Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora

Abstract

BackgroundIsepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2ʹ-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2ʹ hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis.ResultsWe blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2ʹ-NH2 into 2ʹ-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml.ConclusionWe constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0402-6) contains supplementary material, which is available to authorized users.

Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria

“…We have previously demonstrated that Cas9 poses toxicity in cyanobacteria15. We sought to circumvent the toxicity issue by employing an alternative RNA guided DNA nuclease, Fncpf1 from Francisella novicida 16.…”

“…We previously demonstrated CRISPR genome editing in Synechococcus 2973 by adapting pCRISPOmyces, a cas9 based system from Streptomyces, to function in Synechococcus 1315. However, this system was suboptimal because of toxicity issues.…”

“…In recent years, CRISPR genome editing technology has revolutionized the field of biotechnology by enabling precise, efficient modification of DNA sequences in a single step, in a wide variety of organisms from mammals11 to plants12 to bacteria131415. This technology is ideally suited to engineer markerless knock-ins, knock-outs or specific point mutations in numerous species.…”

“…This technology is ideally suited to engineer markerless knock-ins, knock-outs or specific point mutations in numerous species. Unfortunately, CRISPR technology has not been widely used in cyanobacteria due to the apparent toxicity of the Cas9 nuclease in these organisms15. We sought to overcome this obstacle by employing Cpf1 from Francisella novicida , a novel RNA directed dsDNA nuclease that we determined to be nontoxic to cyanobacteria.…”

Current knowledge and perspectives of Paenibacillus: a review

“…Synthesis of this enzyme requires a minimum of three structural genes and three genes for FeMo-cofactor biosynthesis [23]. However, optimum activity requires the presence of additional genes, as the specific activity of a nitrogenase expressed in Escherichia coli was only about 10% of that observed in the Paenibacillus species from which it was derived; and improvements were made with additional transgenes [24]. Alternatives to Nif have active site cofactors that lack Mo, instead containing both vanadium and iron (Vnf), or iron only (Anf) [23].…”