Recombinant DNA Strategies for Industrial Potential of Fungi

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This entry is adapted from the peer-reviewed paper 10.3390/jof9080834

Genetic and metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable molecules produced naturally in fungi, as well as those produced heterologously. Wild-type strains do not often produce these compounds at the levels required in industry, or do not produce the desired enzymes with the required properties or catalytic specificity. To overcome these problems, different genetic engineering approaches have been developed to improve the industrial potential of fungi.

genetic engineering fungal biotechnology fungal strains industrial strains

1. Gene Downregulation or Inactivation

Gene downregulation or inactivation is defined as a genetic engineering approach in which one target gene is made inoperative in an organism. Gene downregulation can be achieved either via deletion, mutation or silencing of the target genes.

1.1. Gene Deletion

Gene deletion or gene knockout is a powerful method to address genetic functionality. In order to completely delete a gene, double crossover homologous recombination events at the genomic level are required. In filamentous fungi, DNA integrates preferably via NHEJ, which results in low frequencies of homologous recombination ^[1]. To obtain gene deletion mutants with high homologous recombination efficiency, defective strains in NHEJ with improved site-specific recombination have been constructed by deletion of the Ku70 or Ku80-encoding genes in numerous filamentous fungi, including the model organisms Neurospora crassa ^[2] and Aspergillus nidulans ^[3], the industrial workhorses Aspergillus niger ^[4], Aspergillus oryzae ^[5] and Trichoderma reesei ^[6], or the phytopathogenic fungi Pycularia oryzae ^[7] and Penicillium digitatum ^[8].

Gene deletions can be applied for the generation of strains with higher production of industrially relevant enzymes than the wild types for example by the inactivation of transcriptional repressor-encoding genes. The deletion of the genes encoding the repressors CreA and CreB (creA and creB) in A. oryzae resulted in an increased production of α-amylases, xylanases and β-glucosidases ^[9], enzymes which are used in various industrial fields such as food, pharmaceuticals, textiles, detergents and pulp and paper ^[10]^[11]. A creA knockout strain of Trichoderma orientalis also enhanced enzyme production, specifically cellulases and hemicellulases ^[12]. In T. reesei deletion of Cre2, an orthologue of creB, resulted in increased cellulase activity ^[13]. In T. reesei deletion of dmm2 (putative DNA methylation modulator-2) had a significant improvement in cellulose production activity (150–200%), when compared to the parental strain RUT-C30, in the presence of microcrystalline cellulose (Avicel) or lactose ^[14]. Additionally, the deletion of ace1, which encodes the negative transcriptional regulator ACE1 in the same species, resulted in an increased expression of all the main cellulase-encoding genes and two xylanase-encoding genes in sophorose- and cellulose-containing cultures ^[15]. Similarly, deletion of bglR, a beta-glucosidase regulator, contributed to improved cellulase production in T. reesei, Penicillium decumbens and Penicillium oxalicum ^[16]^[17]^[18].

Gene knockout strategies have also enabled improved production of interesting metabolites and inhibition of toxic metabolites in filamentous fungi by metabolic engineering. After deletion of two fumarate reductase and the mitochondrial fumarase genes (Mtfr1 and Mtfr2) of Myceliophthora thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer, which is widely used in the food and pharmaceutical industries ^[19]. In Aspergillus fumigatus, deletion of veA and laeA, both encoding velvet complex components, up-regulated the gene cluster responsible for the synthesis of fumagillin ^[20]. Fumagillin has been intensely studied due to its potential in the treatment of amebiasis, microsporidiosis and for its anti-angiogenic activity as inhibitor of the human type 2 methionine aminopeptidase ^[20]. Deletion of the L-galactonic acid dehydratase-encoding genes gaaB and lgd1 in A. niger and T. reesei, respectively, increased extracellular accumulation of L-galactonic acid, with potential applications in the pharmaceutical, cosmetic and other industries ^[21]. Deletion of the fifteen genes involved in the patulin biosynthetic pathway resulted in a decreased ability of Penicllium expansum to produce patulin, a mycotoxin that can be present as a contaminant in food, particularly in fruits and fruit-derived products ^[22]. Meanwhile, in the yeast Saccharomyces cerevisiae simultaneous deletion of GPD2 (glycerol 3-phosphate dehydrogenase 2), FPS1 (Aquaglyceroporin FPS1) and ADH2 (alcohol dehydrogenase 2) increase ethanol production by 0.18% in comparison with the wild-type strain ^[23].

1.2. Point Mutations

Gene inactivation can also be achieved by single DNA base pair deletion and/or single nucleotide changes. Point mutations can result in the exchange of nucleotides (substitution), elimination of nucleotides (deletions) or introduction of nucleotides in the DNA sequence (insertions). Point mutations are the most common source of genetic variation, and although most are neutral or deleterious, some become beneficial for the organisms, giving them novel characteristics, e.g., better adaptation to the environment or improvement of their catalytic performance. Point mutations in filamentous fungi are usually obtained using physical or chemical mutagens ^[24]. However, they can also be generated through side-directed mutagenesis, which allows the precise introduction of the target point mutations.

