High-value metabolites are increasingly identified and isolated from biological organisms, including fungi. However, the amount of these bioactive metabolites is in low concentrations, limiting industrial applications. Moreover, the presence of “cryptic pathways” which are silent and do not produce metabolites is documented and necessitates a requirement of strain improvement approaches [142]. In previous methods, a high-yielding strain was screened among all strains and further improved via mutagenesis and selection [143]. For example, Penicillium chrysogenum was screened for penicillin (an antibiotic) production and it produced 100-fold higher titers than the original strain. Further, developments in screening and strain improvement led to industrial production of penicillin (100,000-fold higher than the original strain) [144]. This method is beneficial in the way that no prior knowledge of the microbe’s genetics and the metabolic pathway is required, and the best-screened strain can be employed for metabolite production. Furthermore, multiple strategies can be adopted for the reorientation of metabolic flux toward specific metabolite production, such as improving precursor supply, monitoring gene expression regulation, improving enzyme functions, and metabolic pathway reconstitution in heterologous systems, including others [142].

For strain improvement in fungi, different mutagenesis approaches were adopted, using chemical or physical agents. To induce specific or random alterations, low/controlled levels were used, since they may lead to harmful mutations [145], and screening of mutants to locate the desired mutants was attempted. While the chemical agents used are base analogs causing base deamination, mainly GC→AT and AT→GC transitions, and impairment [145], physical mutagens comprise ionizing radiation (γ, X-rays) that cause DNA strand breakage, structural modifications, and ultraviolet radiation, which may cause frameshifts mutations and deletions [145]. In this direction, microwave radiations were also employed for strain improvement [146]. Other mutagens including caffeine lead to frameshift mutations with potent effects in fungi and bacteria, and acridine dyes, and ethidium bromide result in deletions and frameshift mutations [145]. Multiple studies in fungi for strain improvement have employed chemical and physical mutagens in Trichoderma reesei Rut C-30, [147], and Penicillium chrysogenum [148]. Random mutagenesis was attempted in fungi for the enhanced yield of polyamines [149]. Yang and coworkers [150] employed mutagenesis in Penicillium oxalicum, phosphate-solubilizing fungi for improved production of organic acid and phosphate solubilization, respectively, which increased significantly on mutagenesis by radiation. In addition, physical and chemical mutagens were used to produce high-value metabolites from fungi, aimed toward a bio-based economy.

When the precursor supply does not affect product titer, the pathway enzyme expression may result in key outcomes. For example, a high-yielding strain of P. chrysogenum BW1890 (with multiple copies of gene clusters) leads to a 64-fold increase in the production of penicillin [151]. The advances in metabolic engineering facilitate the expression of all biosynthetic pathway enzyme(s), providing a solution. Moreover, the expression of single enzymes can be tuned by regulating transcription [152], and protein engineering [153], including other methods. In another key example, penicillin production in A. nidulans showed aminoadipyl-cysteinylvaline synthetase (ACVS) as a limiting enzyme, and the gene overexpression for ACVS resulted in enhanced ACVS expression and increased penicillin production [154]. However, gene overexpression for acyltransferase (ACYT), and iso penicillin N synthetase (IPNS), only slightly improved penicillin production [155]. Malla and coworkers [156] studied enhanced doxorubicin production (anticancer polyketide), aided by gene overexpression for glycosyltransferase and deoxysugar biosynthesis [157]. An important consideration suggests monitoring the toxic effects of a metabolite (if any) at higher concentrations, in the case of doxorubicin production, the overexpression of resistance genes was essential [158].

In some organisms, the production of desired metabolites has been improved by altering the regulatory components of a metabolic pathway. For example, in Streptomyces, gene clusters encode Streptomyces antibiotic regulatory protein (SARP) that positively regulates the production of antibiotics [159]. Moreover, the SARP encoding fredericamycin in Streptomyces griseus ATCC 49344 was overexpressed and led to higher antibiotic production in the engineered strain [159]. Furthermore, SARP MtmR (mithramycin gene cluster in Streptomyces argillaceus) overexpression increased mithramycin titer 16-fold, respectively, and the MTMR-activated actinorhodin-producing pathway when expressed in S. coelicolor [160].

For all the major classes of natural products, increasing the supply of precursor molecules has been a successful method in both native and heterologous systems. These precursors can be primary metabolites or those derived from primary metabolites. For example, malonyl-CoA comprises a key precursor for polyketide biosynthesis; Ryu and colleagues attempted S. coelicolor engineering by overexpression of ACCase genes for enhanced malonyl-CoA production, and the study resulted in enhanced actinorhodin production [161]. Zha and coworkers [162] combined multiple methods for increased malonyl-CoA levels in E. coli, including pathway knockouts, gene overexpression, and limiting pathways for malonyl-CoA degradation, resulting in a 15-fold increase in malonyl-CoA [162]. Substantial initiatives in engineering E. coli for precursor supply have focused on the heterologous expression of the MVA pathway or its improvement for increased isopentenyl pyrophosphate (IPP) production, a precursor in the generation of terpenoids [163,164]. Research initiatives focusing on MEP pathway engineering have shown that 1-deoxy-D-xylulose-5-phosphate reductase (dxr), 1-deoxy-D-xylulose-5-phosphate synthase (dxs), and isopentenyl diphosphate isomerase (idi) overexpression enhanced production of isoprenoids [165]. In primary metabolism, the shikimate pathway is a key component, generating precursors for the biosynthesis of aromatic amino acids, utilized by several classes of natural products as precursors in the biosynthesis of metabolites. The yield of natural products has been considerably increased via increasing shikimate pathway flux and steps in amino acid biosynthesis [166].

