Towards T7 RNA polymerase (T7RNAP)-based expression system in yeast: challenges and opportunities

During the last decades, we have witnessed unprecedented advances in biological engineering and synthetic biology. These disciplines aim to take advantage of gene pathway regulation and gene expression in different organisms, to enable cells to perform desired functions. Yeast has been widely utilized as a model for the study of eukaryotic protein expression while bacteriophage T7RNAP and its promoter constitute the preferred system for prokaryotic protein expression (such as pET-based expression systems). The ability to integrate a T7RNAP-based expression system in yeast could allow for a better understanding of gene regulation in eukaryotic cells, and potentially increase the efficiency and processivity of yeast as an expression system. However, the attempts for the creation of such a system have been unsuccessful to date. This review aims to: (i) summarize the efforts that, for many years, have been devoted to the creation of a T7RNAP-based yeast expression system and ii) provide an overview of the latest advances in knowledge of eukaryotic transcription and translation that could lead to the construction of a successful T7RNAP expression system in yeast. The completion of this new expression system would allow to further expand the toolkit of yeast in synthetic biology and ultimately contribute to boost yeast usage as a key cell factory in sustainable biorefinery and circular economy.The work was supported by the Fundacao para a Ciencia e a Tecnologia [UIDB/04469/2020]; Fundacao para a Ciencia e a Tecnologia [UI/BD/151411/2021].info:eu-repo/semantics/publishedVersio

Investigating the effect of bacteriophages on bacterial FtsZ localisation

Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Several agents that block cell division have been shown to mislocalise FtsZ, including the bacteriophage λ-encoded Kil peptide, resulting in defective cell division and a filamentous phenotype, making FtsZ an attractive target for antimicrobials. In this study, we have used an in vitro meningitis model system for studying the effect of bacteriophages on FtsZ using fluorescent E. coli EV36/FtsZ-mCherry and K12/FtsZ-mNeon strains. We show localisation of FtsZ to the bacterial cell midbody as a single ring during normal growth conditions, and mislocalisation of FtsZ producing filamentous multi-ringed bacterial cells upon addition of the known inhibitor Kil peptide. We also show that when bacteriophages K1F-GFP and T7-mCherry were applied to their respective host strains, these phages can inhibit FtsZ and block bacterial cell division leading to a filamentous multi-ringed phenotype, potentially delaying lysis and increasing progeny number. This occurs in the exponential growth phase, as actively dividing hosts are needed. We present that ZapA protein is needed for phage inhibition by showing a phenotype recovery with a ZapA mutant strain, and we show that FtsI protein is also mislocalised upon phage infection. Finally, we show that the T7 peptide gp0.4 is responsible for the inhibition of FtsZ in K12 strains by observing a phenotype recovery with a T7Δ0.4 mutant

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λP(R) or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures