Table 4 Different strategies generally employed to investigate functional genomic

Selection markers

Drug resistance markers

Host strain can grow in presence of specific concentrations of that drug

The need for expensive antibiotics

A very few number of foreign hetero gene markers can be used as markers

Risk of resistance genes transfer to environment and other microorganisms

Aureobasidin resistance gene; bleomycin resistance gene; hygromycin B resistance gene

Kubodera et al. 2000; Yu et al. 2017;

Zhong et al. 2018; Miura et al. 2018; Jin et al. 2021

Auxotrophic markers

Effective, resulting auxotrophs have similar phenotype with the wild type

The gene pyrG encoding for orotidine-5’-monophosphate (OMP) decarboxylase

Zhu et al. 2013

Transformation

Protoplast-mediated transformation

Main strategy used to introduce DNA into fungi

Difficulty in generalizing protoplast-mediated transformation protocols across different fungi

Introducing aspartic proteinase into A. oryzae from Mucor pusillus; transforming a neutral ceramidase orthologue into A. oryzae

Tada et al. 2009; He et al. 2019

Agrobacterium-mediated transformation

Simple and effective

Fungal spores are used directly

No need to obtain protoplast

Can elevate gene deletion efficiency and targeted integration

Difficulty of developing enough vir genes and heterologous DNA containing binary vectors

Difficulty in generalizing Agrobacterium-mediated transformation protocols

Electroporation

Relatively simple Relatively need short time

Not successful in case of A. oryzae

Protocols require optimization

Has a low DNA transfer efficiency

Genetic manipulations

Successful strategy used to investigate gene functions or enhance the ability of a strain to produce a certain product

Difficulty in isolation of A. oryzae conidia containing only mutated nuclei

Low mutational rates

CRISPR/Cas9 system

(Nødvig et al. 2015; El-Sayed et al. 2017)