Open access peer-reviewed chapter

Random Mutagenesis of Filamentous Fungi Strains for High-Yield Production of Secondary Metabolites: The Role of Polyamines

Written By

Alexander A. Zhgun

Submitted: 04 September 2019 Reviewed: 24 August 2020 Published: 07 October 2020

DOI: 10.5772/intechopen.93702

From the Edited Volume

Genotoxicity and Mutagenicity - Mechanisms and Test Methods

Edited by Sonia Soloneski and Marcelo L. Larramendy

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A filamentous fungus (also called molds or moldy fungus) is a taxonomically diverse organism from phylum Zygomycota and Ascomycota with filamentous hyphae and has the ability to produce airborne spores or conidia. Currently, more than 70,000 molds are known, and some of them contain unique and unusual biochemical pathways. A number of products from such pathways, especially, the secondary metabolite (SM) pathways are used as important pharmaceuticals, including antibiotics, statins, and immunodepresants. Under different conditions, the individual species can produce more than 100 SM. The strain improvement programs lead to high yielding in target SM and significant reduction of spin-off products. The main tool for the strain improvement of filamentous fungi is random mutagenesis and screening. The majority of industrial overproducing SM strains were developed with the help of such technique over the past 50–70 years; the yield of the target SM increased by 100- to 1000-fold or more. Moreover, most of the strains have reached their technological limit of improvement. A new round of mutagenesis has not increased overproduction. Recently, it was shown that that the addition of exogenous polyamines may increase the production of such improved strains of filamentous fungi. The possible molecular mechanism of this phenomenon and its biotechnological applications are discussed.


  • filamentous fungi
  • random mutagenesis and screening
  • strain improvement
  • secondary metabolites
  • polyamines

1. Introduction

Improved strains of filamentous fungi are widely used in the biotechnology industry for recycling of secondary raw materials [1, 2, 3] as biosorbents [4], in fermentation of cheese [5], wine [6, 7], and other food products [8, 9], as well as for the production of enzymes [10, 11, 12, 13], organic acids [14, 15], secondary metabolites (SMs) [16, 17], or for steroid transformation [18, 19]. There are four main tools for fungal strain improvement: (1) sexual crossing [20, 21], (2) somatic crossing (including parasexual recombination [22]), (3) random mutagenesis by physical or/and chemical mutagens and screening [23, 24], and (4) genetic engineering [25, 26, 27]. These methods can be applied separately or in various combinations [28]. The first three tools are referred to as classical strain improvement (CSI) methods and have been used in strain improvement programs for filamentous fungi for SM production since the 1950s of the twentieth century (Figure 1A). The majority of industrial producers of secondary metabolites in fungi were obtained precisely with the use of CSI [25]. The powerful genetic engineering approach has been available since the end of the twentieth century for targeting the particular genetic determinant to introduce novel properties into an organism [28]. Since the improvement of filamentous fungi strains for SM production is a complex and multistage program that radically changes numerous processes, there is no “golden bullet,” any single unique genetic change to produce high yielding (HY) strain from the wild type (WT) strain [29] (Figure 1B). However, introducing of novel targeted features into already improved strains enables to create SM-overproducing strains [30]. For instance, the introduction of the compactin pathway from the Penicillium citrinum, as well as CYP105AS1 (from Amycolatopsis orientalis, for pravastatin hydroxylation) into the β-lactam-negative P. chrysogenum DS50662 strain, yielded more than 6 g/L of pravastatin [25]. This seems to be due to the interaction of different tools that are available to improve strains (Figure 1C).

Figure 1.

The improving of filamentous fungi strains for SM production. (A) CSI programs for fungal strain improvement. (B) The WT strain improvement by the “golden bullet” tool from genetic engineering. (C) The combination of SCI and genetic engineering approaches for the developing of novel HY strain. SM: secondary metabolite, CSI: classical strain improvement, WT: wild type, and HY: high yielding.


2. CSI for SM production in filamentous fungi

For the majority of industrially important filamentous fungi (except members of genera Aspergillus, Claviceps, and Emercicellopsis), the sexual breeding is not available [28]. From the other side, a number of these organisms produce haploid conidia, which provide the ideal material for mutagenic treatment: in the absence of a complementary set of genes, mutations will be easily detected using suitable screening and the stability of the mutant will generally be good [28]. Filamentous fungi turned out to be surprisingly tolerant to strong mutagenic effects, retaining their strength and productivity even after radical rearrangements of their chromosomes [31, 32]. In this case, the main tool of CSI for SM production in filamentous fungi is random mutagenesis mutagens and screening [33].

2.1 The overproduction of target SM in filamentous fungi

The individual species of filamentous fungi under different external and internal signals are able to produce up to 100–150 or more different SMs [34, 35, 36]. This is achieved due to the presence in the genomes of these organisms of 30–80 clusters of genes, responsible for various biosynthetic pathways of SMs, so-called biosynthetic gene clusters (BGCs) [37, 38], and by the fine tuning in the regulation of their expression [39, 40]. Currently, more than 20,000 SMs are known to be produced from more than 1000 characterized gene clusters of filamentous fungi [35, 36]. Normally, gene clusters are “silent,” the expression level of BGCs is extremely low, and there is practically no biosynthesis of any SMs (Figure 2A). For the biosynthesis of particular SM, the corresponding BGC must be “awakened” by some specific signal. For instance, the environmental signal 1 is resulted in the biosynthesis of SM1 (Figure 2A). A number of possible SMs, which can be produced in a particular organism after the “awakening” of corresponding BGCs, constitute its biosynthetic capacity for SMs (Figure 2A). In order to become an industrial producer, the fungus strain must increase the production the target SM 100- to 1000-fold or more (Figure 2B). It is also necessary that under favorable conditions (usually, these are fermentation conditions), the content of spin-off products would be extremely small (Figure 2B).

