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

## TL;DR

This study identifies the gentamicin biosynthesis pathway in Micromonospora purpureochromogenes and uses a D-optimal design to optimize production conditions, significantly increasing antibiotic yield.

## Contribution

The study proposes a gentamicin biosynthetic pathway and applies D-optimal design to optimize production conditions for the first time in this organism.

## Key findings

- The gentamicin biosynthetic pathway was proposed by analyzing identified genes and proteins.
- Optimized conditions increased gentamicin production 13.5-fold compared to the basic medium.
- D-optimal design successfully modeled and validated four key environmental factors for production.

## Abstract

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.

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.

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.

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.

The online version contains supplementary material available at 10.1186/s12866-025-04001-8.