In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depend- ing on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications. . . . . Keywords Fungal biotechnology Inducible promoter systems Heterologous products Functional genomics Strain improvement Introduction accompanied by the development of sophisticated gene ex- pression systems in the early studies, filamentous fungi lag Filamentous fungi possess a highly important role in pharma- behind in providing comparable systems (Alberti et al. ceutical and biotechnical applications. As producers of prima- 2017). This is in part due to the fact that the expression ma- ry and secondary metabolites, they are economically impor- chinery is more complex in filamentous fungi than in bacteria tant for the food, paper, or pharmaceutical industry, among and yeasts. others (Alberti et al. 2017; Dufosse et al. 2014; Kuivanen et As an advantage, filamentous fungi tend to have post- al. 2015). In order to obtain strains with an optimal production translational modification systems that allow the production yield, strain improvement programs were set up to generate of functional mammalian proteins, which are difficult to per- randomly mutagenized derivatives that have passed strict se- form in bacterial and yeast expression systems in many cases lection procedures. With the advent of DNA-mediated trans- (Ward 2012). formation systems for filamentous fungi about 40 years ago, In a recent study, molecular tools, such as homologous in vitro recombinant technologies were invented to genetically recombination and RNA interference systems that are suitable manipulate industrial strains (Case et al. 1979; Stahl et al. for the genetic manipulation of filamentous fungi were 1982). While bacterial and yeast expression systems were reviewed (Kück and Hoff 2010). Here, we will extend this survey by summarizing the available inducible promoter sys- tems to obtain controlled gene expression in industrial strains. * Ulrich Kück Finally, we focus on representative examples, where filamen- firstname.lastname@example.org tous fungi were used for the successful production of heterol- ogous proteins. Nowadays, this is highly influenced by the Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University availability of genomic and functional genomic data sets, Bochum, Universitätsstr. 150, 44780 Bochum, Germany 6358 Appl Microbiol Biotechnol (2018) 102:6357–6372 which are used for novel strategies in strain improvement model organism. Thus, it must be considered that certain in- programs. Another recent valuable tool for directed strain im- ductive or repressive substances or the produced protein has a provement is the CRISPR/Cas9 system, which is suitable for toxic effect in some organisms (Kim et al. 2016; Lee et al. multiple gene editing events. We would like to refer to recent- 2010). In addition, a leakiness of the promoter, which results ly published reviews that cover the development of CRISPR/ in a minimal constitutive activity, leads to uncontrolled gene Cas9-based gene editing in filamentous fungi (Krappmann expression (Meyer et al. 2011). In the subsequent years, sev- 2017; Shi et al. 2017). eral inducible expression systems were discovered and used for applied approaches. Few of them that were used in recent Claims and conditions for the application of inducible biotechnical applications will be summed up in this review. promoters Inducible promoters depending on carbon sources Extensive studies in eukaryotic systems have established the mechanistic key concept of controlled gene regulation (Levine The glaA promoter of the glucoamylase A gene from et al. 2014;Soutourina 2017; Spitz and Furlong 2012). The Aspergillus niger was one of the first inducible systems, which expression of eukaryotic genes is governed primarily at the was commonly used in filamentous fungi. Induction of glaA level of transcriptional initiation, which corresponds to the promoter-driven gene expression occurs on starch-containing complex interplay between the promoter, RNA polymerase growth media or alternatively on media with maltodextrine, II, and transcription factors (Sainsbury et al. 2015; Struhl maltose, or glucose. Repressionis achievedwhen strains are 1987). Promoters are defined as the DNA sequence immedi- grown on xylose-containing minimal media (Siedenberg et al. ately surrounding the transcription start site, which is bound 1999). In another homologous promoter system, promoter se- by the basal transcription machinery, thus allowing transcrip- quences of the glucoamylase gene were fused with the β- tional initiation. Over the years, the knowledge of promoter glucosidase gene from A. niger or endoglucanase IV from architecture expanded and led to the development and estab- Trichoderma reesei for the construction of recombinant lishment of constitutive and inducible promoter systems (Su et Penicillium verruculosum strains (Bulakhov et al. 2017). al. 2012; Woo and Li 2011). In Table 1, we list inducible Compared to the wild type, the strain harboring the recombinant promoter systems that are commonly used in applied research promoter displayed a more efficient hydrolysis of a lignocellu- with filamentous fungi. In the following, some