Figure 2.

The shift in biosynthetic capacity for SM production after CSI programs of filamentous fungi. (A) The response of WT strain to the environmental signal 1 and production of SM1. (B) The overproduction of target SM in the HY strain under fermentation conditions. SM: secondary metabolite, CSI: classical strain improvement, WT: wild type, and HY: high yielding.

2.2 The molecular mechanisms of SM overproduction in filamentous fungi

An increase in the production of the target SM by 100- to 1000-fold and the elimination of spin-off products under the fermentation conditions in the improved fungal strains (Figure 2B) are associated with two main molecular events, the upregulation of genes from target BGC and the knockout of genes from alternative BGC [27, 33, 41, 42]. Since the expression of BGC genes is controlled by the pathway-specific regulation [27, 43, 44], global regulation [45, 46], and global regulation of SM [47, 48, 49, 50], the SCI programs are accompanied by changes in such controls. For instance, during CGI program for penicillin G (PenG) production in HY strain (DS17690) two main events occurred, the shift in global regulation of secondary metabolism by introducing mutations in LaeA and VelA and mutations in key enzymes for spin-off SM [33]. That enabled to escape control from the global regulation of SM and involve more than one gene copy of BGC for PenG.

Usually, an increase in the gene dose (introducing several BGC copies for target SM) does not lead to an increase in gene expression. For instance, in the another PenG-overproducing strain (P2niaD18) the enhanced penicillin titer does not strictly depend on the copy number of the cluster [51]. This phenomenon occurs due to the control from the global regulation of SM, which brings only one cluster to work, the rest are silent [35, 52, 53]. Since there are 8 BGC copies for PenG in DS17690 strain, the escape of global regulation resulted in the significant increase in the yield of the target SM [33]. However, the shift in global regulation of SM could also significantly upregulate the expression from alternative BGC [54, 55]. From this point of view, it becomes clear why the CSI program for DS17690 strain changes in global regulation of SM also accompanied by mutations in central enzymes for spin-off SM [33]. The shift in global regulation of SM not only took out of control additional BGC copies for PenG, but also – BGC for spin-off SM. Since the screening went against spin-off SM, variants with mutations in central enzymes of alternative BGC were selected [33]. It was demonstrated, that delections in such central enzymes (NRPS or PKS megasynthases) lead by a still unknown mechanism to the silencing of all genes from the corresponding gene cluster [56].

Thus, if the improving of filamentous fungi strains for SM production led to duplication of target BGC, the simultaneous changes both in the system of global regulation of SM and at the level of alternative BGC expression are required [33]. However, in many improved strains, industrial producers of SMs, spin-off products are still formed during fermentation [57, 58]. These impurities are intermediates of the target SM biosynthesis; their amount depends on numerous fermentation conditions [58]. For instance, the cephalosporin C (CPC) yield after fermentation of improved Acremonium chrysogenum strains often contaminated with deacetylcephalosporin C (DAC) [57]. DAC is immediate precursor of CPC in the biosynthetic pathway. The conversion from DAC to CPC is catalyzed by deacetylcephalosporin-C acetyltransferase enzyme (CefG; EC by, occurs in the cytoplasm [59] and is utilizes one molecule of cytoplasmic acetyl-CoA per reaction. In HY strains the CPC production increased 200- to 300-fold and the expression from BGC for CPC (cef genes) upregulated 20- to 300-fold [41]. In this case the acetyl-CoA content may be depleted in some HY strains [57, 58]. From the other side the screening during CSI programs went the same way against DAC admixture, events that reduce CPC/DAC ratio were selected. In the A. chrysogenum HY strain RNCM 408D [60], the CPC/DAC does not exceed 10–15% [61]. Thus, to increase in SM production is accompanied not only by changes in the expression and regulation of BGCs, but by reprogramming the whole organism, starting with changes in the primary metabolism (for the needs of target SM biosynthesis), ending with changes in the transport and assimilation of nutrients, the ability to assimilate oxygen, adaptation to fermentation conditions, and much more [42]. That is why the improvement of the filamentous fungi strain is a multi-step process, involving alterations in many spheres of the strain’s vital activity, and there is no “golden bullet,” no one cardinal event that converts WT strain to HY (Figure 1).

