Investigating the Antibiotic Productivity of Streptomyces rimosusA. MACFADYEN1, Z. TANG1,2, R. KIRBY3, R. EDRADA-EBEL1, I. HUNTER1 and P. HERRON1

1Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK; 2State Key Laboratory of Bioreactor Engineering, East ChinaUniversity of Science and Engineering, Shanghai, China; 3Department of Life Sciences and Institute of Genome Science, National Yang-Ming University, Taipei,Taiwan

Conclusions•Loss of production of other secondary metabolites may lead to increased antibiotic production

•Modification of regulatory proteins may influence antibiotic production

•Alteration within metabolic pathways may alter the antibiotic productivity

•Single nucleotide polymorphisms can have a major impact on the production levels of an antibiotic

•Genomic comparisons can lend great insights into the varying contributions to differing antibiotic production levels and allow for targeted genetic engineering of industrial strains

IntroductionStreptomyces rimosus, the industrial strain used in the production of the type-II polyketide antibiotic oxytetracycline (OTC), has undergone extensive strain improvement over the past 50 years. This has resulted in OTC levels increasing from less than 0.5g per litre in the original soil isolate to over 70g per litre for the current production strain. By analysing the genome sequences of four strains that lie within this lineage (Figure 1), each with a different level of OTC productivity, we have begun to investigate the genetic basis behind increased OTC yield.

ObjectivesTo determine the genetic and metabolomic reasons behind increased antibiotic production, by comparing the original soil isolate to the later strains in the lineage

Methodology

Results

G7 M4018 15883S 23383

Cycloheximide 100 mg/mL

-ve Control

Figure 3: Rimocidin assay. Saccharomyces cerevisiae was used as the test organism. The negative control was ethanol.

• A loss of 155 Kb is present in 15883S which is unrelated to the spontaneous OTC cluster deletion

• There is a loss of genes required to encode the Polyketide Synthase of another secondary metabolite in strains M4018, 15883S and 23383 (Figure 3)

M4018 (20g/L) 15883S 23383 (55g/L)0

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200

300

400

500

600

700

Number of Single Nucleotide Polymorphisms Relative to Progenitor Strain G7

Num

ber o

f SN

Ps

Strain Name (OTC g/L)

Strain Improvement

Figure 2: Histogram of the Single Nucleotide Polymorphisms identified using MAQ SNP files generated during alignments.

Paired-end Reads

SNP

DELETION

Roche 454 Sequence M4018

Illumina 30 bp paired-end

sequence – G7, 15883S and

23383

Sequence Strains

Comparative analysis Individual Analysis

SEED ViewerHMMER

RAST

Move from in silico to in vivo

Artemis

CCG to UCG =

Proline to Serine

Figure 4: 23383 aligned with M4018 in Artemis. The SNP highlighted is within a homologue of SCO1937, a zwf gene, which are associated with antibiotic production.

• >600 SNPs identified in the latest strain

• Most noteworthy are Non-Synonymous SNPs in a putative Glucose 6-phosphate 1-dehydrogenase (Figure 4) and other proteins associated with regulation.

Future Work

•Recreate deletions in early strains and mutate the putative Glucose 6-Phosphate 1-Dehydrogenase

• Analyse mutant and wild type strains using metabolomics

Figure 1: Geneology of Strains. Denotes the strains of which we have genome sequences for.

Strain (OTC g/L)