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  • Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Applied microbiology and biotechnology (2017-12-03)
Renata Novakova, Luz Elena Núñez, Dagmar Homerova, Renata Knirschova, Lubomira Feckova, Bronislava Rezuchova, Beatrica Sevcikova, Nuria Menéndez, Francisco Morís, Jesús Cortés, Jan Kormanec
ABSTRACT

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Lithium acetate, 99.95% trace metals basis
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Yeast Nitrogen Base Without Amino Acids, Yeast classification medium used for selecting yeasts based on amino acid and carbohydrate requirements
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Yeast Synthetic Drop-out Medium Supplements, without leucine
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