D-optimal design model and biosynthetic pathway for gentamicin production by Micromonospora purpureochromogenes NRRL B-16094

Abstract

Background: Micromonospora purpureochromogenes NRRL B-16094, a natural producer of gentamicin (GEN), a 5,6-diglycosylated 2-dexoystreptamine-aminoglycoside antibiotic (2DOS-AGA) broad-spectrum bactericidal activity. In literature, limited studies are concerned with the biosynthetic route and various cultural conditions influencing GEN production.

Methods: Therefore, this study aimed to explore the GEN biosynthesis pathway and compare it to that of fortimicin and kanamycin. In addition, four key environmental conditions influencing GEN production were statistically optimized using response surface D-optimal design (DOD). Herein, the biosynthetic pathway of GEN was proposed based on the biochemistry of the identified genes/proteins within the gene cluster. Comparing the GEN-biosynthetic gene cluster to that of kanamycin and fortimicin suggested that gentamicin biosynthesis could have originated from a combination of biosynthetic pathways of both antibiotics.

Results: For the optimization experiments, culture media 4 (CM4) and 6 (CM6) gave the highest specific productivity at 6.36 and 3.80 µg/mg, respectively. A DOD quadratic model was successfully generated to optimize four key environmental factors. Predicted and experimentally confirmed optimized factors were an initial pH of 7, an incubation temperature of 30˚C, and an agitation of 300 rpm for 10 days. This resulted in a 13.5-fold increase (289.5 µg/mL) over that produced by the basic CM1 production medium (21.4 µg/mL) and 2.4 times (over that obtained by CM4 (123.7 µg/mL) as verified by HPLC analysis.

Conclusion: DOD is an efficient tool for optimizing GEN. Accordingly, the optimized conditions are highly advisable during the scaling up of GEN production by M. purpureochromogenes NRRL B-16094.

Keywords: D-optimal design Micromonospora purpureochromogenes NRRL B-16094; Fortimicin; Gentamicin; Kanamycin.

Fig. 1

Biosynthetic gene cluster of gentamicin (GEN) produced by M. purpureochromogenes NRRL B-16,094 compared to that of fortimicin and kanamycin gene clusters. Open reading frames (ORFs) that are conserved in gentamicin (gen), kanamycin (kan), and fortimicin (for) biosynthetic gene clusters are colored red. ORFs conserved in gen- and kan- clusters are colored in yellow. ORFs conserved in gen and for clusters are colored in green. ORFs reside outside the ACAGA gene clusters (dotted blue)

Fig. 2

Chemical structure of gentamicin, fortimicin and kanamycin. Arrows indicate similarities in structure in both sugar and aglycone moieties among three aminoglycoside antibiotics (AGAs)

Fig. 3

The proposed biosynthetic pathway of gentamicin in its natural producer, M. purpureochromogenes NRRL B-16,094. AT, aminotransferase, GT, glycosyltransferase, GEN, Gentamicin, GEN-C (Gentamicin C-complex), SSM, Sisomycin, VDM, Verdamicin

Fig. 4

Three-dimensional (3D) surface plots for the effects of: a) initial pH versus incubation temperature; b) incubation temperature versus agitation; c) agitation versus incubation time; d) incubation time versus initial pH on gentamicin (GEN) produced by M. purpureochromogenes NRRL B-16094

Fig. 5

Model Diagnostics of the D-optimal design (DOD): (a) Box-Cox plot for Power Transforms; (b) Residuals vs. Run plot; c); Predicted vs. actual plot; d) Normal plot of Residuals on GEN produced by M. purpureochromogenes NRRL B-16094

Fig. 6

Graphical presentation of the GEN produced by M. purpureochromogenes NRRL B-16,094 under un-optimized, optimized culture medium (CM4) and optimized condition using the D-optimal model