Deciphering hygromycin B biosynthetic pathway and D-optimal design for production optimization

Abstract

Hygromycin B (HYG-B) is a 5-glycosylated 2-dexoystreptamine- aminoglycoside antibiotic-(2DOS-AGA) produced by Streptomyces hygroscopicus subspecies hygroscopicus NRRL ISP-5578 with broad-spectrum activity against many pathogenic bacteria, fungi and helminths. In the literature, limited studies are concerned with the biosynthetic pathway and different cultural conditions affecting the production of HYG-B. This study aimed to optimize key environmental conditions influencing its production as one-factor-at-a-time (OFAT) and as a statistical model of response surface D-optimal design (DOD). Moreover, the biosynthetic pathway of HYG-B in light of the identified genes/proteins of the HYG-B gene cluster was proposed and elucidated. The effect of culture media composition and incubation time were studied OFAT, and the results showed that both culture media 6 (CM6) and CM4 gave the highest specific productivity, 5.88 and 3.99 µg/mg, respectively, and 7 days as incubation time. So, using CM6 and 7 days incubation resulted in a sevenfold increase (190 µg/mL) compared to the original unoptimized condition (CM1 and 6 days incubation; 26.9 µg/mL). Three important factors-initial pH, incubation temperature, and agitation-were tested using a DOD quadratic model generating 20 experimental runs. An initial pH of 6.4, an incubation temperature, of 28 ℃, and agitation. of 295 rpm were predicted and experimentally verified, resulting in a 13-fold increase (371.5 µg/mL) compared to the unoptimized condition and a sevenfold increase compared to that obtained as OFAT. In conclusion, DOD design is an efficient tool for optimizing HYG-B. However, the optimized conditions should be scaled up in a bioreactor for industrial production of HYG-B by S. hygroscopicus NRRL ISP-5578.