Another prospective approach in this direction is to delete certain genes for pathway silencing so that associated metabolic pathways and their unnecessary intermediates can be avoided. A key example highlights that squalene synthase in yeast is encoded by erg9 and utilizes farnesyl-pyrophosphate (FPP), a sesquiterpene precursor, and amorphadiene production is increased by knocking out the erg9 gene, respectively. In another example discussing doxorubicin biosynthesis, multiple genes encoded by the dxr cluster were removed to improve desired protein production [167]. In addition, the efficiency of the heterologous system can be increased via the deletion of specific genes. NADPH-dependent enzymes are encoded by several natural pathways: for instance, oxidoreductases create metabolic pressure on the cell as the pathway metabolic flux gradually increases [167]. Chemler and coworkers [168] showed NADPH as a limiting factor in flavonoid (+)-catechins production in E. coli. In the study, gene knockouts were identified utilizing a metabolic modeling approach, for improving NADPH availability and thereby, flavonoid production [168]. Komatsu and coworkers [169] reported a ‘genome-minimized’ approach (deletion of non-essential elements) in Streptomyces avermillitis, in which the genome was reduced to 83% of its original size, creating space for the introduction of a gene cluster of streptomycin. The minimized genome of Streptomyces produced higher amounts of streptomycin, highlighting a prospective approach to enhance the production of biochemicals [169].

In this direction, the existence of divergence and homologies among genes between related metabolic pathways have led to switching genes and modules between interlinked pathways for novel microbial chassis [142]. The metabolic pathways of aromatic polyketide biosynthesis, namely, the macrolides, the teicoplanin, lipopeptides daptomycin/A54145, and aminocourmarins, highlight some examples. Hopwood and coworkers [170] used genes for related polyketides for combinational biosynthesis. In a key study, Streptomyces species (producing dihydrogranaticin and medermycin) were engineered by introducing actinorhodin pathway genes, resulting in hybrid antibiotics, dihydrogranatihordin, and mederrhodin, respectively [170]. For pathway engineering in a microbial system, genetic manipulation/switching biosynthetic genes downstream of the pathway defines higher success owing to the involvement of a few downstream enzymes [171]. The biosynthetic enzymes act on similar substrates/intermediates in closely related pathways. The creation of novel chimeras by gene switching showcases higher success potential and defines new research initiatives in the discovery and engineering of natural product pathways [142]. Novel fungal chimeras can be created via the reshuffling of genes and modules among linked pathways aimed at new combinations. Furthermore, a metabolic pathway can be engineered by altering a combination of genes in the biosynthetic pathway, for the creation of new chemical entities, subject to the tolerance of the downstream enzyme to substrate alteration [171]. One key concern in pathway engineering includes the non-disruption of the main scaffold, and the introduction of changes in the latter pathway steps has better chances of success, with the involvement of few downstream enzymes. Moreover, pathway engineering in the native host is carried out by new gene insertion/gene deletion or combinational pathway reconstitution [142].

An emerging genome editing tool, CRISPR/Cas has witnessed key success in genome editing of filamentous fungi to produce high-value metabolites including pigments, enzymes, secondary metabolites, compounds of industrial importance, and agriculture, respectively. The CRISPR/Cas tool has been widely employed in improving fungal strains including Aspergillus, Trichoderma, and Penicillium sp. having industrial importance. Moreover, studies have documented the genetic manipulation of fungal strains for heterologous protein production. Manganese peroxidase, classified in the family of heme-containing peroxidases, degrades lignin and is produced by white-rot fungi, which has relevance in chemical industries. The two proteins, manganese peroxide and Interleukin 6, were produced in Aspergillus species [172]. Besides, socially important fungal strains, namely, Mortierella alpinis [173], Fusarium veneratum [174], A. japonicas [175], Chrysosporium lucknowense [176], have been developed for metabolites and protein production. Genetic manipulation strategies were attempted for Cordyceps militaris (edible medicinal mushroom) chassis; codon-optimized cas9 was used with promoter Pcmlsm3, and terminator Tcmura3 was expressed in the system. A CRISPR-Cas9 system comprising a single-strand DNA template, Cas9 DNA endonuclease, and RNA pre-synthesized in vitro was employed for insertion and site-specific deletion. The study aimed at genome editing of edible mushrooms for increasing genomic chassis and rapid development as ‘functional food’, respectively [177]. Chen and coworkers [178] employed CRISPR/Cas-mediated genome editing tools in C. militaris for enhanced ergothioneine production by discovering and regulating the metabolic pathway for ergothioneine biosynthesis [178]. In Fusarium fujikuroi, genetic manipulation methods were employed for enhanced gibberellic acid production [179].

Metabolic engineering of microbes has witnessed good translational success, with multiple bacterial and fungal species engineered for food additives production in recent times [180]. For the production of malic acid (used in food and beverages), overexpression of genes was attempted in A. flavus, A. oryzae, S. cerevisiae, etc. [181]. The CRISPR-Cas 9 engineering tool was employed to alter the molecular structure and colors of pigments, by introducing change in the desired sites. Another key study discussed the genetic manipulation of Y. lipolytica for β-hydroxylase and β-ketolase production by gene introduction in fungal species and increased astaxanthin production [182]. This study provided key inputs to produce astaxanthin, with a high commercial value. Research initiatives attempted in fungal chassis have substantially enhanced the production of high-value metabolites, thanks to contributions of transcriptome-based analysis, cloning, and mutational approaches in non-yielding species [77]. Moreover, emerging insights into different transcription stages and their manipulations have considerably increased the production of cellulases, amylases, and xylanases in filamentous fungi via gene overexpression [183].