Research

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Research
on the Post-PKS Modification
Steps of Rifamycin B Biosynthesis
in
Amycolatopsis
mediterranei S699
Dr. Jun Xu
22 December 2006

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1. INTRODUCTION
1.1 Ansamycins
Common Characteristics of Structure
Ansamycins are a class of macrocyclic lactam.
In the ansamycins structure, there is
non-adjacent positions of an aromatic ring
system spanned by a long aliphatic bridge.
Ansamycins structure is rigid and compact.
Bioactivities
Ansamycins have wide range of bioactivity.
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1.2 Structures and Biological Activities of
Rifamycins
Rifamycins were the earliest reported
ansamycins.

The rifamycins (except for rifamycin B and
rifamycin W) inhibit the growth of
Gram-positive bacteria.
Rifamycin SV was the first compound applied
clinically.
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Structure of some important rifamycins and their
relation
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1.4  Rifampicin Its Mechanism of Action and
Resistance

Rif-RNAP cocrystal structure and a schematic
drawing of RNAP ß subunit interaction with Rif
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2. PROGRESS OF THE RESEARCH ON RIFAMYCIN
BIOSYNTHESIS
2.1  Earlier Studies on 3-Amino-5-hydroxybenz
oic Acid (AHBA) Biosynthesis

mC7N unit is a common moiety of ansamycins,
and it consists of a six-member carbocyclic
ring carrying an extra carbon and a nitrogen atom
in a meta arrangement.
AHBA was determined to be the source of this
mC7N unit in Amycolatopsis mediterranei.
AHBA was established to be the precursor for
the mC7N moiety in other ansamycins as well, such
as ansamitocin, actamycin, ansatrienin, and
streptovaricin, as well as the structurally
unrelated mitomycin.
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2.2 Rifamycin B Biosynthetic Gene Cluster
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The genetic organization of rif PKS genes and
active domains
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Research Strategies
Plasmid construction
Plasmid transformation
Mutant screening
Mutant fermentation
Product purification
Structure elucidation
Enzyme over-expression
Enzyme characterization
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3. RESULTS
3.1 Construction of Plasmid DNA
Cosmid FKN 108, covering part of the rifamycin B
PKS genes, the AHBA biosynthetic genes,
regulatory genes, and all of the post-PKS
modification genes, was used to construct suicide
plasmids for the inactivation or deletion of
target post-PKS modification genes.
Plasmid pTM 0031 AB
Plasmid pTM4502
Plasmid pTM 1401 and pTM 1901
Plasmid pTM0035
Plasmid pTM 0035S
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Disruption of orf14Deduced Function
Methyltransferase and
orf19Deduced Function 3-(3-Hydroxy-phenyl)propi
onate Hydroxylase
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3.2 Marking Plasmid DNA by Resistance Gene
All constructed plasmid DNAs were marked for
selection by the hygromycin B resistance gene
(hygR), which was obtained from plasmid pIJ 5607.
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  3.3 Transformation of Foreign Plasmid
DNA into Amycolatopsis
mediterranei S699
Timing of competent cell harvest
The cell cycle
Growth curve
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Plasmid DNA methylation
1. Numerous strain-specific restrication-modifica
tion barriers of organisms is the self-defense of
Nature.
2. Endonucleases can digest intruding foreign
DNA.
Foreign DNA that acquires the same methylation
pattern of the restriction sites characteristic
of the genomic DNA of Amycolatopsis mediterranei
S699 can evade, in some degree, the numerous
strain-specific restrication-modification
barriers of the organism.
3.
Plasmid DNA methylation is to incubate foreign
plasmid DNA with a combination of cell-free
extracts from different growth stages of
Amycolatopsis mediterranei S699, in the presence
of S-adenosylmethionine (AdoMet) and
ethylenediamine tetraacetic acid (EDTA).
4.
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Cell Growth Timing
 
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3.4 The Selection of Single Cross-over Mutants
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3.5 The Screening for double Cross-over Mutants
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3.6 Purification of the Secondary Metabolites of
the Mutants
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3.6.4 Products of orf 19--3-(3-Hydroxyphenyl)prop
ionate Hydroxylase Knockout and Deletion
of All PKS Genes
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Products of orf14--Methyltransferase Knockout
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H-27
OCH3
H-27
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Cu2
DTT Or Cell free extract
Effect of DTT and cell-free extract on the
oxidation of DMRSV to DMRS
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3.7 Overexpression of orf14
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Gel behavior of Orf14
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All Possible Substrates of Orf14
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Substrates Specificity
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Catalytic Properties of Orf14
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3.8 Crystallization
3.7.4 Crystallization of Orf14
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4. DISCUSSION
4.1 General
Orf3 may play a regulatory role in rifamycin
biosynthesis.
Orf4, orf9, orf10, orf11, and orf18 do not
affect rifamycn B production.
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4.2 Orf14 The Gene, the Mutants Secondary
Metabolites and the Enzyme
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4.3 Orf5 The Gene, the Mutants Secondary
Metabolites
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4.4 Orf19 the gene, the mutants secondary
metabolites
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Acknowledgement
Dr. Heinz G. Floss Dr. Taifo Mahmud Dr. Wenqing
Xu All Floss lab members
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