of these are losic substrate. In Aspergillus oryzae,the glaA promoter was described in detail. recently used for the production of L-malate, which is widely Inducible promoters were initially developed to synthesize utilized in the food and beverage industry as an acidulant and exactly one additional gene product under precisely defined flavor enhancer (Liu et al. 2017b; Thakker et al. 2015). A fur- conditions within a certain time interval. Therefore, these sys- ther improvement of promoter strength was attained when five tems find application in the functional characterization of es- copies of the -427 to -331 upstream region of glaA carrying at sential genes in the laboratory, as well as in the biotechnolog- least one CAAT-Box were integrated to efficiently increase L- ical production of heterologous protein products, where a fine- malate production directly from corn starch (Liu et al. 2017b). tunable expression is often desired, especially when the pro- This cis-element arrangement was also shown to improve pro- tein is toxic to the cell (Lee et al. 2010; Nevalainen et al. moter strength and inducibility of the Trichoderma reesei cbh1- 2005). An ideal inducible promoter is primarily characterized and xyn1 promoters. Detailed analysis of the first 850 bp of both by a strong and tight controllable regulation, a cost-efficient promoter sequences identified binding sites for Xyr1 (XBS), the induction, and an effective expression of the gene of interest essential transactivator of cbh1 and xyn1 gene expression. In being placed downstream of the inducible promoter sequence. these promoters, 14 and 8 XBS, respectively, are arranged in In general, a distinction takes place between chemically and tandem or in the inverted repeat direction. Further improvement physiologically induced promoters. Chemically regulated sys- of these promoters was obtained when additional XBS were tems are induced or repressed by the presence or absence of integrated in inverted repeat orientation. In the case of cbh1 chemical compounds, such as alcohols, antibiotics, hormones, promoter, an induction of gene expression by xylan or wheat or carbon sources (Matsuzawa et al. 2013; Meyer et al. 2011). straw was received by modifying the XBS inverted repeats In contrast, the regulation of physiologically controlled pro- (Kiesenhofer et al. 2018). Also, in a recent attempt, the cbhI moters is determined by abiotic environmental factors. In this promoter was further improved to control the heterologous ex- context, systems were established, whose regulation takes pression of two feruloyl esterase genes to produce ferulic acid, a place depending on the osmotic stress, temperature, light, food additive or potential anti-inflammatory therapeutic agent etc. (Fischer et al. 2016; Zhang et al. 2016). from wheat bran (Long et al. 2018). The selection of a suitable promoter system should be spe- Xylose-inducible promoters have been established in some cifically adapted to experimental approaches because not ev- industrial host systems for some time, including the ery system brings along optimal conditions for the selected abovementioned xyn1 promoter from T. reesei and xylP from Appl Microbiol Biotechnol (2018) 102:6357–6372 6359 Table 1 Inducible promoters in filamentous fungi Promoter Gene product Induction by/repression by Donor Reference alcA Alcohol dehydrogenase I Ethanol/glucose Aspergillus nidulans Waring et al. (1989) amyB TAKA-amylase A Starch or maltose/glucose Aspergillus oryzae Tada et al. (1991), Tsuchiya et al. (1992) bli-3 Blue light-inducible gene Blue light/darkness Neurospora crassa Eberle and Russo (1994) bphA Benzoate p-hydrolase Benzoic acid (benzoate)/deficiency Aspergillus niger Antunes et al. (2016) of benzoate catR Catalase H O and CaCO /n.d. Aspergillus niger Sharma et al. (2012) 2 2 3 cbhI Cellobiohydrolase I Various saccharides/glucose Trichoderma reesei Nyyssönen and Keränen (1995) cre1 Glucose repressor Glucose/deficiency of glucose Acremonium Janus et al. (2008) chrysogenum exylA Endoxylanase Xylose/sucrose Aspergillus awamori Gouka et al. (1996) gas 1,3-beta-glucanosyltransferase Low pH value/pH > 5.0 Aspergillus niger Yin et al. (2017) glaA Glucoamylase A Glucose/xylose Aspergillus niger Boel et al. (1984) gla1 Glucoamylase Glucose/xylose Penicillium verruculosum Bulakhov et al. (2017) mir1 Siderophore transporter Iron starvation/Iron sufficiency Acremonium Gsaller et al. (2013) chrysogenum niiA Nitrite reductase Nitrate/ammonium Aspergillus oryzae Müller et al. (2002) qa-2 Catabolic 3-dehydroquinase Quinic acid/low quinic acid or high Neurospora crassa Giles et al. (1985) sugar concentration Smxyl Endoxylanase Xylose/glucose Acremonium Bloemendal et al. (2014) chrysogenum tcu-1 Copper transporter Copper depletion/copper availability Neurospora crassa Lamb et al. (2013) thiA Thiamine thiazole synthase Thiamine/deficiency of thiamine Aspergillus oryzae Shoji et al. (2005) vvd Blue light receptor Light/darkness Neurospora crassa Hurley et al. (2012) xyl1 Endoxylanase Xylose (and xylan)/glucose Acremonium Blatzer et al. (2014) chrysogenum xylP Endoxylanase Xylose (and xylan)/glucose Penicillium chrysogenum Zadra et al. (2000) xyn1 Endoxylanase Low concentration