2.3 The technological limit of CSI of filamentous fungi for SM production

Filamentous fungi are a good facility for the improving of SM production by random mutagenesis and screening [2, 28, 62, 63]. Among the most popular mutagens used for fungal strain improvement are DNA alkylating NTG (N′-methyl-N′-nitro-N′-nitrosoguanidine) which typically produces a variety of point mutations and UV irradiation at 254 nm, which causes the formation of pyrimide dimers leading to point mutations and deletions [28]. In general, the CSI program for SM production in filamentous fungi looks as shown in Figure 3. The WT strain produce target SM in most cases at a low level, usually it does not exceed 30–50 μg/ml of fermentation medium [27, 41]. On order to convert WT to HY strain a number of independent events, involving BGCs regulation, changes in primary metabolism, strain physiology and so on, must occur. Moreover, all these events do not have to happen simultaneously. There are a number of ways in which the production of target SM gains added benefit. The first round of mutagenesis against WT strain results in a series of mutants, some of them have shift in the production of target SM (Figure 3). The majority of alterations lead to decrease or lack of the production change, but some mutants may show the increase in SM production. They are used as origins for the next mutagenesis round, followed by the next stage of screening. For example, on the A′ round of mutagenesis, the production level of SM was increased by A%, on the B′ round of mutagenesis, the production level of SM was increased by B% (Figure 3). Thus the CSI gradually leads to the emergence of a whole set of changes leading to an increase in the production. However, along with beneficial changes that increase the production of the target SM, reduce the amount of spin-off products, and others, numerous side changes begin to accumulate in the fungal strain. They can appear in a slow growth on agar and liquid media [27, 64, 65], a decrease in stress resistance [66], reduction in the conidia formation [64] and many other properties, expressed in a decrease in the overall viability of the strain [66, 67]. Finally, the stage comes when the next mutagenic effect no longer leads to further strain improvement. This is the technological limit of CSI method, it comes for each improvement program for a particular strain of filamentous fungus and is usually found at the 10–50th round of mutagenesis [60, 68].

Figure 3.

Random mutagenesis and screening for the improving of filamentous fungi strains for SM production. SM: secondary metabolite, CSI: classical strain improvement, WT: wild type, and HY: high yielding.


3. Role of polyamines in filamentous fungi HY strains (after SCI for SM production)

Aliphatic polyamines (PAs) such as putrescine, spermidine, and spermine are widespread in nature; they are present in all living organisms and are also present in viral particles [69]. Despite the fact that these compounds have long been known as components of biological systems, there is still no clear understanding of their role in various bioprocesses [70]. The most studied functions of PAs are associated with stimulating the growth of microorganisms, increasing membrane stability, interacting with nucleic acids, and regulating the level of heterochromatin in the cell [71, 72, 73, 74]. The roles of PAs in fungi cell have also been discussed [71, 75]. The main topics correspond to stress resistance [76], phytopathogenicity [77] and fungal development, including sporulation, growth and other stages of lifecycle [78, 79]. There is tight control of polyamine homeostasis in the cell [80]. For a particular organism, there is a certain content of PAs. For this, there are both biosynthetic and catabolic enzymes of polyamines (Figure 4), moreover, the amount of key biosynthetic enzymes, such as ornithine decarboxylase (ODC), or S-adenosylmethionine decarboxylase (AdoMetDC) is regulated at the levels of transcription, translation and turnover rate (half-life) [80, 81].

Figure 4.

The metabolism of polyamines in filamentous fungi. PAs: polyamines, ODC: ornithine decarboxylase, ADC: arginine decarboxylase, AdoMetS: S-adenosylmethionine synthetase, AdoMetDC: S-adenosylmethionine decarboxylase, SpdS: spermidine synthase, SpmS: spermine synthase, PAO: polyamine oxidase, SpmO: spermine oxidase, SSAT: spermidine/spermine-N1-acetyltransferase, APAO: N-acetylpolyamine oxidase, DFMO: α-difluoromethylornithine, APA: 1-aminooxy-3-aminopropane, DFMA: α-difluoromethylarginine, and AO-Agm: 1-aminooxy-3-guanidinopropane.

3.1 Influence of PAs on SM production in improved strains

Recently it was demonstrated, aliphatic PAs, such as 1,3-Diaminopropane (DAP) or spermidine (Spd) may increase the production of target SMs in HY fungi strains [82]. The production level of PenG in P. chrysogenum increased by 10–15% [82], the CPC production in A. chrysogenum HY strain increased by 10–15% [83] and the production of lovastatin (LOV) by Aspergillus terreus HY strain at the particular timepoints of fermentation increased by 20–45% [84]. The addition of 5 mM PAs to agar media increased the survival of HY strains, as demonstrated by the drop and dilution assay [83, 84]. The PAs addition during the fermentation of improved strains also led to upregulation of corresponding BGCs (pen, cef and lov genes) [82, 83, 84]. This is important because A. chrysogenum and A. terreus HY strains have reached their technological limit after CSI programs [60, 68] and the possibility of further increasing the production of valuable pharmacologically significant substances as a result of the addition of relatively cheap PAs may be significant in the biotechnology industry.

3.2 Possible mechanisms of influencing of exogenous PAs on SM production

The addition of exogenous PAs also accompanied by the laeA upregulation in all studied improved strains [82, 83, 84]. LaeA is global regulator of SM in filamentous fungi [50]. It is S-adenosylmethionine (SAMe)-dependent histone methylate, which acts epigenetically, through the chromatin remodeling [85]. Since the biosynthesis of PAs and the work of LaeA require the same substrate, SAMe, the addition of exogenous PAs can lead to a shift in the global regulation of the studied HY strains. It is also known that in all these strains, P. chrysogenum Wis 54-1255, A. chrysogenum RNCM 408D and A. terreus No. 44-62, the only one copy of corresponding BGC is present, one copy of pen genes [86], one copy of “early” and “late” cef genes [3241] and one copy on lov genes [84] respectively. In this regard, the CSI programs for these strains could follow a rather different pathway than P. chrysogenum DS17690 with eight copies of pen genes [30], without significant shifting and removing the global regulation of SM and mutation in LaeA.

