Effects of the Methylmalonyl-CoA Metabolic Pathway on Ansamitocin Production in Actinosynnema pretiosum

Ansamitocins, which may have antitumor activity, are important secondary metabolites produced by Actinosynnema pretiosum sp. auranticum ATCC 31565. As one of the precursors for ansamitocin biosynthesis, methylmalonyl-CoA may be a critical metabolic node for secondary metabolism in A. pretiosum. In this study, we investigated two key enzymes related to the methylmalonyl-CoA metabolic pathway: methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC). For MCM, inactivation of the asm2277 gene (encoding the large subunit of MCM) resulted in 3-fold increase in ansamitocin P-3 (AP-3) production (reaching 70 mg/L) compared with that in wild-type A. pretiosum. The three genes responsible for PCC were asm6390, encoding propionyl-CoA carboxylase beta chain, and asm6229 and asm6396, which encoded biotin carboxylases, respectively. Heterogeneous overexpression of the amir6390 gene alone and concurrent overexpression of amir6390 with both amir6396 and amir6229 were carried out, and the resulting engineered strains could produce AP-3 at levels that were 1.6-fold and 3-fold (28.3 and 51.5 mg/L in flask culture, respectively) higher than that in the wild-type strain. These results suggested that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively increase ansamitocin production in A. pretiosum.

[1]  I. Jacobsen,et al.  Candida albicans Utilizes a Modified β-Oxidation Pathway for the Degradation of Toxic Propionyl-CoA* , 2014, The Journal of Biological Chemistry.

[2]  L. Bai,et al.  The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  I. Brikun,et al.  Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production. , 2007, Metabolic engineering.

[4]  D. Hopwood,et al.  A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome , 1992, Journal of bacteriology.

[5]  Q. Hua,et al.  Enhancement of ansamitocin P-3 production in Actinosynnema pretiosum by a synergistic effect of glycerol and glucose , 2013, Journal of Industrial Microbiology & Biotechnology.

[6]  C. Gilmore,et al.  Maytansine, a novel antileukemic ansa macrolide from Maytenus ovatus. , 1972, Journal of the American Chemical Society.

[7]  H. Floss,et al.  3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics* , 1998, The Journal of Biological Chemistry.

[8]  Yong-qiang Zhang,et al.  Connection of Propionyl-CoA Metabolism to Polyketide Biosynthesis in Aspergillus nidulans , 2004, Genetics.

[9]  Linquan Bai,et al.  Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B. , 2002, Journal of the American Chemical Society.

[10]  Richard H. Baltz,et al.  Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes , 2001, Antonie van Leeuwenhoek.

[11]  T. Hasegawa,et al.  Ansamitocin, a group of novel maytansinoid antibiotics with antitumour properties from Nocardia , 1977, Nature.

[12]  Yimeng Kong,et al.  Genotyping of amino acid-producing Corynebacterium glutamicum strains based on multi-locus sequence typing (MLST) scheme , 2015, Bioresources and Bioprocessing.

[13]  Linquan Bai,et al.  Enhancement of UDPG synthetic pathway improves ansamitocin production in Actinosynnem pretiosum , 2015, Applied Microbiology and Biotechnology.

[14]  A. Kumar UV mutagenesis treatment for improved production of endoglucanase and β-glucosidase from newly isolated thermotolerant actinomycetes, Streptomyces griseoaurantiacus , 2015, Bioresources and Bioprocessing.

[15]  Christine J. Martin,et al.  Increasing the efficiency of heterologous promoters in actinomycetes. , 2002, Journal of molecular microbiology and biotechnology.

[16]  Pietro Alifano,et al.  Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering. , 2014, Metabolic engineering.

[17]  Z. Deng,et al.  Two pHZ1358-derivative vectors for efficient gene knockout in streptomyces. , 2010, Journal of microbiology and biotechnology.

[18]  John A. Robinson,et al.  Insertional Inactivation of Methylmalonyl Coenzyme A (CoA) Mutase and Isobutyryl-CoA Mutase Genes in Streptomyces cinnamonensis: Influence on Polyketide Antibiotic Biosynthesis , 1999, Journal of bacteriology.

[19]  Won Seok Jung,et al.  A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in Streptomyces hygroscopicus ATCC 29253 , 2011, Applied Microbiology and Biotechnology.

[20]  I. Brikun,et al.  Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea , 2006, Journal of Industrial Microbiology and Biotechnology.

[21]  I. Brikun,et al.  Engineering precursor flow for increased erythromycin production in Aeromicrobium erythreum. , 2004, Metabolic engineering.

[22]  Q. Hua,et al.  Heterologous expression and characterization of soluble recombinant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Actinosynnema pretiosum ssp. auranticum ATCC31565 through co-expression with Chaperones in Escherichia coli. , 2012, Protein expression and purification.

[23]  A. Demain,et al.  Genetic improvement of processes yielding microbial products. , 2006, FEMS microbiology reviews.

[24]  Jun Chen,et al.  Process optimization with alternative carbon sources and modulation of secondary metabolism for enhanced ansamitocin P-3 production in Actinosynnema pretiosum. , 2014, Journal of biotechnology.

[25]  Weiwen Zhang,et al.  Molecular analysis and heterologous expression of the gene encoding methylmalonyl—coenzyme a mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32 , 1999, Applied biochemistry and biotechnology.

[26]  Young Ji Yoo,et al.  Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor , 2009, Journal of Industrial Microbiology & Biotechnology.

[27]  S. Akiyama,et al.  Biosynthetic origin of aminobenzenoid nucleus (C7N-unit) of ansamitocin, a group of novel maytansinoid antibiotics. , 1982, The Journal of antibiotics.

[28]  L. Bai,et al.  The post-polyketide synthase modification steps in the biosynthesis of the antitumor agent ansamitocin by Actinosynnema pretiosum. , 2003, Journal of the American Chemical Society.

[29]  Lei Tang,et al.  Insights into the Role of Glucose and Glycerol as a Mixed Carbon Source in the Improvement of ε-Poly-l-Lysine Productivity , 2014, Applied Biochemistry and Biotechnology.

[30]  H. Xiang,et al.  Propionyl Coenzyme A (Propionyl-CoA) Carboxylase in Haloferax mediterranei: Indispensability for Propionyl-CoA Assimilation and Impacts on Global Metabolism , 2014, Applied and Environmental Microbiology.

[31]  Kenji Watanabe,et al.  Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Dana J Morrone,et al.  Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering , 2009, Applied Microbiology and Biotechnology.

[33]  L. Bai,et al.  Identification of asm19 as an acyltransferase attaching the biologically essential ester side chain of ansamitocins using N-desmethyl-4,5-desepoxymaytansinol, not maytansinol, as its substrate. , 2002, Journal of the American Chemical Society.

[34]  J. Nielsen,et al.  Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics. , 2004, Advances in biochemical engineering/biotechnology.