcatgc cttaaggagg
ctcggtctcc 120 atcatgatga ttccgatctt tttaatttcg tttcgctggg
gtgtcaaagc aggtcttact 180 acaggtttgt tgacaggtct agtacaaata
gcaatcggaa acttgtttgc tcaacatcct 240 gtacagctat tgttagatta
cattgtcgct ttcgcagcaa tcggcataag cggctgtttc 300 gcttcttctg
tccgtaaagc cgctgtatca aaaacaaaag ggaaattgat tgtttcagtg 360
gtcagcgcag tttttatcgg cagtttgctg cgctatgccg cgcatgtcat ttcaggagct
420 gtgtttttcg gcagctttgc tccaaaagga acaccggtat ggatttattc
tttaacttat 480 aatgcgactt acatggttcc ttcattcatt atttgtgcaa
ttgtcctatg tttattattt 540 atgacagcac cccgtctgct taaaagtgac aaagcg
576 34 34 PRT Bacillus subtilis 34 Met Asn Gln Ser Lys Gln Leu Val
Arg Leu Ile Glu Ile Ala Ile Met 1 5 10 15 Thr Ala Ala Ala Val Ile
Leu Asp Ile Val Ser Gly Met Phe Leu Ser 20 25 30 Met Pro 35 192 PRT
Bacillus subtilis 35 Met Asn Gln Ser Lys Gln Leu Val Arg Leu Ile
Glu Ile Ala Ile Met 1 5 10 15 Thr Ala Ala Ala Val Ile Leu Asp Ile
Val Ser Gly Met Phe Leu Ser 20 25 30 Met Pro Gln Gly Gly Ser Val
Ser Ile Met Met Ile Pro Ile Phe Leu 35 40 45 Ile Ser Phe Arg Trp
Gly Val Lys Ala Gly Leu Thr Thr Gly Leu Leu 50 55 60 Thr Gly Leu
Val Gln Ile Ala Ile Gly Asn Leu Phe Ala Gln His Pro 65 70 75 80 Val
Gln Leu Leu Leu Asp Tyr Ile Val Ala Phe Ala Ala Ile Gly Ile 85 90
95 Ser Gly Cys Phe Ala Ser Ser Val Arg Lys Ala Ala Val Ser Lys Thr
100 105 110 Lys Gly Lys Leu Ile Val Ser Val Val Ser Ala Val Phe Ile
Gly Ser 115 120 125 Leu Leu Arg Tyr Ala Ala His Val Ile Ser Gly Ala
Val Phe Phe Gly 130 135 140 Ser Phe Ala Pro Lys Gly Thr Pro Val Trp
Ile Tyr Ser Leu Thr Tyr 145 150 155 160 Asn Ala Thr Tyr Met Val Pro
Ser Phe Ile Ile Cys Ala Ile Val Leu 165 170 175 Cys Leu Leu Phe Met
Thr Ala Pro Arg Leu Leu Lys Ser Asp Lys Ala 180 185 190 36 1470 DNA
Bacillus subtilis mutation (538)..(538) Mutation tx26-2 in ykoD 36
atgcaagcct ttgatgagct tctgacggtt gagcagctca gcttctctta tgaagaagac
60 gagaaaccgg tttttcaaga catttcgttt gagcttcaaa aaggagaatg
tgttttatta 120 ttaggaccga gcggatgcgg taaaagctcg ctcgcccttt
gtttaaacgg tctatatccg 180 gaggcttgcg acggcattca gtccggacat
gtatttctat ttcaaaagcc ggtcacagat 240 gctgaaacct ccgaaacgat
tactcagcat gccggggtcg tttttcagga tcctgatcag 300 cagttctgca
tgctgacggt ggaggacgaa atagcgttcg ggctggaaaa tctgcaaatt 360
ccaaaagaag aaatgacaga gaaaatcaac gccgtattag gaaaattacg cattacccat
420 ttaaaagaaa aaatgatctc aaccctttca ggaggacaaa agcagaaagt
ggctctcgcc 480 tgtattttgg cgatggagcc tgagcttatt attttagatg
agccgacctc tcttttaaac 540 cctttctcag ctcgggagtt cgttcatctg
atgaaggatc ttcagcggga aaaaggtttc 600 agcctcctcg tcattgagca
ccagcttgat gaatgggcgc cttggattga gagaacgatc 660 gtactcgaca
aatcaggcaa aaaggcactg gatggcctga cgaaaaatct atttcagcat 720
gaagcggaga cactaaagaa attgggcatc gcaattccaa aggtctgtca tctgcaggaa
780 aagctgagta tgccgtttac tttatcaaaa gagatgctgt tcaaagagcc
