Abstract
Polyketides, such as erythromycin, are complex natural products with diverse therapeutic applications. They are synthesized by multi-modular megaenzymes, so-called polyketide synthases (PKSs). The macrolide core of erythromycin, 6-deoxyerythronolide B (6dEB), is produced by the deoxyerythronolide B synthase (DEBS) that consists of three proteins each with a size of 330–370 kDa. We cloned and investigated the expression of the corresponding gene cluster from Saccharopolyspora erythraea, which comprises more than 30 kb, in Bacillus subtilis. It is shown that the DEBS genes are functionally expressed in B. subtilis when the native eryAI–III operon was separated into three individual expression cassettes with optimized ribosomal binding sites. A synthesis of 6dEB could be detected by using the acetoin-inducible acoA promoter and a fed-batch simulating EnBase-cultivation strategy. B. subtilis was capable of the secretion of 6dEB into the medium. In order to improve the 6dEB production, several genomic modifications of this production strain were tested. This included the knockout of the native secondary metabolite clusters of B. subtilis for the synthesis of surfactin (26 kb), bacillaene (76 kb), and plipastatin (38 kb). It is revealed that the deletion of the prpBD operon, responsible for propionyl-CoA utilization, resulted in a significant increase of the 6dEB product yield when exogenous propionate is provided. Although the presented B. subtilis 6dEB production strain is not competitive with established Escherichia coli 6dEB production strains, the results of this study indicate that B. subtilis is a suitable heterologous host for the secretory production of a complex polyketide.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6990-6) contains supplementary material, which is available to authorized users.
Highlights
The broad-spectrum antibiotic erythromycin, produced by the actinomycete Saccharopolyspora erythraea, is a well-studied representative of a class of complex natural products called polyketides (Corcoran 1981)
In order to investigate the expression of the eryI-III gene cluster from Saccharopolyspora erythraea, which comprises more than 30 kb, in B. subtilis, the deoxyerythronolide B synthase (DEBS) genes were localized to the B. subtilis JK13 chromosome in three different cluster organizations: (A) native operon, (B) native operon with optimized ribosomal binding sites, and (C) the three native ery gene sequences as separate cassettes with optimized ribosomal binding sites
The DEBS genes could be localized in the chromosome of B. subtilis in three different cluster organizations (A, Fig. 3 Comparison of modified B. subtilis JK59-1 strains with engineered genetic background. a Growth curves (OD600) during cultivation for 64 h (16 h pre- plus 48 h post-boostering) in EnBase® medium. b Quantitative multi-reaction monitoring (MRM) analysis of secreted 6deoxyerythronolide B (6dEB) of modified B. subtilis strains (t=48 h after boostering)
Summary
The broad-spectrum antibiotic erythromycin, produced by the actinomycete Saccharopolyspora erythraea, is a well-studied representative of a class of complex natural products called polyketides (Corcoran 1981). Appl Microbiol Biotechnol (2016) 100:1209–1220 natural products shows an enormous degree of structural diversity. This in turn results in the extraordinary variety of biological properties and is the reason for the significance of this group, especially for pharmaceutical applications. The corresponding gene cluster for 6dEB synthesis comprises three genes of approximately 10 kb (eryAI–III) They encode for three large proteins, denoted DEBS1, DEBS2, and DEBS3, that show a size of 330– 370 kDa each and form an enzymatic complex. With regard to the industrial production of drugs, many safety requirements which are regulated and controlled by the European Medicine Agency (EMA) or the Food and Drug Administration (FDA) have to be considered
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