As examples of industrial relevance, a point mutation in the hemi-cellulolytic transcriptional activator Xyr1 introduced via UV mutagenesis in T. reesei was found to result in a constitutively active form of this regulator, resulting in constitutive expression of cellulase and xylanase encoding genes, even in the presence of a repressing carbon source ^[25]. In addition, many T. reesei strains that are used in industry underwent point mutations leading to catabolite de-repression, resulting in increased extracellular enzyme and protein levels compared to their parent strain ^[26]. Point mutations also resulted in improved cysteine biosynthesis in Penicillium rubens by the inactivation of enzymatic conversions that compete with the cysteine biosynthetic pathway, which plays a key role in penicillin production ^[27]. By site-directed mutagenesis, the thermostability of an A. niger xylanase has been improved, showing up to 80% of its maximal activity after incubation for 2 h at 50 °C in the presence of xylan, compared to only 15% activity for the wild-type enzyme ^[28]. Additionally, using the CRISPR/Cas9 system, a modified gaaR gene carrying a single point mutation causing a W361R amino acid change was introduced in A. niger, which causes constitutive activation of GaaR and therefore constitutive production of pectinases under non-inducing conditions ^[29]^[30].

1.3. RNA Interference (RNAi)

The possibility to inactivate genes or metabolic pathways is not restricted to DNA-based approaches. RNA interference (RNAi) is an evolutionarily conserved mechanism found in most eukaryotic organisms, including fungi. It was originally described as a mechanism that confers protection against exogenous and endogenous genetic threats (virus, transposons...) and regulates gene expression by means of non-coding RNA of around 30 nucleotides, mainly short interfering RNAs (siRNAs), microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs) ^[31]^[32]. This mechanism has been adapted as a potential biotechnological tool to improve fungal strains. It is particularly advantageous when target genes are present in multiple copies or when deletion of the target gene(s) is lethal. The resulting knock-down transformants still carry the target gene; however, RNAi leads to a reduction of the transcription level, which can be close to zero in some transformants ^[33]. Double-stranded RNA (dsRNA) induces the inactivation of cognate sequences by mRNA degradation, translation inhibition, chromatin remodeling or DNA elimination ^[34].

RNAi strategy was used to attenuate the expression of the creA gene in Penicillium chrysogenum for higher penicillin production ^[35]. In A. oryzae, RNAi-based inactivation of three α-amylase encoding genes improved heterologous protein production in this species ^[36]. In A. niger, RNAi was used to knockdown chitin synthase activator (CHS3). A. niger chs3 mutants exhibited better citric acid production potential compared to that of the parent strain in scale-up fermentation ^[37]. RNAi was also applied to silence the expression of hydroxymethyl glutaryl coenzyme A reductase (hmgR) and farnesyl pyrophosphate synthase (fpps) genes in Fusarium sp., resulting in higher levels of bikaverin, a known antimicrobial and antitumor compound ^[38].

2. Gene Up-Regulation

Gene up-regulation is a genetic engineering approach aimed to increase the expression level of a target gene. In filamentous fungi, up-regulation of specific genes may increase the production of a metabolite or enzyme of interest or improve the conversion rate of a given substrate. Additionally, greater production of metabolites or enzymes can be achieved by the overexpression of the genes encoding regulatory proteins that control the expression of the corresponding genes. In the next sections, different gene up-regulation strategies in filamentous fungi for different purposes are reviewed.

2.1. Promoter Swap

Gene expression in eukaryotic organisms is mainly governed at the level of transcriptional initiation, which corresponds to the complex interplay between the promoter, RNA polymerase II and transcription factors ^[39]. There are different types of promoter sequences. Constitutive promoters are always active regardless of environmental or internal signals, leading to overexpression of their target genes, and thus resulting in high production titers. In contrast, inducible promoters are controlled by transcription factors after the recognition of specific environmental signals, e.g., pH, presence of sugars or catabolic enzymes ^[40]. One possible genetic approach to (over)produce enzymes or specific metabolites relies on the substitution of the original promoter sequences for a constitutively active one. However, overexpression of some genes might lead to overburdening of the cellular mechanisms or the accumulation of (toxic) side compounds, which can be detrimental to cells. In this context, inducible promoters are a great alternative to control gene expression over time ^[40] and are the preferred choices at the industrial level, where a fine-tunable expression with cost-efficient induction is desired ^[41].

