New knowledge about the biosynthesis of lovastatin and its production by fermentation of Aspergillus terreus.

Lovastatin, and its semisynthetic derivative simvastatine, has great medical and economic importance, besides great potential for other uses. In the last years, a deeper and more complex view of secondary metabolism regulation has emerged, with the incorporation of cluster-specific and global transcription factors, and their relation to signaling cascades, as well as the new level of epigenetic regulation. Recently, a new mechanism, which regulates lovastatin biosynthesis, at transcriptional level, has been discovered: reactive oxygen species (ROS) regulation; also new unexpected environmental stimuli have been identified, which induce the synthesis of lovastatin, like quorum sensing-type molecules and support stimuli. The present review describes this new panorama and uses this information, together with the knowledge on lovastatin biosynthesis and genomics, as the foundation to analyze literature on optimization of fermentation parameters and medium composition, and also to fully understand new strategies for strain genetic improvement. This new knowledge has been applied to the development of more effective culture media, with the addition of molecules like butyrolactone I, oxylipins, and spermidine, or with addition of ROS-generating molecules to increase internal ROS levels in the cell. It has also been applied to the development of new strategies to generate overproducing strains of Aspergillus terreus, including engineering of the cluster-specific transcription factor (lovE), global transcription factors like the ones implicated in ROS regulation (or even mitochondrial alternative respiration aox gen), or the global regulator LaeA. Moreover, there is potential to apply some of these findings to the development of novel unconventional production systems. KEY POINTS: • New findings in regulation of lovastatin biosynthesis, like ROS regulation. • Induction by unexpected stimuli: autoinducer molecules and support stimuli. • Recent reports on culture medium and process optimization from this stand point. • Applications to molecular genetic strain improvement methods and production systems.

Penicillin and cephalosporin biosyntheses are also regulated by reactive oxygen species.

In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.