In order to confirm this hypothesis we carried out fermentation with PAs for WT, HY and E6 strains of A. terreus [44]. The LOV production in A. terreus is under the control of two major positive regulators, the LovE pathway-specific regulator and LaeA global regulator of fungi SM [44]. The A. terreus E6 strain derived from WT by the genetic engineering introduction the additional copy of lovE gene under the control of constitutive promotor [27]. LovE is Zn2Cys6 transcription factor for pathway-specific regulation of lov genes; in A. terreus OE::lovE the LOV production increased 10- to 12-fold [27]. Surprisingly, the addition of PAs during the fermentation of the E6 strain led, on the contrary, to a decrease in LOV production (Figure 5). But it is also known that pathway-specific regulators can negatively regulate LaeA [49]. For instance, AflR, a sterigmatocystin pathway-specific transcription factor, negatively regulate the expression of laeA [49]. E6 strain has the only one targeted change in the genome of WT, that led to constitutive (which also means LaeA-independent) overexpression of lovE. The effects of laeA downregulation (due to an increase in the dose of the negative regulator gene) on LOV production in E6 strain are compensated by lovE upregulation [49]. However, LovE, unlike LaeA, upregulate only lov genes for biosynthesis, not for transport and resistance Therefore, the PAs addition during fermentation of E6 strain causes a toxic effect and the LOV production decreases (Figure 5).

Figure 5.

Effect of exogenous polyamines on lovastatin production in the WT, E6, and HY A. terreus strains. WT: wild type, E6: OE::lovE, HY: high yielding, and Spd: spermidine. Adapted with permission from [44].

3.3 The endogenous polyamines content in A. chrysogenum HY strain

Since exogenous PAs are able to influence the production of SM in HY strains, it is important to know if there have been any changes in the metabolism and homeostasis of polyamines into the cells of improved fungi strains. Recently it was demonstrated, that A. chrysogenum HY strain shows increased resistance to inhibitors of ODC, the key enzyme of PAs biosynthesis, on minimal agar media [65]. The addition of 5 mM α-difluoromethylornithine (DFMO) or 5 mM of 1-aminooxy-3-aminopropane (APA) completely inhibited the growth of the WT strain, unlike HY strain (Figure 4) [65]. Such kind of resistance against inhibiters of key enzyme for PAs production turned out to be rather strange since HY strain is significantly weakened after SCI program [32, 61, 64, 66, 67]. The only previously observed advantage over the WT strain was expressed in CPC overproduction [41]. To explain the phenomenon of the resistance of HY strain to ODC inhibitors, an inhibitory analysis of A. chrysogenum WT and HY strains was performed against all pathways of putrescine biosynthesis (Figure 3). In filamentous fungi, in addition to the main pathway of putrescine (Put) biosynthesis, via ODC, there is also an additional pathway through arginine decarboxylase (ADC) and biosynthesis of agmatine (Figure 3). The inhibitory analysis demonstrated shift from the ADC-dependent to ODC-dependent biosynthesis of Put. During the fermentation for CPC production the total PAs content into HY strain has been increased by about fivefold [65].

The reasons for the increased production of PAs in the HY strain were discussed [65]. One on the reasons may be related to strain improvement techniques. The increasing in PAs content may be spin-o result of mutagenesis and DNA damage. Recently it was demonstrated that PAs can maintain the genome integrity via homology-directed DNA repair, enhancing the DNA strand exchange activity of RAD51 recombinase [87]. PAs also can protect DNA from free-radical damage by reacting direct with the reactive oxygen species [88, 89, 90].


4. Conclusions

As a result of CSI programs for filamentous fungi, a number of pharmaceutically significant SMs have been overproduced. One of the side effects of the high yielding strains improvement may be an increase in the content of polyamines (PAs). An increase in the PAs’ content could occur as a response to mutagenesis during CSI. The recently discovered increase in the production of targeted SM in some HY strains after the addition of exogenous PAs may occur due to a decrease in endogenous biosynthesis of PAs and the release of additional resources for the biosynthesis of the target SM.



This research was funded by the Russian Foundation for Basic Research (grant number 19-04-01173).


Conflict of interest

The author declares no conflict of interest.


Notes/thanks/other declarations

This work is dedicated to the author’s teacher and the scientific consultant of doctoral dissertation, Yuri Eduardovich Bartoshevich (passed away in 2014), one of the pioneers in the field of fungi chemical mutagenesis, the author of numerous scientific works, including publications in the Nature journal [90].


