Positive selection in cytochrome P450 genes is associated with gonad phenotype and mating strategy in social bees.

The honey bee, Apis mellifera differs from all other social bees in its gonad phenotype and mating strategy. Honey bee queens and drones have tremendously enlarged gonads, and virgin queens mate with several males. In contrast, in all the other bees, the male and female gonads are small, and the females mate with only one or very few males, thus, suggesting an evolutionary and developmental link between gonad phenotype and mating strategy. RNA-seq comparisons of A. mellifera larval gonads revealed 870 genes as differentially expressed in queens versus workers and drones. Based on Gene Ontology enrichment we selected 45 genes for comparing the expression levels of their orthologs in the larval gonads of the bumble bee Bombus terrestris and the stingless bee, Melipona quadrifasciata, which revealed 24 genes as differentially represented. An evolutionary analysis of their orthologs in 13 solitary and social bee genomes revealed four genes with evidence of positive selection. Two of these encode cytochrome P450 proteins, and their gene trees indicated a lineage-specific evolution in the genus Apis, indicating that cytochrome P450 genes may be involved in the evolutionary association of polyandry and the exaggerated gonad phenotype in social bees.

Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Mining novel cis-regulatory elements from the emergent host Rhodosporidium toruloides using transcriptomic data.

The demand for robust microbial cell factories that produce valuable biomaterials while resisting stresses imposed by current bioprocesses is rapidly growing. Rhodosporidium toruloides is an emerging host that presents desirable features for bioproduction, since it can grow in a wide range of substrates and tolerate a variety of toxic compounds. To explore R. toruloides suitability for application as a cell factory in biorefineries, we sought to understand the transcriptional responses of this yeast when growing under experimental settings that simulated those used in biofuels-related industries. Thus, we performed RNA sequencing of the oleaginous, carotenogenic yeast in different contexts. The first ones were stress-related: two conditions of high temperature (37 and 42°C) and two ethanol concentrations (2 and 4%), while the other used the inexpensive and abundant sugarcane juice as substrate. Differential expression and functional analysis were implemented using transcriptomic data to select differentially expressed genes and enriched pathways from each set-up. A reproducible bioinformatics workflow was developed for mining new regulatory elements. We then predicted, for the first time in this yeast, binding motifs for several transcription factors, including HAC1, ARG80, RPN4, ADR1, and DAL81. Most putative transcription factors uncovered here were involved in stress responses and found in the yeast genome. Our method for motif discovery provides a new realm of possibilities in studying gene regulatory networks, not only for the emerging host R. toruloides, but for other organisms of biotechnological importance.

Recent advances in plasmid-based tools for establishing novel microbial chassis.

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with "build-your-own" microbial host.

Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi.

We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.

A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. RESULTS: This data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides. CONCLUSIONS: A set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

A Novel Cys2His2 Zinc Finger Homolog of AZF1 Modulates Holocellulase Expression in Trichoderma reesei.

Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.

The art of vector engineering: towards the construction of next-generation genetic tools.

When recombinant DNA technology was developed more than 40 years ago, no one could have imagined the impact it would have on both society and the scientific community. In the field of genetic engineering, the most important tool developed was the plasmid vector. This technology has been continuously expanding and undergoing adaptations. Here, we provide a detailed view following the evolution of vectors built throughout the years destined to study microorganisms and their peculiarities, including those whose genomes can only be revealed through metagenomics. We remark how synthetic biology became a turning point in designing these genetic tools to create meaningful innovations. We have placed special focus on the tools for engineering bacteria and fungi (both yeast and filamentous fungi) and those available to construct metagenomic libraries. Based on this overview, future goals would include the development of modular vectors bearing standardized parts and orthogonally designed circuits, a task not fully addressed thus far. Finally, we present some challenges that should be overcome to enable the next generation of vector design and ways to address it.