Since the advent of systems and synthetic biology, many studies have sought to harness microbes as cell factories through genetic and metabolic engineering approaches. Yeast and filamentous fungi have been successfully harnessed to produce fine and high value-added chemical products. In this review, we present some of the most promising advances from recent years in the use of fungi for this purpose, focusing on the manipulation of fungal strains using systems and synthetic biology tools to improve metabolic flow and the flow of secondary metabolites by pathway redesign. We also review the roles of bioinformatics analysis and predictions in synthetic circuits, highlighting in silico systemic approaches to improve the efficiency of synthetic modules.

As the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad hostrange plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

Bacterial promoters are usually formed by multiple cis-regulatory elements recognized by a plethora of transcriptional factors (TFs). From those, global regulators are key elements since these TFs are responsible for the regulation of hundreds of genes in the bacterial genome. For instance, Fis and IHF are global regulators that play a major role in gene expression control in Escherichia coli, and usually, multiple cis-regulatory elements for these proteins are present at target promoters. Here, we investigated the relationship between the architecture of the cis-regulatory elements for Fis and IHF in E. coli. For this, we analyze 42 synthetic promoter variants harboring consensus cis-elements for Fis and IHF at different distances from the core −35/−10 region and in various numbers and combinations. We first demonstrated that although Fis preferentially recognizes its consensus cis-element, it can also recognize, to some extent, the consensus-binding site for IHF, and the same was true for IHF, which was also able to recognize Fis binding sites. However, changing the arrangement of the cis-elements (i.e., the position or number of sites) can completely abolish the non-specific binding of both TFs. More remarkably, we demonstrated that combining cis-elements for both TFs could result in Fis and IHF repressed or activated promoters depending on the final architecture of the promoters in an unpredictable way. Taken together, the data presented here demonstrate how small changes in the architecture of bacterial promoters could result in drastic changes in the final regulatory logic of the system, with important implications for the understanding of natural complex promoters in bacteria and their engineering for novel applications.

Here, we investigated novel interactions of three global regulators of the network that controls biofilm formation in the model bacterium Escherichia coli using computational network analysis, an in vivo reporter assay and physiological validation experiments. We were able to map critical nodes that govern planktonic to biofilm transition and identify 8 new regulatory interactions for CRP, IHF or Fis responsible for the control of the promoters of rpoS, rpoE, flhD, fliA, csgD and yeaJ. Additionally, an in vivo promoter reporter assay and motility analysis revealed a key role for IHF as a repressor of cell motility through the control of FliA sigma factor expression. This investigation of first stage and mature biofilm formation indicates that biofilm structure is strongly affected by IHF and Fis, while CRP seems to provide a fine-tuning mechanism. Taken together, the analysis presented here shows the utility of combining computational and experimental approaches to generate a deeper understanding of the biofilm formation process in bacteria.