  1. 1. Meyer V, Basenko EY, Benz JP, Braus GH, Caddick MX, Csukai M, et al. Growing a circular economy with fungal biotechnology: A white paper. Fungal Biology and Biotechnology. 2020;7:5. DOI: 10.1186/s40694-020-00095-z
  2. 2. Meyer V, Andersen MR, Brakhage AA, Braus GH, Caddick MX, Cairns TC, et al. Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: A white paper. Fungal Biology and Biotechnology. 2016;3:6. DOI: 10.1186/s40694-016-0024-8
  3. 3. Zhgun A, Avdanina D, Shumikhin K, Simonenko N, Lyubavskaya E, Volkov I, et al. Detection of potential biodeterioration risks for tempera painting in 16th century exhibits from State Tretyakov Gallery. PLoS One. 2020;15:e0230591. DOI: 10.1371/journal.pone.0230591
  4. 4. Siegel SM, Galun M, Siegel BZ. Filamentous fungi as metal biosorbents: A review. Water, Air, and Soil Pollution. 1990;53:335-344. DOI: 10.1007/BF00170747
  5. 5. Kure CF, Skaar I. The fungal problem in cheese industry. Current Opinion in Food Science. 2019;29:14-19. DOI: 10.1016/j.cofs.2019.07.003
  6. 6. Ly S, Kakahi FB, Mith H, Phat C, Fifani B, Kenne T, et al. Engineering synthetic microbial communities through a selective biofilm cultivation device for the production of fermented beverages. Microorganisms. 2019;7:206. DOI: 10.3390/microorganisms7070206
  7. 7. Anupma A, Tamang JP. Diversity of filamentous fungi isolated from some amylase and alcohol-producing starters of India. Frontiers in Microbiology. 2020;11:905. DOI: 10.3389/fmicb.2020.00905
  8. 8. Geisen R, Färber P. New aspects of fungal starter cultures for fermented foods. Applied Microbiology. 2005:13-29. DOI: 10.1007/0-306-46888-3_1
  9. 9. Sugiharto S. A review of filamentous fungi in broiler production. Annals of Agricultural Science. 2019:1-8. DOI: 10.1016/j.aoas.2019.05.005
  10. 10. Arnau J, Yaver D, Hjort CM. Strategies and challenges for the development of industrial enzymes using fungal cell factories. Grand Challenges in Fungal Biotechnology. 2020:179-210. DOI: 10.1007/978-3-030-29541-7_7
  11. 11. Gudynaite-Savitch L, White TC. Fungal biotechnology for industrial enzyme production: Focus on (hemi)cellulase production strategies, advances and challenges. Fungal Biology. 2016:395-439. DOI: 10.1007/978-3-319-27951-0_19
  12. 12. Hu HL, van den Brink J, Gruben BS, Wösten HAB, Gu JD, de Vries RP. Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi. International Biodeterioration and Biodegradation. 2011;65:248-252. DOI: 10.1016/j.ibiod.2010.11.008
  13. 13. Jun H, Kieselbach T, Jönsson LJ. Enzyme production by filamentous fungi: Analysis of the secretome of Trichoderma reesei grown on unconventional carbon source. Microbial Cell Factories. 2011;10:68. DOI: 10.1186/1475-2859-10-68
  14. 14. Magnuson JK, Lasure LL. Organic acid production by filamentous fungi. Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. 2004:307-340. DOI: 10.1007/978-1-4419-8859-1_12
  15. 15. Yang L, Lübeck M, Lübeck PS. Aspergillus as a versatile cell factory for organic acid production. Fungal Biology Reviews. 2017;31:33-49. DOI: 10.1016/j.fbr.2016.11.001
  16. 16. Calvo AM, Wilson RA, Bok JW, Keller NP. Relationship between secondary metabolism and fungal development. Microbiology and Molecular Biology Reviews. 2002;66:447-459. DOI: 10.1128/mmbr.66.3.447-459.2002
  17. 17. Alberti F, Foster GD, Bailey AM. Natural products from filamentous fungi and production by heterologous expression. Applied Microbiology and Biotechnology. 2017;101:493-500. DOI: 10.1007/s00253-016-8034-2
  18. 18. Nassiri-Koopaei N, Faramarzi MA. Recent developments in the fungal transformation of steroids. Biocatalysis and Biotransformation. 2015;33:1-28. DOI: 10.3109/10242422.2015.1022533
  19. 19. Kristan K, Rižner TL. Steroid-transforming enzymes in fungi. The Journal of Steroid Biochemistry and Molecular Biology. 2012;129:79-91. DOI: 10.1016/j.jsbmb.2011.08.012
  20. 20. Kwon-Chung KJ, Sugui JA. Sexual reproduction in Aspergillus species of medical or economical importance: Why so fastidious? Trends in Microbiology. 2009;17:481-487. DOI: 10.1016/j.tim.2009.08.004
  21. 21. Krijgsheld P, Bleichrodt R, van Veluw GJ, Wang F, Müller WH, Dijksterhuis J, et al. Development in Aspergillus. Studies in Mycology. 2013;74:1-29. DOI: 10.3114/sim0006
  22. 22. Clutterbuck AJ. Parasexual recombination in fungi. Journal of Genetics. 1996;75:281-286. DOI: 10.1007/BF02966308
  23. 23. Guzmán-Chávez F, Zwahlen RD, Bovenberg RAL, Driessen AJM. Engineering of the filamentous fungus Penicillium chrysogenumas cell factory for natural products. Frontiers in Microbiology. 2018;15:2768. DOI: 10.3389/fmicb.2018.02768
  24. 24. Dikshit R, Tallapragada P. Development and screening of mutants from Monascus sanguineus for secondary metabolites production. Beni-Suef University Journal of Basic and Applied Sciences. 2018;7:235-240. DOI: 10.1016/j.bjbas.2018.03.001
