Evasion of Cas9 toxicity to develop an efficient genome editing system and its application to increase ethanol yield in Fusarium venenatum TB01

Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01

“…Dry mycelial weight was determined as described [ 15 ]. Briefly, conidial suspensions (300 μL, 5 × 10 6 conidia/mL) derived from 10 days GYA plates were cultured in 50 mL fermentation medium (glucose 60 g/L, yeast extract 0.5 g/L, (NH 4 ) 2 SO 4 6 g/L, MgSO 4 1.5 g/L, KCl 0.7 g/L, Na 2 SO4 0.5 g/L, KH 2 PO 4 2 g/L, CaCO 3 0.5 g/L) at 28 °C and 180 rpm for 4 days.…”

“…To further promote the development of F. venenatum through genetic engineering, efficient genetic manipulation tools are necessary. Recently, a classical gene deletion method (homologous recombination) based on homology arms [ 14 ] and an efficient DNA-free CRISPR/Cas9 system based on the AMA1-based Cas9 expression vector [ 15 ] were established by our group in F. venenatum TB01. Using the above system, the knockout or deletion of target genes can be successfully accomplished.…”

“…Currently, the expression of target genes in fungi mainly depends on episomal vectors and genome integration. The episomal vectors can replicate autonomously in the host based on a self-replicator such as AMA1 and CEN/ARS [ 16 , 17 ]; however, the obvious disadvantage is that they are readily lost without selective pressure, including in F. venenatum TB01 [ [ 15 , [18] , [19] , [20] , [21] , [22] ]]. Compared to self-replicating vectors, integration of target genes into the genome for overexpression is a more stable strategy [ 22 ].…”

Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality

“…After Cas9-encoding gene is transformed into host strains using common delivery strategies, the cas9 gene is generally randomly integrated into the genome, which may affect the host genome structure, leading to unexpected phenotypes, such as growth delay, further rearrangements or off-target mutations, and toxic effects on the host . Therefore, it is important to ensure that Cas9 protein has no adverse effects on filamentous fungi by testing the biological properties, such as development, stress resistance and virulence of Cas9-expressing strains. − …”

Efficient Mycoprotein Production with Low CO2 Emissions through Metabolic Engineering and Fermentation Optimization of Fusarium venenatum

“…22−24 Mycoprotein production in F. venenatum can, in theory, be improved by adopting the above strategy, in particular, large quantities of the byproduct ethanol have been found during the fermentation process. 25 Pyruvate decarboxylase, located upstream of the ethanol synthesis pathway in fungi, catalyzes the conversion of pyruvate to CO 2 and acetaldehyde. 26 Considering that CO 2 escape is also an important factor limiting the efficient conversion of carbon sources, 27 deleting the major pyruvate decarboxylase gene is expected to significantly improve the carbon conversion ratio of mycoprotein synthesis, attributed to the simultaneous blocking or reducing of ethanol synthesis and CO 2 emissions.…”