tattcctgcc 840 gggcatgtca aaaagaagaa agccccttct ggggagagtg
tgcttgaagt cagcagcctt 900 tcgttcgcga gaggacagca ggcgattttc
aaagacatca gcttttcgtt gcgcgaaggc 960 tctttaacgg cgcttgtcgg
tccgaacgga actggaaaat cgacgctcct atcagttctg 1020 gccagtctca
tgaaaccgca aagcggcaaa atccttctct atgatcagcc gctgcagaaa 1080
tataaagaaa aagaattgcg taaacggatg ggatttgttt ttcaaaaccc tgagcatcaa
1140 ttcgtcaccg atacggtgta tgacgagctt ctgttcggcc agaaagcaaa
tgctgaaact 1200 gagaaaaaag cgcaacacct gctgcagcgt tttggtcttg
cgcatttggc tgatcatcat 1260 ccgtttgcga tcagccaagg gcaaaaacgg
cgactgagcg tagctactat gctcatgcat 1320 gacgtaaagg ttttattatt
agacgaacca acctttggcc aggatgcccg cacggcggct 1380 gaatgcatgg
aaatgattca acgtatcaag gcagagggaa ctgctgtcct tatgattaca 1440
caaggatatg gagcaagtct cttcgtatgc 1470 37 490 PRT Bacillus subtilis
37 Met Gln Ala Phe Asp Glu Leu Leu Thr Val Glu Gln Leu Ser Phe Ser
1 5 10 15 Tyr Glu Glu Asp Glu Lys Pro Val Phe Gln Asp Ile Ser Phe
Glu Leu 20 25 30 Gln Lys Gly Glu Cys Val Leu Leu Leu Gly Pro Ser
Gly Cys Gly Lys 35 40 45 Ser Ser Leu Ala Leu Cys Leu Asn Gly Leu
Tyr Pro Glu Ala Cys Asp 50 55 60 Gly Ile Gln Ser Gly His Val Phe
Leu Phe Gln Lys Pro Val Thr Asp 65 70 75 80 Ala Glu Thr Ser Glu Thr
Ile Thr Gln His Ala Gly Val Val Phe Gln 85 90 95 Asp Pro Asp Gln
Gln Phe Cys Met Leu Thr Val Glu Asp Glu Ile Ala 100 105 110 Phe Gly
Leu Glu Asn Leu Gln Ile Pro Lys Glu Glu Met Thr Glu Lys 115 120 125
Ile Asn Ala Val Leu Gly Lys Leu Arg Ile Thr His Leu Lys Glu Lys 130
135 140 Met Ile Ser Thr Leu Ser Gly Gly Gln Lys Gln Lys Val Ala Leu
Ala 145 150 155 160 Cys Ile Leu Ala Met Glu Pro Glu Leu Ile Ile Leu
Asp Glu Pro Thr 165 170 175 Ser Leu Leu Asn Pro Phe Ser Ala Arg Glu
Phe Val His Leu Met Lys 180 185 190 Asp Leu Gln Arg Glu Lys Gly Phe
Ser Leu Leu Val Ile Glu His Gln 195 200 205 Leu Asp Glu Trp Ala Pro
Trp Ile Glu Arg Thr Ile Val Leu Asp Lys 210 215 220 Ser Gly Lys Lys
Ala Leu Asp Gly Leu Thr Lys Asn Leu Phe Gln His 225 230 235 240 Glu
Ala Glu Thr Leu Lys Lys Leu Gly Ile Ala Ile Pro Lys Val Cys 245 250
255 His Leu Gln Glu Lys Leu Ser Met Pro Phe Thr Leu Ser Lys Glu Met
260 265 270 Leu Phe Lys Glu Pro Ile Pro Ala Gly His Val Lys Lys Lys
Lys Ala 275 280 285 Pro Ser Gly Glu Ser Val Leu Glu Val Ser Ser Leu
Ser Phe Ala Arg 290 295 300 Gly Gln Gln Ala Ile Phe Lys Asp Ile Ser
Phe Ser Leu Arg Glu Gly 305 310 315 320 Ser Leu Thr Ala Leu Val Gly
Pro Asn Gly Thr Gly Lys Ser Thr Leu 325 330 335 Leu Ser Val Leu Ala
Ser Leu Met Lys Pro Gln Ser Gly Lys Ile Leu 340 345 350 Leu Tyr Asp
Gln Pro Leu Gln Lys Tyr Lys Glu Lys Glu Leu Arg Lys 355 360 365 Arg
Met Gly Phe Val Phe Gln Asn Pro Glu His Gln Phe Val Thr Asp 370 375
380 Thr Val Tyr Asp Glu Leu Leu Phe Gly Gln Lys Ala Asn Ala Glu Thr
385 390 395 400 Glu Lys Lys Ala Gln His Leu Leu Gln Arg Phe Gly Leu