  25. 25. McLean KJ, Hans M, Meijrink B, van Scheppingen WB, Vollebregt A, Tee KL, et al. Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proceedings of the National Academy of Sciences. 2015;112:2847-2852. DOI: 10.1073/pnas.1419028112
  26. 26. Anyaogu DC, Mortensen UH. Heterologous production of fungal secondary metabolites in Aspergilli. Frontiers in Microbiology. 2015;6:77. DOI: 10.3389/fmicb.2015.00077
  27. 27. Zhgun AA, Dumina MV, Voinova TM, Dzhavakhiya VV, Eldarov MA. Role of acetyl-CoA synthetase and LovE regulator protein of polyketide biosynthesis in lovastatin production by wild-type and overproducing Aspergillus terreus strains. Applied Biochemistry and Microbiology. 2018;54:188-197. DOI: 10.1134/S0003683818020138
  28. 28. Nevalainen KMH. Strain improvement in filamentous fungi - An overview. Applied Mycology and Biotechnology. 2001;1:289-304. DOI: 10.1016/S1874-5334(01)80013-7
  29. 29. Nielsen JC, Nielsen J. Development of fungal cell factories for the production of secondary metabolites: Linking genomics and metabolism. Synthetic and Systems Biotechnology. 2017:5-12. DOI: 10.1016/j.synbio.2017.02.002
  30. 30. Skellam E. Strategies for engineering natural product biosynthesis in fungi. Trends in Biotechnology. 2019;37:416-427. DOI: 10.1016/j.tibtech.2018.09.003
  31. 31. Zolan ME. Chromosome-length polymorphism in fungi. Microbiological Reviews. 1995;59:686-698. DOI: 10.1128/mmbr.59.4.686-698.1995
  32. 32. Dumina MV, Zhgun AA, Domracheva AG, Novak MI, El’darov MA. Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C. Russian Journal of Genetics. 2012;48:778-784. DOI: 10.1134/S1022795412050067
  33. 33. Salo OV, Ries M, Medema MH, Lankhorst PP, Vreeken RJ, Bovenberg RAL, et al. Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum. BMC Genomics. 2015;16:937. DOI: 10.1186/s12864-015-2154-4
  34. 34. Brakhage AA. Regulation of fungal secondary metabolism. Nature Reviews. Microbiology. 2013;11:21-32. DOI: 10.1038/nrmicro2916
  35. 35. Kjærbølling I, Mortensen UH, Vesth T, Andersen MR. Strategies to establish the link between biosynthetic gene clusters and secondary metabolites. Fungal Genetics and Biology. 2019:107-121. DOI: 10.1016/j.fgb.2019.06.001
  36. 36. Brakhage AA, Schroeckh V. Fungal secondary metabolites - Strategies to activate silent gene clusters. Fungal Genetics and Biology. 2011;48:15-22. DOI: 10.1016/j.fgb.2010.04.004
  37. 37. Macheleidt J, Mattern DJ, Fischer J, Netzker T, Weber J, Schroeckh V, et al. Regulation and role of fungal secondary metabolites. Annual Review of Genetics. 2016;50:371-392. DOI: 10.1146/annurev-genet-120215-035203
  38. 38. Drott MT, Bastos RW, Rokas A, Ries LNA, Gabaldón T, Goldman GH, et al. Diversity of secondary metabolism in Aspergillus nidulans clinical isolates. mSphere. 2020;5:e00156-20. DOI: 10.1128/msphere.00156-20
  39. 39. Yu JH, Keller N. Regulation of secondary metabolism in filamentous fungi. Annual Review of Phytopathology. 2005;43:437-458. DOI: 10.1146/annurev.phyto.43.040204.140214
  40. 40. Keller NP. Fungal secondary metabolism: Regulation, function and drug discovery. Nature Reviews Microbiology. 2019;17:167-180. DOI: 10.1038/s41579-018-0121-1
  41. 41. Dumina MV, Zhgun AA, Novak MI, Domratcheva AG, Petukhov DV, Dzhavakhiya VV, et al. Comparative gene expression profiling reveals key changes in expression levels of cephalosporin C biosynthesis and transport genes between low and high-producing strains of Acremonium chrysogenum. World Journal of Microbiology and Biotechnology. 2014;30:2933-2941. DOI: 10.1007/s11274-014-1721-1
  42. 42. Terfehr D, Dahlmann TA, Kück U. Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs. BMC Genomics. 2017;18:272. DOI: 10.1186/s12864-017-3663-0
  43. 43. Yin W, Keller NP. Transcriptional regulatory elements in fungal secondary metabolism. Journal of Microbiology. 2011;49:329-339. DOI: 10.1007/s12275-011-1009-1
  44. 44. Zhgun AA, Nuraeva GK, Eldarov M. The role of LaeA and LovE regulators in lovastatin biosynthesis with exogenous polyamines in Aspergillus terreus. Applied Biochemistry and Microbiology. 2019;55:626-635. DOI: 10.1134/S0003683819060176
  45. 45. Alkan N, Meng X, Friedlander G, Reuveni E, Sukno S, Sherman A, et al. Global aspects of pacC regulation of pathogenicity genes in Colletotrichum gloeosporioides as revealed by transcriptome analysis. Molecular Plant-Microbe Interactions. 2013;26:1345-1358. DOI: 10.1094/MPMI-03-13-0080-R
  46. 46. Jekosch K, Kück U. Loss of glucose repression in an Acremonium chrysogenum beta-lactam producer strain and its restoration by multiple copies of the cre1 gene. Applied Microbiology and Biotechnology. 2000;54:556-563. Available from:
