Development of an Efficient and Stable Transformation System for Aspergillus Oryzae Based on the pyrG Gene

Background Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. An efficient and stable transformation system is the key to the successful expression of the gene of interest in A. oryzae.Results To improve the expression efficiency of the gene of interest in A. oryzae, we constructed the uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG by Ultraviolet (UV) mutagenesis of pyrG gene deletion which would be used as a host for further transformation. In addition, a novel and efficient expression vector pBC-hygro.4 was constructed, including the pyrG cassette gene, His-Tag, amyB promoter and terminator,and green fluorescent protein GFP marker. pBC-hygro.4 transformed A. oryzae RIB40ΔpyrG efficiently via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was tested by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, we successfully obtained a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG. At the same time, the developed vectors are fully functional for heterologous expression of the GFP fluorescent proteins in the A. oryzae RIB40ΔpyrG.Conclusion Our work provides a new method that can be applied to other filamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.

Microbial Transformation of Licochalcones

Microbial transformation of licochalcones B (1), C (2), D (3), and H (4) using the filamentous fungi Aspergillus niger and Mucor hiemalis was investigated. Fungal transformation of the licochalcones followed by chromatographic separations led to the isolation of ten new compounds 5–14, including one hydrogenated, three dihydroxylated, three expoxidized, and three glucosylated metabolites. Their structures were elucidated by combined analyses of UV, IR, MS, NMR, and CD spectroscopic data. Absolute configurations of the 2″,3″-diols in the three dihydroxylated metabolites were determined by ECD experiments according to the Snatzke’s method. The trans-cis isomerization was observed for the metabolites 7, 11, 13, and 14 as evidenced by the analysis of their 1H-NMR spectra and HPLC chromatograms. This could be useful in better understanding of the trans-cis isomerization mechanism of retrochalcones. The fungal transformation described herein also provides an effective method to expand the structural diversity of retrochalcones for further biological studies.

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

Background Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation. Results To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin. Conclusion By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094. Graphic abstract