Ala His Leu 405 410 415 Ala Asp His His Pro Phe Ala Ile Ser Gln Gly
Gln Lys Arg Arg Leu 420 425 430 Ser Val Ala Thr Met Leu Met His Asp
Val Lys Val Leu Leu Leu Asp 435 440 445 Glu Pro Thr Phe Gly Gln Asp
Ala Arg Thr Ala Ala Glu Cys Met Glu 450 455 460 Met Ile Gln Arg Ile
Lys Ala Glu Gly Thr Ala Val Leu Met Ile Thr 465 470 475 480 Gln Gly
Tyr Gly Ala Ser Leu Phe Val Cys 485 490 38 490 PRT Bacillus
subtilis 38 Met Gln Ala Phe Asp Glu Leu Leu Thr Val Glu Gln Leu Ser
Phe Ser 1 5 10 15 Tyr Glu Glu Asp Glu Lys Pro Val Phe Gln Asp Ile
Ser Phe Glu Leu 20 25 30 Gln Lys Gly Glu Cys Val Leu Leu Leu Gly
Pro Ser Gly Cys Gly Lys 35 40 45 Ser Ser Leu Ala Leu Cys Leu Asn
Gly Leu Tyr Pro Glu Ala Cys Asp 50 55 60 Gly Ile Gln Ser Gly His
Val Phe Leu Phe Gln Lys Pro Val Thr Asp 65 70 75 80 Ala Glu Thr Ser
Glu Thr Ile Thr Gln His Ala Gly Val Val Phe Gln 85 90 95 Asp Pro
Asp Gln Gln Phe Cys Met Leu Thr Val Glu Asp Glu Ile Ala 100 105 110
Phe Gly Leu Glu Asn Leu Gln Ile Pro Lys Glu Glu Met Thr Glu Lys 115
120 125 Ile Asn Ala Val Leu Gly Lys Leu Arg Ile Thr His Leu Lys Glu
Lys 130 135 140 Met Ile Ser Thr Leu Ser Gly Gly Gln Lys Gln Lys Val
Ala Leu Ala 145 150 155 160 Cys Ile Leu Ala Met Glu Pro Glu Leu Ile
Ile Leu Asp Glu Pro Thr 165 170 175 Ser Leu Leu Asp Pro Phe Ser Ala
Arg Glu Phe Val His Leu Met Lys 180 185 190 Asp Leu Gln Arg Glu Lys
Gly Phe Ser Leu Leu Val Ile Glu His Gln 195 200 205 Leu Asp Glu Trp
Ala Pro Trp Ile Glu Arg Thr Ile Val Leu Asp Lys 210 215 220 Ser Gly
Lys Lys Ala Leu Asp Gly Leu Thr Lys Asn Leu Phe Gln His 225 230 235
240 Glu Ala Glu Thr Leu Lys Lys Leu Gly Ile Ala Ile Pro Lys Val Cys
245 250 255 His Leu Gln Glu Lys Leu Ser Met Pro Phe Thr Leu Ser Lys
Glu Met 260 265 270 Leu Phe Lys Glu Pro Ile Pro Ala Gly His Val Lys
Lys Lys Lys Ala 275 280 285 Pro Ser Gly Glu Ser Val Leu Glu Val Ser
Ser Leu Ser Phe Ala Arg 290 295 300 Gly Gln Gln Ala Ile Phe Lys Asp
Ile Ser Phe Ser Leu Arg Glu Gly 305 310 315 320 Ser Leu Thr Ala Leu
Val Gly Pro Asn Gly Thr Gly Lys Ser Thr Leu 325 330 335 Leu Ser Val
Leu Ala Ser Leu Met Lys Pro Gln Ser Gly Lys Ile Leu 340 345 350 Leu
Tyr Asp Gln Pro Leu Gln
Lys Tyr Lys Glu Lys Glu Leu Arg Lys 355 360 365 Arg Met Gly Phe Val
Phe Gln Asn Pro Glu His Gln Phe Val Thr Asp 370 375 380 Thr Val Tyr
Asp Glu Leu Leu Phe Gly Gln Lys Ala Asn Ala Glu Thr 385 390 395 400
Glu Lys Lys Ala Gln His Leu Leu Gln Arg Phe Gly Leu Ala His Leu 405
410 415 Ala Asp His His Pro Phe Ala Ile Ser Gln Gly Gln Lys Arg Arg
Leu 420 425 430 Ser Val Ala Thr Met Leu Met His Asp Val Lys Val Leu
Leu Leu Asp 435 440 445 Glu Pro Thr Phe Gly Gln Asp Ala Arg Thr Ala
Ala Glu Cys Met Glu 450 455 460 Met Ile Gln Arg Ile Lys Ala Glu Gly
Thr Ala Val Leu Met Ile Thr 465 470 475 480 Gln Gly Tyr Gly Ala Ser
Leu Phe Val Cys 485 490
* * * * *