Anthracyclines: biosynthesis, engineering and clinical applications.

Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been widely used as an anticancer agent in the clinic for several decades, but its use is restricted due to severe side-effects such as cardiotoxicity. Recent studies into the mode-of-action of anthracyclines have revealed that effective cardiotoxicity-free anthracyclines can be generated by focusing on histone eviction activity, instead of canonical topoisomerase II poisoning leading to double strand breaks in DNA. These developments have coincided with an increased understanding of the biosynthesis of anthracyclines, which has allowed generation of novel compound libraries by metabolic engineering and combinatorial biosynthesis. Coupled to the continued discovery of new congeners from rare Actinobacteria, a better understanding of the biology of Streptomyces and improved production methodologies, the stage is set for the development of novel anthracyclines that can finally surpass doxorubicin at the forefront of cancer chemotherapy.

Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels.

Engineered strains of Saccharomyces cerevisiae are used for industrial production of succinic acid. Optimal process conditions for dicarboxylic-acid yield and recovery include slow growth, low pH, and high CO2 . To quantify and understand how these process parameters affect yeast physiology, this study investigates individual and combined impacts of low pH (3.0) and high CO2 (50%) on slow-growing chemostat and retentostat cultures of the reference strain S. cerevisiae CEN.PK113-7D. Combined exposure to low pH and high CO2 led to increased maintenance-energy requirements and death rates in aerobic, glucose-limited cultures. Further experiments showed that these effects were predominantly caused by low pH. Growth under ammonium-limited, energy-excess conditions did not aggravate or ameliorate these adverse impacts. Despite the absence of a synergistic effect of low pH and high CO2 on physiology, high CO2 strongly affected genome-wide transcriptional responses to low pH. Interference of high CO2 with low-pH signaling is consistent with low-pH and high-CO2 signals being relayed via common (MAPK) signaling pathways, notably the cell wall integrity, high-osmolarity glycerol, and calcineurin pathways. This study highlights the need to further increase robustness of cell factories to low pH for carboxylic-acid production, even in organisms that are already applied at industrial scale.

Metabolic engineering of Streptomyces peucetius for biosynthesis of N,N-dimethylated anthracyclines.

Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius, are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N-dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N-dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001. To achieve this, we introduced genes from the aclarubicin biosynthetic pathway encoding the sugar N-methyltransferases AclP and AknX2. Furthermore, the native gene for glycosyltransferase DnrS was replaced with genes encoding the aclarubicin glycosyltransferases AknS and AknT. Additionally, the gene for methylesterase RdmC from the rhodomycin biosynthetic pathway was introduced. Results: A new host was engineered successfully, whereby genes from the aclarubicin pathway were introduced and expressed. LC-MS/MS analysis of the engineered strains showed that dimethylated sugars were efficiently produced, and that these were incorporated ino the anthracycline biosynthetic pathway to produce the novel dimethylated anthracycline N,N-dimethyldaunorubicin. Further downstream tailoring steps catalysed by the cytochrome P450 monooxygenase DoxA exhibited limited efficacy with N,N-dimethylated substrates. This resulted in only low production levels of N,N-dimethyldaunorubicin and no N,N-dimethyldoxorubicin, most likely due to the low affinity of DoxA for dimethylated substrates. Discussion: S. peucetius G001 was engineered such as to produce N,N-dimethylated sugars, which were incorporated into the biosynthetic pathway. This allowed the successful production of N,N-dimethyldaunorubicin, an anticancer drug with reduced cytotoxicity. DoxA is the key enzyme that determines the efficiency of the biosynthesis of N,N-dimethylated anthracyclines, and engineering of this enzyme will be a major step forwards towards the efficient production of more N,N-dimethylated anthracyclines, including N,N-dimethyldoxorubicin. This study provides valuable insights into the biosynthesis of clinically relevant daunorubicin derivatives, highlighting the importance of combinatorial biosynthesis.