Cpf1 enables fast and efficient genome editing in Aspergilli

Additional file 1: Fig. S1. Cloning procedure of tRNA based gRNA expression. a Primer pair sets for amplifying the bipartite gRNA biobricks. b Bipartite gRNA biobricks after PCR amplification. We note that one biobrick (P1 + 2) is constant for all experiments, as only the PCR fragment (Px + 4) containing the protospacer needs to be specifically produced for each new experiment. c Design of the primer tails for USER fusion of bipartite gRNA biobricks. d Cpf1-CRISPR vector fragment (pAC1430) after linearization with PacI and Nt.BbvCI. e Insertion of gRNA biobrick into Cpf1-CRISPR vector (pAC1430) by USER cloning in E. coli

Indirect and direct routes to <i>C</i>-glycosylated flavones in <i>Saccharomyces cerevisiae</i>

Upon publication of this article [1], it was brought to our attention that revised Fig. 1 supplied by the author during proof correction was unfortunately not presented in the original version of the article. The revised Fig. 1 is given in this erratum

Advances in Recombinant Lipases: Production, Engineering, Immobilization and Application in the Pharmaceutical Industry

Lipases are one of the most used enzymes in the pharmaceutical industry due to their efficiency in organic syntheses, mainly in the production of enantiopure drugs. From an industrial viewpoint, the selection of an efficient expression system and host for recombinant lipase production is highly important. The most used hosts are Escherichia coli and Komagataella phaffii (previously known as Pichia pastoris) and less often reported Bacillus and Aspergillus strains. The use of efficient expression systems to overproduce homologous or heterologous lipases often require the use of strong promoters and the co-expression of chaperones. Protein engineering techniques, including rational design and directed evolution, are the most reported strategies for improving lipase characteristics. Additionally, lipases can be immobilized in different supports that enable improved properties and enzyme reuse. Here, we review approaches for strain and protein engineering, immobilization and the application of lipases in the pharmaceutical industry

Indirect and direct routes to C-glycosylated flavones in Saccharomyces cerevisiae

Abstract Background C-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health. These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. In contrast to O-glycosyltransferases, few C-glycosyltransferases (CGTs) have so far been characterized. Two different biosynthetic routes for C-glycosylated flavones have been identified in plants. Depending on the type of C-glycosyltransferase, flavones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyflavanone intermediate formed by a flavanone 2-hydroxylase (F2H). Results In this study, we reconstructed the pathways in the yeast Saccharomyces cerevisiae, to produce some relevant CGT substrates, either the flavanones naringenin and eriodictyol or the flavones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyflavanone intermediates into the 6C-glucoside flavones isovitexin and isoorientin, and the 8C-glucoside flavones vitexin and orientin. Furthermore, we established direct glycosylation of flavones using the recently identified GtUF6CGT1 from Gentiana triflora. The ratio between 6C and 8C glycosylation depended on the CGT used. The indirect route resulted in mixtures, similar to what has been reported for in vitro experiments. In this case, hydroxylation at the flavonoid 3′-position shifted the ratio towards the 8C-glucosylated orientin. The direct flavone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides. Conclusions The current study features yeast as a promising host for production of flavone C-glycosides, and it provides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation

A Mad7 System for Genetic Engineering of Filamentous Fungi

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories

A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering

[Image: see text] Efficient homologous recombination in baker’s yeast allows accurate fusion of DNA fragments via short identical sequence tags in vivo. Eliminating the need for an Escherichia coli cloning step speeds up genetic engineering of this yeast and sets the stage for large high-throughput projects depending on DNA construction. With the aim of developing similar tools for filamentous fungi, we first set out to determine the genetic- and sequence-length requirements needed for efficient fusion reactions, and demonstrated that in nonhomologous end-joining deficient strains of Aspergillus nidulans, efficient fusions can be achieved by 25 bp sequence overlaps. Based on these results, we developed a novel fungal in vivo DNA assembly toolbox for simple and flexible genetic engineering of filamentous fungi. Specifically, we have used this method for construction of AMA1-based vectors, complex gene-targeting substrates for gene deletion and gene insertion, and for marker-free CRISPR based gene editing. All reactions were done via single-step transformations involving fusions of up to six different DNA fragments. Moreover, we show that it can be applied in four different species of Aspergilli. We therefore envision that in vivo DNA assembly can be advantageously used for many more purposes and will develop into a popular tool for fungal genetic engineering