of xylose/high Trichoderma reesei Mach et al. (1996) concentration of xylose zeaR Transcription factor Zearalenone/Deficiency of Fusarium grainearum Lee et al. (2010) zearalenone Penicillium chrysogenum (Zadra et al. 2000). Interestingly, Metabolism-independent inducible promoter systems the latter was proven to be functional in other filamentous fungi from the order Sordariomycetes (Bloemendal et al. A few years ago, the thiamine-regulatable thiA promoter was 2014; Kopke et al. 2010). This includes the well-established established for A. oryzae. The expression level of this promot- β-lactam producer Acremonium chrysogenum for which three er was controlled by the concentration of external thiamine in controllable promoter systems were developed in recent years. the media. This promoter was recently used in an interesting One is the mir1 promoter, encompassing a 1700-bp region approach, where autophagy-related genes were shown to af- upstream of a siderophore transporter gene, which was shown fect the heterologous expression of the bovine chymosin gene to induce gfp gene expression during iron starvation, while in A. oryzae. The transformation of A. oryzae strains, lacking iron sufficiency results in repression (Gsaller et al. 2013). autophagy genes Aoatg1, Aoatg13, Aoatg4, Aoatg8,or Two other inducible promoters stem from the xylanase genes Aoatg15 with a bovine chymosin (CHY) expression construct from Sordaria macrospora and A. chrysogenum and drive resulted in a threefold enhanced production level of CHY in gene expression on xylose-containing media. On the contrary, comparison to the control strain. However, conidiation was glucose results in gene repression. Both promoters are very significantly reduced in these strains. Since huge amounts of effective in A. chrysogenum and contribute to the rather lim- conidia are necessary for the inoculation of large-scale cul- ited number of molecular tools for this highly important in- tures, gene-conditional expression strains of four Aoatg genes dustrial fungus. Furthermore, the Smxyl promoter was suc- were constructed in which the promoter region of the corre- cessfully used for the application of a one-step FLP/FRT re- sponding genes was substituted by the thiA promoter. combination system in order to construct marker-free trans- Conidiation was clearly increased in the absence of thiamine, genic strains (Blatzer et al. 2014; Bloemendal et al. 2014). whereas autophagy was still repressed under growth 6360 Appl Microbiol Biotechnol (2018) 102:6357–6372 conditions with thiamine. Nevertheless, the production level protein is responsible for an antiport of tc, which leads to of CHY was comparable to that of the ΔAoatg strains (Yoon et resistance to this antibiotic. The tetA gene is under the tran- al. 2013). The fact that thiamine content does not alter fungal scriptional control of the tc-dependent Tet repressor (TetR), morphology makes the thiA promoter a useful alternative to which negatively controls tc resistance. In the absence of tc, conventionally used promoters. For example, alcA and amyB TetR binds to the tet operator (tetO) resulting in a transcrip- promoters depend on the available carbon sources, which tional shutdown of the operon. In contrast, in the presence of might have an impact on fungal physiology or morphology tc, the interaction between the repressor and the operator is (Shoji et al. 2005). In submerged cultures from A. nidulans, efficiently prevented, leading to a dissociation of TetR from expression of the abaA gene under the control of the alcA tetO. For use in eukaryotic organisms, the modification of promoter resulted in vacuolated hyphae with abnormal regulatory elements was necessary and a differentiation be- septation (Mirabito et al. 1989). A comparable effect was ob- tween a Tet-On and the Tet-Off system was made. served when a copper-inducible promoter was used for heter- Transgenic mice were the first eukaryotes in which Tet-Off ologous expression since copper has physiological and toxic was established (Gossen and Bujard 1992). A tc-dependent effects on fungal growth and development (Lee et al. 2010). transactivator (tTA) was generated by the fusion of TetR with A completely new promoter system was established for A. the transcriptional activator domain from herpes simplex virus niger. This was adapted by the medium conditions of citric protein 16 (VP16). By the addition of tc or its analog doxy- acid fermentation. During this process, the pH of the growth cycline (Dox), tTA cannot specifically bind to tetO; thus, tran- medium decreased from 5.0 below 2.0 and was nearly consis- scription of the gene of interest is disrupted (Tet-Off state) tent at a value below 2.0 in later cultivation phases. Therefore, (Fig. 1a). In contrast, for the Tet-On system, a reverse hybrid a pH-responsive promoter, Pgas, which efficiently enhances tTA (rtTA) was constructed, which, in the presence of tc or gene expression at pH 2.0, was developed for dynamic meta- Dox, functions as an activator of gene expression. The inser- bolic engineering. The gas gene was found in an expression tion of additional mutations resulted in the reverse hybrid data-set and encodes the 1,3-β-glucanosyltransferase