Primary Metabolism

Microbial life strategies

~1 mm~3 cm

ActinomycetesCyanobacteria

Filamentous Fungi

E. coli Salmonella

Streptococcus

“Nomads” “Settlers”

~10 cm

Settlers have to protect themselves from the Nomads

AntibioticsSiderophores

Signaling moleculesSpecialized cell walls/lipids

Secondary metabolites, or natural products

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Chemical Genetics

O

O

O

OO

OHO

O

HO

OH

HON

Secondarymetabolite

Genes (Open Reading Frames)

Gene Cluster

ATACP KS

ACP

KR

mAT KSACP

KR

mAT KSACPmAT KS

ACPmAT

ERDH KR

KSACP

KR

AT KSACP

KR

mAT TE

Domains

DNA sequence

5-module NRPSFAS/PKS-related

FAS/PKS-related

Enediyne PKS

Rifamycin

NRPS-related

Calicheamycin – part1

Calicheamycin – part2sid2

Staurosporine2-module PKS/NRPS

pks2sid1

FAS/PKS-related

4-module NRPS

FAS/PKS-relatedType I PKS

Lym

Cyclomarin

pks4nrps2

Lycopene/Iterative Type I PKS

S. arenicola5.68 Mb

The GenusSalinispora

Jensen, Fenical

Udwary, Ziegler, Lapidus, Moore, Jensen

A Yersiniabactin Cluster?

27698 bpunknown transport

Yersiniabactin clusterfrom Yersinia pestis

Yersiniabactin clusterfrom Yersinia pestis

The quick brown fox jumped over the lazy dog

A fast tan dingo leaped above some sleepy muttLow sequence

identity,Similar “product”

Yoda speak: Over the lazy dog the quick brown fox jumped.

The quick brown fox jumped over the lazy dog

S

N

S

NH OH

S

N

OH

COOH

S

N

OHOH

S

NH S

N

COOH

The quick brown fox jumped over the lazy dogHigh “sequenceidentity”,

But different“product”

Would lead to different,but similar, chemical products

that may have different activity

BLAST can’t reallytell the difference

Project #1: Operon/Cluster identification tool

Goal: Develop a rapid automated method to identify conserved gene clusters or operons across genomes.

Personnel needs: Programmer/scripter (Java or Perl is ideal)Biologist with some knowledge of gene structure or genomics

Rationale: I have a need to locate conserved or related operons or gene clusters across multiple species. Important to understanding of genome evolution, and functional analysis.

Identifying Gene Clusters

PKS: KSATDHERKRACPMethyl transferase MTTE

NRPS: CAT (PCP)EpimerizationC-cyclizationN-methyltransferaseTE

Other: Prenyl transferaseCytochrome P450Type III PKSKAS III

BLAST vs contig sequences

Evaluate proximity of hits

Examine predicted function ofgenes around “clusters” of hits.Determine boundaries for putative clusters

DNA sequence(s)

Putative clusters

The hard part

NP Chemistry

Biochemistry

Enzymology

Automation?

Genome

Library of query genes and

desirability scoresGene

extract

Score for each gene

KSATDHERKRACPMethyl transferase MTTECAT (PCP)EpimerizationC-cyclizationN-methyltransferaseTEPrenyl transferaseCytochrome P450KAS III

High scores

Low scores

DNA sequence(s)

Putative clusters

GenomeGenome(s)

Map across chromosome

Project #2: Synthetic Sequence Generator

Goal: Develop a tool to generate organism-specific DNA sequences suitable for DNA synthesis from protein sequences

Personnel needs: Programmer/scripterBiologist with some knowledge of molecular biology and genetics

Rationale: Synthesis of large DNA sequences is commercially feasible. There is need for an organism-agnostic design tool.

S.coelicolor SCO7671 region6901 bp

SCO7667 - phosphohydrolase

SCO7668 - regulatory

SCO7669 - oxidoreductase

SCO7670 - Isoprenylcysteine carboxyl methyltransferase

SCO7671 - Type III PKS

SCO7672 - prenyl transferase

SCO7673 - lipoprotein

SCO7674 - Cu-binding plastocyanin

SCO7675 - unknown membrane protein

Cluster 12 - Type III region - pks411301 bp

STRO2873

STRO2874 - DNA-binding

STRO2875 - DNA repair

STRO2876

STRO2877 - UbiA (SCO7672)

STRO2878 - UbiG benzoquinol methyltransferase

STRO2879 - Acyl-CoA dehydrogenase

STRO2880 - Type III PKS

STRO2881

STRO2882 - transcription factor

STRO2883

Old Way Dan’s WayType a DNA sequence

Email sequence to acompany to synthesize it

Transform into expression host

Culture organism

Evaluate activity in vivo

Type a DNA sequence

Email sequence to acompany to synthesize it

Transform into expression host

Culture organism

Evaluate activity in vivo

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

PCR-amplify gene(s)

Purify PCR product(s)

Clone into expression plasmid(s)

Confirm accuracy of sequence(s)

Transform into expression host

Culture organism

Purify proteins

Evaluate activity in vitro

Problem: I want to understand what the four-gene operon from each organism does, biochemically. NEED all four proteins active and in the same place. Plus, one organism is not available to me.

Protein sequence Codon usage table of host

organism

Reversetranslate

Codon-adjusted DNA sequence

Codon-adjusted DNA sequence

Re-engineered, optimized DNA

sequence

Re-engineered, optimized DNA

sequence

Optional modifications

Add expression tag(s)

Remove restriction sites

Remove or alter otherundesirable motifs

Engineered, optimized expressible DNA operonEngineered, optimized

expressible DNA operon

Adjust for expressible multi-gene transcript