Directed evolution, natural products for cancer chemotherapy, and micro‐biosensing robots

Abstract

Directed evolution is an effective and powerful method for protein engineering. To date, several different methods have been designed to create sequence diversity; unfortunately most of them are confined to the model bacterium Escherichia coli, and this constitutes a challenging limitation for the improvement of many industrially relevant biocatalysts. A clear example of this restriction is the production of new secretory enzymes that are of interest in the food, textile and pharmaceutical industries. In this case, the most common procedure used involves two organisms (E. coli and Bacillus subtilis), two transformations and several intermediate DNA manipulation steps. In addition, DNA transfer into B. subtilis is usually highly inefficient. Zhang and Zhang (2011) present a new methodology in Microbial Biotechnology that overcomes this problem by addressing it from two sides simultaneously. On one hand, they have increased the receptivity of B. subtilis to foreign DNA by increasing expression of the competence transcription factor ComK. On the other hand, they have improved the DNA material to be transformed so that candidate sequences are cloned into multimeric plasmids, by a combination of two PCR procedures, one error-prone for the target gene and one error-free for the vector. The authors demonstrate the efficiency of the protocol obtaining mutants of endoglucanase BsCel5 with increased hydrolase activity and improved secretion properties. Finally, we would like to highlight that apart from the time saving facet of this methodology, this protocol can also avoid bias as a result of multiple steps and the limitations of cloning mutants by traditional methods (restriction enzymes and ligation); thus broadening the range of potential candidates. Bacteria from the genus Streptomyces produce more than 70% of commercially available antibiotics, plus many other metabolites of therapeutic interest such as anti-tumour agents or immunosuppressive drugs. The production of these compounds is tightly controlled by means of γ-butyrolactones; microbial hormones that are also involved in the regulation of other processes such as morphological differentiation or pathogenesis. The article presented by D'Alia and co-workers focuses on the regulation of these hormones by the ScbA regulator, a homologue of the A-factor biosynthesis afsA gene from Streptomyces coelicolor (D'Alia et al., 2011). For this purpose, they studied the differences in transcript levels between the wild-type strain and a ΔscbA mutant using global microarray analysis, and the results were confirmed by more accurate qRT-PCR. The authors show that ScbA not only controls antibiotic production but also interferes with intracellular iron levels modulating siderophore desferrioxamine E biosynthesis. Interestingly, this study also shows that in the scbA mutant genes coding for enzymes related to primary metabolism are upregulated just before antibiotic production, placing butanolides upfront of general metabolic regulation in S. coelicolor. When S. coelicolor A3(2) grows under nutrient limited conditions, it triggers a gamut of nutrient–stress responses, mediated by a number of global regulators including PhoP, GlnR, AfsR and others. The action of these regulators is also integrated at the molecular level to control secondary metabolite biosynthesis and differentiation. As a result of the potential uses of these microbes in biotechnology, nutritional control has received relevant attention in the field and, in this issue, Martin and colleagues (2011) describe how phosphate control of primary and secondary metabolism in Streptomyces species is mediated by the two-component regulatory system PhoR-PhoP. PhoP controls secondary metabolism by binding to the PHO box in the afsS promoter. They illustrate that this PHO Box overlaps with the AfsR binding site (D'Alia et al., 2011), and based on this physical organization, they suggest that the afsS promoter serves to integrate the PhoP-med