  47. 47. Lind AL, Smith TD, Saterlee T, Calvo AM, Rokas A. Regulation of secondary metabolism by the velvet complex is temperature-responsive in Aspergillus. G3: Genes, Genomes, Genetics. 2016;6:4023-4033. DOI: 10.1534/g3.116.033084
  48. 48. Kosalková K, García-Estrada C, Ullán RV, Godio RP, Feltrer R, Teijeira F, et al. The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum. Biochimie. 2009;91:214-225. DOI: 10.1016/j.biochi.2008.09.004
  49. 49. Bok JW, Keller NP. LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryotic Cell. 2004;3:527-535. Available from:
  50. 50. Sarikaya-Bayram Ö, Palmer JM, Keller N, Braus GH, Bayram Ö. One Juliet and four Romeos: VeA and its methyltransferases. Frontiers in Microbiology. 2015;6:1. DOI: 10.3389/fmicb.2015.00001
  51. 51. Ziemons S, Koutsantas K, Becker K, Dahlmann T, Kück U. Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes. BMC Biotechnology. 2017;17:16. DOI: 10.1186/s12896-017-0335-8
  52. 52. Gacek A, Strauss J. The chromatin code of fungal secondary metabolite gene clusters. Applied Microbiology and Biotechnology. 2012;95:1389-1404. DOI: 10.1007/s00253-012-4208-8
  53. 53. Strauss J, Reyes-Dominguez Y. Regulation of secondary metabolism by chromatin structure and epigenetic codes. Fungal Genetics and Biology. 2011;48:62-69. DOI: 10.1016/j.fgb.2010.07.009
  54. 54. Collemare J, Seidl MF. Chromatin-dependent regulation of secondary metabolite biosynthesis in fungi: Is the picture complete? FEMS Microbiology Reviews. 2019;43:591-607. DOI: 10.1093/femsre/fuz018
  55. 55. Wang B, Lv Y, Li X, Lin Y, Deng H, Pan L. Profiling of secondary metabolite gene clusters regulated by LaeA in Aspergillus niger FGSC A1279 based on genome sequencing and transcriptome analysis. Research in Microbiology. 2017;169:67-77. DOI: 10.1016/j.resmic.2017.10.002
  56. 56. Chen G, Chu J. Characterization of two polyketide synthases involved in sorbicillinoid biosynthesis by Acremonium chrysogenum using the CRISPR/Cas9 system. Applied Biochemistry and Biotechnology. 2019;188:1134-1144. DOI: 10.1007/s12010-019-02960-z
  57. 57. Gutiérrez S, Velasco J, Marcos AT, Fernández FJ, Fierro F, Barredo JL, et al. Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum. Applied Microbiology and Biotechnology. 1997;48:606-614. DOI: 10.1007/s002530051103
  58. 58. Fujisawa Y, Shirafuji H, Kida M, Nara K, Yoneda M, Kanzaki T. New findings on cephalosporin C biosynthesis. Nature: New Biology. 1973;246:154-155. DOI: 10.1038/newbio246154a0
  59. 59. Martín JF. Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi. Fungal Biology and Biotechnology. 2020;7:6. DOI: 10.1186/s40694-020-00096-y
  60. 60. Patent RU2426793 C12P35/06, C07D501/02, C12R1/75. Method of cephalosporin C biosynthesis by using new Acremonium chrysogenum strain; 2011
  61. 61. Dumina MV, Zhgun AA, Kerpichnikov IV, Domracheva AG, Novak MI, Valiachmetov AY, et al. Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae. Applied Biochemistry and Microbiology. 2013;49:368-377. DOI: 10.1134/S0003683813040042
  62. 62. Kramer A, Paun L, Imhoff JF, Kempken F, Labes A. Development and validation of a fast and optimized screening method for enhanced production of secondary metabolites using the marine Scopulariopsis brevicaulis strain LF580 producing anti-cancer active scopularide A and B. PLoS One. 2014;9:e103320. DOI: 10.1371/journal.pone.0103320
  63. 63. Domratcheva AG, Zhgun AA, Novak NV, Dzhavakhiya VV. The influence of chemical mutagenesis on the properties of the cyclosporine a high-producer strain Tolypocladium inflatum VKM F-3630D. Applied Biochemistry and Microbiology. 2018;54:53-57. DOI: 10.1134/S0003683818010027
  64. 64. Zhgun AA, Ivanova MA, Domracheva AG, Novak MI, Elidarov MA, Skryabin KG, et al. Genetic transformation of the mycelium fungi Acremonium chrysogenum. Applied Biochemistry and Microbiology. 2008;44:600-607. DOI: 10.1134/S0003683808060070
  65. 65. Hyvönen MT, Keinänen TA, Nuraeva GK, Yanvarev DV, Khomutov M, Khurs EN, et al. Hydroxylamine analogue of agmatine: Magic bullet for arginine decarboxylase. Biomolecules. 2020;10:1-16. DOI: 10.3390/biom10030406
  66. 66. Kalebina TS, Selyakh IO, Gorkovskii AA, Bezsonov EE, El’darov MA, Novak MI, et al. Structure peculiarities of cell walls of Acremonium chrysogenum-an autotroph of cephalosporin C. Applied Biochemistry and Microbiology. 2010;46:614-619. DOI: 10.1134/S0003683810060098
  67. 67. Valiakhmetov AI, Trilisenko LV, Vagabov VM, Bartoshevich IE, Kulaev IS, Novak MI, et al. The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum. Prikladnaia Biokhimiia i Mikrobiologiia. 2010;46:198-204. Available from:
  68. 68. Patent RU2261901 C12N1/14, C12P17/06, C07D309/30. Fungal strain Aspergillus terreus No. 44-62 - Producer of lovastatin, industrial method of isolation of lovastatin and method of lactonization of statins; 2005
  69. 69. Cohen SS. A Guide to the Polyamines. 1st ed. NY: Oxford University Press; 1998
