Recent Progress on Systems and Synthetic Biology Approaches to Engineer Fungi As Microbial Cell Factories

Abstract

Filamentous fungi are remarkable organisms naturally specialized in deconstructing plant biomass and this feature has a tremendous potential for biofuel production from renewable sources. The past decades have been marked by a remarkable progress in the genetic engineering of fungi to generate industry-compatible strains needed for some biotech applications. In this sense, progress in this field has been marked by the utilization of high-throughput techniques to gain deep understanding of the molecular machinery controlling the physiology of these organisms, starting thus the Systems Biology era of fungi. Additionally, genetic engineering has been extensively applied to modify wellcharacterized promoters in order to construct new expression systems with enhanced performance under the conditions of interest. In this review, we discuss some aspects related to significant progress in the understating and engineering of fungi for biotechnological applications, with special focus on the construction of synthetic promoters and circuits in organisms relevant for industry. Different engineering approaches are shown, and their potential and limitations for the construction of complex synthetic circuits in these organisms are examined. Finally, we discuss the impact of engineered promoter architecture in the single-cell behavior of the system, an often-neglected relationship with a tremendous impact in the final performance of the process of interest. We expect to provide here some new directions to drive future research directed to the construction of high-performance, engineered fungal strains working as microbial cell factories.

Keywords: Fungal engineering; Regulatory networks; Synthetic biology; Synthetic promoters.; Systems biology.

Fig. (1)

Organism engineering strategies (native and recombinant) and related approaches to reach a successful CBP microorganism. Examples of the most promising candidates for starting each strategy are given, such as fungi and thermophilic bacteria for the native approach and yeasts for the recombinant one. While Systems Biology approaches supply the basis for understating metabolic microorganism functioning, Synthetic Biology provides new tools to implement the preferred features for effective production of biofuels.

Fig. (2)

Engineering synthetic promoters in fungi. The combination of functional regulatory elements has a tremendous potential for the construction of new expression systems for the applications of interest. As summarized in the figure, a pool of such elements with well-characterized functions can be used as a start point. At first, the category of cis-regulatory is formed by upstream activation sequences (UAS), upstream repression sequences (URS) and nucleosome destabilizing sequences (dA-dT). Next, core promoters from different systems (AOX1, GAP, HIS, etc.) provide the elements recognized by the basal transcriptional machinery, such as the TATA-box. Finally, strong termination signals are used to isolate the transcriptional unit constructed. Once assembled, multicloning sites (MCS) can be used to insert the gene of interest, generating thus the final functional system.

Fig. (3)

Construction of complex circuits and pathways. The COMPACTER approach is represented as an example. In this strategy, series of expression systems with flanking homologous regions (in this case, termination signals) are recombined to assemble complex networks in vivo [142]. This approach can be used in cycles to generate systems with increasing complexity, as long as sufficient unique regulatory elements are available [142].

Fig. (4)

Single-cell behavior of expression systems. In the deterministic (also called graded) expression behavior, cells respond simultaneously to a given signal, and the expression level in each cell increase as the signal gets stronger. In a stochastic (alternatively known as all-or-none) behavior, cells randomly switch from OFF to ON state when the signal is present. Thus, the switching rate of cells is directly proportional to the concentration of the inducer.