GelD, transactivator rtTA2 -M2 to increase the rtTA binding sensi- which is necessary for maintenance of the fungal cell wall tivity to Dox and the expression level of rtTA in eukaryotes. due to the synthesis of glucan. This study showed that Pgas An improvement of the tetracycline-responsive promoter was is tightly expressed at pH 2.0. In terms of promoter strength, achieved by the insertion of seven copies of the tetO sequence Pgas is comparable to PgpdA. Furthermore, the promoter (tetO7) upstream of a minimal promoter Pmin (Fig. 2a, b). strength varied by using different types of acids. While induc- tion was strictly pH dependent, promoter strength was higher Previous and recent applications of the Tet system using organic acids compared to inorganic acids. Pgas was used to express the cis-aconitate decarboxylase (CAD)gene Previously, the Tet-On/Tet-Off system was shown to be func- from Aspergillus terreus in A. niger. Due to the fact that A. tional in Aspergillus fumigatus (Vogt et al. 2005). In this study, niger produces large amounts of citrate (180 g/l), which is the three plasmids were constructed, one harboring the expression precursor of itaconate, the aim was to modify the natural cit- cassette consisting of the transactivator rtTA2 -M2 under the rate producer into an itaconate producer. Itaconic acid is an control of the constitutive gpdA promoter (PgpdA::rtTA2 - unsaturated dicarbonic acid with a high industrial potential M2), the other comprising PgpdA::tTA, and the third one with because it can be used as a monomer for the production of a a tetO7 sequence linked to a minimal promoter sequence of plethora of products including resins, plastics, paints, and syn- PgpdA upstream of an E. coli hygromycin resistance gene thetic fibers. Since constitutive expression of the CAD gene (tetO7::Pmin::hyg ). The corresponding plasmid pairs were resulted in only a small yield of itaconate, the use of the Pgas transferred in parallel into a single A. fumigatus strain and promoter led to a gradually increased expression level of CAD hygromycin-resistant transformants were obtained in the pres- parallel to a decreasing pH value. Finally, a titer of 5 g/l was ence and absence of Dox indicating that the Tet system func- obtained, which was fivefold higher than comparable experi- tions in A. fumigatus. However, the tightness of the system ments in which the PgpdA promoter was used to drive expres- was not examined in detail. Especially the Tet-On system was sion of the CAD gene (Yin et al. 2017). further developed for an application in A. niger. It was also established as a metabolism-independent gene expression sys- Adaption of the Tet system for eukaryotes tem in this study, but all components of the Tet-On system were combined on one plasmid. For testing its functionality, a Another frequently used expression mechanism, which was minimal gpdA promoter, which was fused to the tetO7 se- originally identified in Escherichia coli is the tetracycline quence, was used to control luciferase gene expression. (tc) expression system (Tet system). The central components While in the absence of Dox no luminescence was detectable, of this system stem from the Tn10 tetracycline resistance op- the presence of Dox led to the induction of luciferase gene eron. In Gram-negative bacteria, the membrane-residing TetA expression. It was clearly shown that the system is tight, Appl Microbiol Biotechnol (2018) 102:6357–6372 6361 Fig. 1 Tet-Off expression system. a Basic version of the Tet-Off depending on the model organism (PgeneX). Seven copies of tetO expression system in eukaryotes. The fusion of TetR with VP16 leads sequence were inserted (tetO7) upstream of a minimal promoter in to the generation of the tetracyclin-dependent transactivator tTA. order to increase tTA2 binding. In the absence of tetracycline or its When tetracycline or its analog doxycycline is added, tTA is unable analog doxycycline tTA2 is able to specifically bind tetO7. Thus, the to specifically bind to tetO due to a conformational change. As a transcriptional induction of the gene of interest, which is controlled result, transcription of the gene of interest, which is controlled by by Pmin takes place. Addition of drug leads to transcriptional shut- Pmin, is disrupted. Thus, transcriptional activation takes place in down. For further details, see text. Abbreviations: Dox, doxycycline; the absence of tetracycline or doxycycline. Addition of the either VP16, transcriptional activator domain from herpes simplex virus tc, drug leads to transcriptional shutdown. Dox, doxycycline; VP16, tetracycline; TetR, tetracycline-dependent Tet repressor; tetO, tetra- transcriptional activator domain from herpes simplex virus; tc, tetra- cycline operator sequence; tetO7, seven copies of tetracycline oper- cycline; TetR, tetracycline-dependent Tet repressor; tetO; tetracycline ator sequence; tTA, tetracyclin-dependent transactivator; tTA2 , operator sequence; tTA, tetracyclin-dependent transactivator; TtrpC, mammalian tetracyclin-dependent transactivator; TcrgA, crgA termi- trpC terminator from Aspergillus nidulans; Pmin, minimal promoter. nator from Aspergillus fumigatus;TtrpC, trpC terminator from b Optimized