  70. 70. Miller-Fleming L, Olin-Sandoval V, Campbell K, Ralser M. Remaining mysteries of molecular biology: The role of polyamines in the cell. Journal of Molecular Biology. 2015;427:3389-3406. DOI: 10.1016/j.jmb.2015.06.020
  71. 71. Rocha RO, Wilson RA. Essential, deadly, enigmatic: Polyamine metabolism and roles in fungal cells. Fungal Biology Reviews. 2019;33:47-57. DOI: 10.1016/j.fbr.2018.07.003
  72. 72. Chen D, Shao Q , Yin L, Younis A, Zheng B. Polyamine function in plants: Metabolism, regulation on development, and roles in abiotic stress responses. Frontiers in Plant Science. 2019;9:1945. DOI: 10.3389/fpls.2018.01945
  73. 73. Pegg AE. Mammalian polyamine metabolism and function. IUBMB Life. 2009;61:880-894. DOI: 10.1002/iub.230
  74. 74. Mounce BC, Olsen ME, Vignuzzi M, Connor JH. Polyamines and their role in virus infection. Microbiology and Molecular Biology Reviews. 2017;81:e00029-17. DOI: 10.1128/mmbr.00029-17
  75. 75. Valdés-Santiago L, Ruiz-Herrera J. Polyamines in Fungi: Their Distribution, Metabolism, and Role in Cell Differentiation and Morphogenesis. (Mycology Book 30) 1st Ed. CRC Press; 19 December 2019. p. 186. ISBN: 9780367377106
  76. 76. Valdés-Santiago L, Ruiz-Herrera J. Stress and polyamine metabolism in fungi. Frontiers in Chemistry. 2013;1:42. DOI: 10.3389/fchem.2013.00042
  77. 77. Valdés-Santiago L, Cervantes-Chávez JA, León-Ramírez CG, Ruiz-Herrera J. Polyamine metabolism in fungi with emphasis on phytopathogenic species. Journal of Amino Acids. 2012;2012:1-13. DOI: 10.1155/2012/837932
  78. 78. Crespo-Sempere A, Estiarte N, Marín S, Sanchis V, Ramos AJ. Targeting Fusarium graminearum control via polyamine enzyme inhibitors and polyamine analogs. Food Microbiology. 2015;49:95-103. DOI: 10.1016/
  79. 79. Guevara-Olvera L, Calvo-Mendez C, Ruiz-Herrera J. The role of polyamine metabolism in dimorphism of Yarrowia lipolytica. Journal of General Microbiology. 1993;139:485-493. DOI: 10.1099/00221287-139-3-485
  80. 80. Minois N, Carmona-Gutierrez D, Madeo F. Polyamines in aging and disease. Aging (Albany NY). 2011;3:716-732. DOI: 10.18632/aging.100361
  81. 81. Iwami K, Wang JY, Jain R, McCormack S, Johnson LR. Intestinal ornithine decarboxylase: Half-life and regulation by putrescine. American Journal of Physiology. Gastrointestinal and Liver Physiology. 1990;258:G309. DOI: 10.1152/ajpgi.1990.258.2.g308
  82. 82. Martín J, García-Estrada C, Kosalková K, Ullán RV, Albillos SM, Martín J-F. The inducers 1,3-diaminopropane and spermidine produce a drastic increase in the expression of the penicillin biosynthetic genes for prolonged time, mediated by the laeA regulator. Fungal Genetics and Biology. 2012;49:1004-1013. DOI: 10.1016/j.fgb.2012.10.001
  83. 83. Zhgun AA, Kalinin SG, Novak MI, Domratcheva AG, Petukhov DV, Dzhavakhiya VV, et al. The influence of polyamines on cephalosporine C biosynthesis in Acremonium chrysogenum strains. Izvestiya Vuzov. Prikladnaya Khimiya i Biotekhnologiya. 2015;14:47-54
  84. 84. Zhgun AA, Nuraeva GK, Dumina MV, Voinova TM, Dzhavakhiya VV, Eldarov MA. 1,3-Diaminopropane and spermidine upregulate lovastatin production and expression of lovastatin biosynthetic genes in Aspergillus terreus via LaeA regulation. Applied Biochemistry and Microbiology. 2019;55:244-255. DOI: 10.1134/S0003683819020170
  85. 85. Martín JF. Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in Penicillium chrysogenum: Cross-talk regulation of secondary metabolite pathways. Journal of Industrial Microbiology & Biotechnology. 2016;44:525-535. DOI: 10.1007/s10295-016-1830-y
  86. 86. Peng Q, Yuan Y, Gao M, Chen X, Liu B, Liu P, et al. Genomic characteristics and comparative genomics analysis of Penicillium chrysogenum KF-25. BMC Genomics. 2014;15:144. DOI: 10.1186/1471-2164-15-144
  87. 87. Lee C-Y, Su G-C, Huang W-Y, Ko M-Y, Yeh H-Y, Chang G-D, et al. Promotion of homology-directed DNA repair by polyamines. Nature Communications. 2019;10:65. DOI: 10.1038/s41467-018-08011-1
  88. 88. Murray Stewart T, Dunston TT, Woster PM, Casero RA. Polyamine catabolism and oxidative damage. The Journal of Biological Chemistry. 2018;293:18736-18745. DOI: 10.1074/jbc.TM118.003337
  89. 89. Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, Casero RA. The natural polyamine spermine functions directly as a free radical scavenger. Proceedings of the National Academy of Sciences of the United States of America. 1998;95:11140-11145. DOI: 10.1073/pnas.95.19.11140
  90. 90. Tikchonenko TI, Velikodvorskaya GA, Bobkova AF, Bartoshevich YE, Lebed EP, Chaplygina NM, et al. New fungal viruses capable of reproducing in bacteria. Nature. 1974;249:454-456. DOI: 10.1038/249454a0

Written By

Alexander A. Zhgun

Submitted: 04 September 2019 Reviewed: 24 August 2020 Published: 07 October 2020