Promoter sequences of industrial relevance are for example the constitutively active promoter from the glycer-aldehyde-3-phosphate dehydrogenase (gpdA) from Aspergillus nidulans ^[42], or the inducible cellobiohydrolase I (cbh1) gene promoter from T. reesei. Pcbh1 is strongly induced in the presence of cellulose and has been widely applied for heterologous protein production in T. reesei and other filamentous fungi ^[43]. The promoter of the glucoamylase A gene (glaA) from A. niger was one of the first inducible systems applied in filamentous fungi, and drives gene expression in the presence of starch and starch-related compounds, while xylose represses gene expression ^[44]. PglaA has been reported to work efficiently in Aspergillus terreus to overexpress acetyl-CoA carboxylase for the increase of lovastatin production, a cholesterol-lowering compound ^[45]. In A. oryzae, the glaA promoter was used for the production of L-malate, a flavor enhancer which is widely utilized in the food and beverage industries ^[46]. Xylose-inducible promoters have also been established in some industrial hosts, such as the xyn1 promoter from T. reesei and xylP from P. chrysogenum ^[47]. Another inducible promoter is the thiamine-regulatable thiA promoter, which was established in A. oryzae and is controlled by different concentrations of thiamine in the medium culture ^[48]. In the industrial workhorse A. niger, a promoter system was established in order to adapt to the medium conditions during citric acid fermentation ^[49]. During the process, the pH decreases dramatically, and therefore, the pH-responsive promoter from the 1,3-β-glucanosyltransferase GelD (Pgas) was used, which enhanced gene expression at very low pH. Pgas was also used to express the cis-aconitate decarboxylase encoding gene from A. terreus in A. niger. Since A. niger produces large amounts of citrate, which is the precursor of itaconate, the aim was to modify the natural citrate producer into an itaconate producer, with high potential for the production of resins, plastics, paints and fibers. Furthermore, itaconate, which is an immunomodulatory metabolite highly expressed in activated macrophages, has potential application in the treatment of inflammatory diseases ^[50]. The use of the Pgas promoter led to a gradually increased production of itaconate, correlating with decreasing pH values ^[49]. The xylP promoter (PxylP) controlling expression of a xylanase from P. chrysogenum allows high induction by xylose and xylan with low basal activity in the absence of the inducer. PxylP was demonstrated to permit conditional gene expression of diverse genes in various mold species including P. chrysogenum, A. nidulans and Aspergillus fumigatus, among others. In A. fumigatus, it has been shown that PxylP mediates not only inducer-dependent activation but also repression in the absence of inducer. Furthermore, PxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of PxylP ^[51].

An alternative type of promoters are bidirectional promoters (BDPs), which allow the expression of two genes at the same time, such as the gene of interest and the selection marker, two copies of the gene of interest, or two different genes. Recently, natural BDPs have been found in filamentous fungi. It has been shown that BDPs are always intergenic regions regulating the flanking two genes that encode proteins relevant to the same biological process ^[52]. The histone H4.1 and H3 promoters can act as natural BDPs, allowing simultaneous expression of enzyme encoding genes in the case of metabolic engineering of Aspergillus sp. ^[53]. Additionally, A. niger D-galacturonic acid reductase promoter shows bidirectional transcription, thus allowing its application as both bidirectional and inducible promoter ^[54]. In T. reesei a 767-bp intergenic region served as a bidirectional promoter. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. This promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the sole carbon source ^[52].

In some cases, yeast expression systems have been shown to work more efficiently for the biotechnical production of mammalian proteins with pharmaceutical relevance. Common hosts are S. cerevisiae, Pichia pastoris and Hansenula polymorpha ^[55]^[56]^[57]. However, filamentous fungi are still used for the heterologous production of different mammalian proteins, for instance, the human hormone peptide obestatin using recombinant T. reesei strains ^[58].

2.2. Increase of Gene Copy Number

Increasing gene copy number often results in increased protein production ^[59]. High gene copy numbers can be achieved by either adding high amounts of DNA during transformation; applying a strong antibiotic pressure during selection; or using bidirectional promoters. However, genomic loci can affect gene expression, leading to some extra copies remaining silent ^[60]. Moreover, increasing the number of copies can sometimes compromise the stability of the resulting strains, which is especially undesired in industries where prolonged cultivations are needed ^[61]. This strategy has been applied for instance in the production of penicillin by inserting multiple copies of the penicillin biosynthesis cluster in the genome of P. chrysogenum ^[62]. Similarly, increasing copies of the glaA gene in A. niger from one to twenty resulted in an increase of secreted glucoamylase levels ^[63]. Alternative ways of inducing high protein production can be associated not only with increased gene copy number but also with increased promoter strength through a multi-copy strategy. Enhancement of promoter strength was achieved when five copies of the −427 to −331 upstream region of glaA gene from A. niger were integrated to efficiently increase L-malate production directly from corn starch ^[64].

In order to achieve heterologous β-carotene synthesis in the yeast Yarrowia lipolytica, which cannot indigenously produce β-carotene, the structural genes responsible for β-carotene synthesis were overexpressed. β-carotene is a kind of high-value tetraterpene compound, which shows various applications in medical, agricultural and industrial areas owing to its antioxidant, antitumor and anti-inflammatory activities. The strain obtained (Y. lipolytica-C (Yli-C)) reached 34.5 mg/L β-carotene ^[65].

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Subjects: Biotechnology & Applied Microbiology

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Sonia Salazar-Cerezo

Ronald P. de Vries

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Update Date: 30 Aug 2023

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