module of the Tet-Off expression system for A. niger. Aspergillus nidulans;Pmin, minimal promoter (adopted from The transactivator tTA2 ,optimized formammals,was used down- Brockamp et al. 2002, Wanka et al. 2016) stream of PgpdA promoter. The promoter can be selected individually induction takes place immediately after addition of the induc- benzyl-1H-pyrrole-2-carboxylic acid, an unknown novel nat- er, and fine-tuning of the system is achieved by inducer con- ural compound, named fumipyrrole (Macheleidt et al. 2015). centration and gene copy number (Meyer et al. 2011). In A. niger, the Tet-On system was used successfully for the In recent years, the Tet system was also used for the con- expression of polycistronic genes from the Asp-melanin bio- trolled expression of secondary metabolite clusters, such as synthetic pathway from Aspergillus terreus (Geib and Brock silent gene clusters in Aspergilli.In A. fumigatus strains grown 2017). This is a further good example that the expression under induced conditions, the Tet-On overexpression of a pu- genes from secondary metabolite clusters can be tightly tative transcription factor resulted in the activation of a sec- expressed in heterologous hosts. Another remarkable example ondary metabolite cluster, which led to the biosynthesis of 5- was reported recently for the rice pathogen Fusarium 6362 Appl Microbiol Biotechnol (2018) 102:6357–6372 Fig. 2 Tet-On expression system. a Basic version of the Tet-On expres- to increase the rtTA binding sensitivity and the expression level of sion system in eukaryotes. The rtTA fusion protein comprises of TetR rtTA. The used promoter can be selected individually depending on and VP16 activation domain. A four amino acid change in the TetR the model organism (PgeneX). An improvement of tetO was achieved DNA binding motif alters rtTA’s binding characteristics, such that it can by the insertion of seven copies of tetO sequence (tetO7) upstream of only recognize tetO sequence in the presence of tetracycline or its the minimal promoter. In the presence of tetracycline or its analog analog doxycycline. Thus transcriptional induction of the gene of inter- doxycycline, rtTA2 -M2 is able to specifically bind tetO7 in order to est, which is controlled by a minimal promoter, takes place. b induce the transcription of the selected gene of interest. For further Optimized version of the Tet-On expression system for A. niger.The details, see text. Abbreviations: rtTA2 -M2, mutated reverse hybrid insertion of additional mutations resulted in the reverse hybrid transactivator. All other abbreviations are given in the legend of transactivator rtTA2 -M2, which is controlled by the PgpdA promoter, Fig. 1. (adopted from Brockamp et al. 2002, Wanka et al. 2016) fujikuroi. In this fungus, constitutive overexpression of the plasmid was integrated into the wild type and five mutants, transcription factor TF22 drives the activation of the complete carrying deletions of different cluster genes. All transformants trichosetin gene cluster. Trichosetin is a PKS-NRPS-derived showed a strong activity of TF22 expression dependent on the tetramic acid possessing biological activity against a broad added Dox concentration. However, a leakiness of the system range of organisms, encompassing plants and bacteria. was observed because TF22 showed a basal expression with- Constitutive overexpression resulted in severe growth reduc- out adding the inducer. As this background expression did not tion. To circumvent this undesired morphological defect, the drive activation of the gene cluster, the Tet-On system was Tet-On system from A. niger was established for F. fujikuroi to suitable as a controllable induction tool. The inducible over- obtain the controlled production of trichosetin for further the expression of TF22 results in activation of the three cluster functional characterization of each gene within the trichosetin genes indicating that this transcription factor directs the bio- cluster (Janevska et al. 2017). One part of the Tet construct synthesis of trichosetin. contains the already described transactivator rtTA2 -M2 un- Optimization of the Tet-Off system in A. niger was der the control of the constitutive PoliC promoter from A. achieved by using the present plasmid for the Tet-On system S S 2 nidulans, while the other part encompasses the rtTA2 -M2- and the reverse transactivator rtTA2 -M2 against the tTAS dependent promoter in front of TF22. The pTET::TF22 transactivator was exchanged, which is optimized for Appl Microbiol Biotechnol (2018) 102:6357–6372 6363 mammals, in order to construct a functional Tet-Off cassette expression was verified and the timing of Dox supplementa- (Fig. 1b) (Wanka et al. 2016). Strains with a single copy of the tion dramatically impaired the survival rate of mice. This modulated Tet-Off showed a strong luciferase activity, where- study provides evidence that the Tet-Off system is used to as the addition of different Dox concentrations resulted in a identify genes required for fungal growth within the host fol- downregulation of the luciferase gene expression. However, lowing the initiation of infection (Peng et al. 2018). during the establishment of Tet-Off, it