Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6′-deoxy-bleomycin Z production

Abstract

The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6'-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6'-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6'-deoxy-BLM at above 70 mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6'-deoxy-BLM via pilot-scale microbial fermentation.

Efficiently activated ε‐poly‐L‐lysine production by multiple antibiotic‐resistance mutations and acidic pH shock optimization in Streptomyces albulus

“…Researchers tended to choose higher antibiotic concentrations (20–50 MIC) to select the strains with mutations in ribosome gene rpsL or rplF , which further contributed to the aberrant protein synthesis activity and high production (Wang, Hosaka, & Ochi, ). Some studies also showed a positive correlation between antibiotic concentration and target product production (Zhu et al., ). However, we concluded from our previous study that colonies cultured on plates containing lower antibiotics exhibited better growth states and higher ε‐PL production.…”

“…Ribosome engineering is a novel and remarkably efficient tool for screening high-producing strains (Funane et al, 2018;Hu & Ochi, 2001). It can modulate the ribosome by simply inducing mutations that confer resistance to antibiotics (streptomycin, paromomycin, etc.)…”

Streptomycin‐induced ribosome engineering complemented with fermentation optimization for enhanced production of 10‐membered enediynes tiancimycin‐A and tiancimycin‐D

“…Although metabolic engineering drove by synthetic biology has shown great potential in strain improvement, its successful application strictly depends on the genetic amenability of the target strain and comprehensive understanding about metabolic pathways (Baltz, ; Smanski et al, ). In contrast, ribosome engineering has circumvented labor‐intensive random mutagenesis or demanding genetic manipulation (Ochi, ), and could be complemented by further medium optimization and fermentation regulation (Y. Zhang et al, ; Zhu et al, ), therefore, it provided us a simple but practical strategy for titer improvement of various natural products.…”

“…In contrast, ribosome engineering has circumvented laborintensive random mutagenesis or demanding genetic manipulation (Ochi, 2017), and could be complemented by further medium optimization and fermentation regulation (Y. Zhang et al, 2015;Zhu et al, 2018), therefore, it provided us a simple but practical strategy for titer improvement of various natural products.…”

“…Therefore, specific ribosome engineering mutations differ in their impact on gene transcription and metabolic changes including the production of antibiotics. Moreover, ribosome engineering could complement with subsequent medium optimization and fermentation regulation to further exploit the biosynthetic potential of mutants (Zhu et al, ).…”