was apparent that not all A. niger transformants containing the Tet-Off construct show a luciferase downregulation after induction. Additionally, most Production of heterologous products of the transformants lost the Tet-Off cassette during storage on minimal medium. Genetic analysis of the transformants de- Filamentous fungi are often used to produce endogenous com- tected 176 bp sequence homology between the minimal gpdA pounds which are generated during the primary or secondary promoter and the constitutive gpdA promoter and this resulted metabolism. Often, these enzymes are active within distinct in intra-molecular recombinations and loss of function. Thus, metabolic pathways. Primary metabolites such as organic constitutive PgpdA was replaced by the endogenous promoter acids are used in diverse industrial applications. For example, of the fraA gene, encoding a putative ribosomal subunit. citric, gluconic, fumaric, koji, lactic, and itaconic acids are Using the PfraA Tet-Off system, genetically stable valuable byproducts that are used in the chemical or food transformants were generated, and, in the presence of Dox, a industry (Becker et al. 2015). Citric acid is within these fungal rapid reduction in reporter luminescence was observed. In organic acids, the one with the highest yearly production yield order to demonstrate a putative application for the characteri- (1.75 million tons), and has a market value of US$1.6 billion zation of industrially relevant genes, the luciferase reporter (Demain 2007). Besides organic acids, vitamins are important gene was substituted by the gfaA gene, which encodes a metabolites, which are produced with the help of filamentous glutamine:fructose-6-phosphate aminotransferase. This en- fungi, e.g., Ashbya gossypii produces riboflavin (vitamin B2) zyme is involved in the first step of chitin synthesis and chitin at a yield of 20 g/l. In addition to primary metabolites, fila- synthesis-defective strains cannot grow on media without the mentous fungi are potent producers of enzymes of which supplementation of glucosamine. The functionality of PfraA about 100 are used for industrial applications. Most of these Tet-Off system-gfaA fusion was finally demonstrated when a enzymes show a hydrolytic activity, such as amylases, prote- gfaA-deficient strain was transformed with this construct. In ases, lipases, xylanases, or catalases and are mostly used for the presence and absence of both Dox and glucosamine in the the degradation of organic material. Amylases have the media, transformants are viable. However, the sole induction highest importance in this context, with an annual production of the PfraATet-Off system by Dox, in the absence of glucos- of 95,000 tons and a market value of US$2.5 billion (Demain amine, results in the lethality of transformants. Thus, this sys- and Vaishnav 2009). Many of these enzymes are heterolo- tem seems to be broadly suitable for industrial application gously produced in diverse microbial host systems to circum- (Wanka et al. 2016). Very recently, the Tet expression system vent difficulties in the cultivation and genetic manipulation of was used to characterize genes that are involved in the viabil- exotic microbes (Schmidt-Dannert 2015). Many fungal en- ity of A. fumigatus during the infection process in order to zymes are synthesized in heterologous fungal host that are identify new antifungal drug targets. For an in vivo approach, already established as industrial producers for a wide range a Tet-Off system was designed where the presence of Dox of different enzymes or primary- and secondary metabolites. resulted in the downregulation of gene expression. As poten- A list of commercial enzymes produced using filamentous tial target genes, those for inosine 5′-monophosphate dehydro- fungi can be found at the Association of Manufacturers and genase (IMPDH), which catalyzed the first step in de novo Formulators of Enzyme Products (AMFEP, www.amfep.org). guanine biosynthesis, and L-ornithine N -oxygenase (sidA), Fungi usually have a high secretion capacity and many catalyzing the initial step of siderophore biosynthesis, were production strains have a GRAS (generally recognized as tested in a murine invasive pulmonary aspergillosis model. safe) status. Furthermore, the post-translational modification The immunosuppression of mice was induced by injection of proteins is distinct in eukaryotic and prokaryotic host sys- of cyclophosphamide for 4 days and 1 day before inoculation tems. Established fungal host systems that are amendable for and injection of cortisone acetate 1 day before inoculation. On genetic manipulation and with a GRAS status are A. niger, A. inoculation day, 40 μl of a fresh A. fumigatus conidia suspen- oryzae, and T. reesei (Schmidt 2006; Sharma et al. 2009). sion was intranasally inoculated under anesthesia. The addi- Therefore, filamentous fungi were used in several efforts for tion of 200 μl Dox (10 mg/ml) was carried out twice a day by the production of mammalian enzymes and proteins. gastric lavage. The downregulation of impdh resulted in re- Particularly, the post-translational modification in eukaryotic duced virulence in vivo, while switching off the gene expres- microbes has supported attempts to obtain functional proteins sion of sidA led to the generation of avirulent A. fumigatus from fungal hosts. The first successful approaches with fila- mentous fungi demonstrated the production of chymosin and strains. Additionally, a time-dependent control of sidA 6364 Appl Microbiol Biotechnol (2018) 102:6357–6372 lactoferrin (Archer 2000). Chymosin from calf vells is used in functional genome analysis, the large data amounts of data the food industry for the production of cheese, while from different high-throughput techniques are used to assign lactoferrin, a multifunctional transferrin from cow’smilk, gene functions with the overall goal of analyzing the relation has an anti-inflammatory activity. Meanwhile, yeast expres- between the genotype and phenotype of an organism on a sion systems have been shown to be superior for the biotech- genome-wide level. nical production of mammalian proteins with pharmaceutical The still-progressing development of the next-generation relevance. Most common host organisms are Saccharomyces sequencing techniques allows a fast and low-cost effective cerevisiae, Pichia pastoris,and Hansenula polymorpha sequencing of complete fungal genomes for which the costs (Ganeva et al. 2017; Mallu et al. 2016;Shibuietal. 2013). were reduced by a factor of about 50,000 (Goodwin et al. Nevertheless, filamentous fungi are still used for the heterol- 2016). In parallel, efficient sequencing techniques have in- ogous production of different mammalian proteins, for exam- creased the output of sequences per run by a factor of 100 to ple, the human hormone peptide obestatin was successfully 1000. The newest sequencing platforms, such as Pacific produced using recombinant T. reesei strains (Sun et al. 2016). Biosciences (PacBio) or Oxford Nanopore Technologies Recently, two fusion proteins each consisting of a highly an- (ONT), provide continuous sequences with a size of up to tigenic protein from Leishmania sp.,anantibody specific for a 200 kb. This is of major importance when repetitive regions receptor of dendritic cells, and a fragment of native carrying telomers or centromeres have to be sequenced. glucoamylase A were generated in A. niger (Magana-Ortiz et Currently, about 2700 genomes of fungal species are pub- al. 2018). Both recombinant proteins may be used for immu- lished and are represented, for example, in the 1000 Fungal nization against Leishmaniasis and point to the potential of Genome Project (http://1000.fungalgenomes.org/home/). The filamentous fungi as hosts for the generation of pharmaceuti- sequenced genomes possess a large number of cally relevant peptides. In this context, it has to be mentioned uncharacterized genes, whose functions can be sometimes that well-established organisms with beneficial traits are deduced from orthologous sequences known from other redeveloped for recombinant protein production. Neurospora species. Valuable classification libraries to evaluate the crassa is a model organism with suitable traits for heterolo- function of proteins include the Functional Catalogue gous production but has not widely been used for industrial (FunCat), Gene Ontology (GO), or the Kyoto Encyclopedia applications. In order to evaluate the usability of N. crassa as a of Genes and Genomes (KEGG) (Rüpp et al. 2004, Ashburner host system, the biosynthesis of the human antibody fragment et al. 2000, Karnehieser and Goto 2000). A common charac- HT186-D11 was tested and production yields of about 3 mg/L teristic of these databases is that classifications of genes are were achieved (Havlik et al. 2017). Although the achieved done on the basis of hierarchical structures, in which major yields cannot compete with established production hosts like categories are split up into sub-categories. Each sub-category provides more precise information, e.g., about the cellular P. pastoris, which is able to produce up to 100-fold higher yields, N. crassa seems to be a promising new host system function of the protein. An alternative approach in predicting for the production and secretion of heterologous proteins. the function of an unknown protein is the use of conserved protein domains for a homology-based search. These domains can be used to search against databases like Protein families Strain improvement using genomics (Pfam), Clusters of Orthologous Groups of proteins (COGs), and functional genomics and The Institute for Genomic Research’s database of protein families (TIGRFAM) to identify other proteins with similar The strain improvement programs of A. chrysogenum, P. domains or domain structure and to predict protein functions chrysogenum, and T. reesei are suitable examples for the effi- (Finn et al. 2016; Galperin et al. 2015;Haft etal. 2003). With ciency of classical strain improvement by random mutagene- this kind of homology-based functional analysis, even ge- sis (Hu and Zhu 2016; Peterson and Nevalainen 2012). nomes of uncharacterized organisms, metagenomes, and However, the random introduction of point mutations also metatranscriptomes can be analyzed. Representative examples has disadvantages. Improved production strains can acquire from the field of filamentous fungi are the genomes from point mutations over several rounds of random mutagenesis Lichtheimia corymbifera, a member of the basal Mucorales, leading to a reduced growth rate, sporulation defects or geno- or various Penicillium species (Lessard et al. 2014;Nielsen et mic instability (Künkel et al. 1992;Lein 1986). In this context, al. 2017; Schwartze et al. 2014). The genome sequences from directed genome manipulation can help to overcome the effect 24 Penicillium species revealed that a total of 1317 putative of deleterious point mutations. However, an essential prereq- biosynthetic gene clusters exist. This survey indicates the po- uisite for this approach is the knowledge of the genome se- tential of natural products that can be synthesized by members quence, the encoded genes and the derived metabolic path- of this genus (Nielsen et al. 2017). After identification, inter- ways. Therefore, functional genome analysis is an important esting genes or even whole gene clusters can be expressed heterologously in suitable host systems (Bok et al. 2015). tool with which to obtain the required information. Within Appl Microbiol Biotechnol (2018) 102:6357–6372 6365 Similar to metagenomes, which are generated by using pathways and potential bottlenecks within them can also be total DNA from the sample for high throughput sequencing, identified using omics approaches. In A. nidulans,tran- metatranscriptomes are generated from the RNA of a given scriptome analysis combined with flux and physiology data sample. The obtained sequencing data will identify active was used to propose a model describing the competition be- metabolic pathways that are effective in biotechnical process- tween biomass formation and polyketide production for the es. For example, the metatranscriptome of Penicillium available acetyl coenzyme A (Panagiotou et al. 2009). camemberti and Geotrichum candidum,which areusedfor the production of Camembert-type cheese, was taken over Comparison of production and wild-type strains 77 days of ripening. The functional annotation identified pro- cesses during Camembert ripening, such as carbohydrate or The extensive random mutagenesis in strain improvement protein metabolic processes, which are most active in the first programs generated production strains that differ substantially 2 weeks of ripening. Based on these data, attempts can be from their ancestral wild type at the genomic level. Genome undertaken to improve cheese quality assessment (Lessard et rearrangements were previously demonstrated by pulsed-field al. 2014). gel electrophoresis (Walz and Kück 1991). The current avail- ability of genome sequences from wild-type and production Identification of biosynthesis genes and their related strains of a broad range of industrial filamentous fungi identi- metabolic pathways fied genome differences down to nucleotide resolution. Identified changes can be valuable indicators for targeted An essential aspect for biotechnological applications is knowl- strain improvement using molecular genetic tools. edge of the genes involved in the biosynthesis of a given Comparison of the genome sequences from the T. reesei product, as well as detection of the metabolic pathways that production strains NG14 and RUT-C30 with the wild-type provide the required precursors. However, when the function- strain QM6a revealed 223 SNPs, 15 small deletions or inser- al assignment of genes and the related pathways are unknown, tions, and 18 larger deletions responsible for the loss of more it can be achieved by functional genomics approaches. than 100 kb (Le Crom et al. 2009). More than half of the single Transcriptomic data of A. oryzae strains inoculated in media nucleotide polymorphisms (SNPs) introduced within coding with different kojic acid biosynthesis rates were used for com- sequences are non-synonymous and can therefore lead to parative analysis, in order to identify genes involved in the changes in protein functionality. A few of these changes were biosynthesis of this important organic acid (Terabayashi et analyzed in more detail, for example, truncation of the gls2α al. 2010). Identification of the involved genes allows targeted gene encoding a β-glucosidase subunit in the production strain improvement by the overexpression of the related bio- strain RUT-C30 (Geysens et al. 2005). Furthermore, the trun- synthesis genes. Most biosynthesis pathways contain one or cation of the transcription factor Cre1, caused by a premature more rate-limiting steps representing interesting adjustment stop codon introduced in the encoding gene leads to a loss of screws for product titers. For example, the biosynthesis of carbon catabolite repression (Ilmen et al. 1996). This enables the phytohormone gibberellic acid produced by F. fujikuroi the production of cellulases using glucose as a carbon source, was enhanced by the overexpression of two genes encoding which prevents cellulase production in wild-type strains. a geranylgeranyl diphosphate synthase 2 and a bifunctional These findings are applicable to other industrially relevant ent-copalyldiphosphate synthase/ent-kaurene synthase. Both fungi. It was, for instance, shown that deletion of the cre1 are key enzymes of the gibberellic acid pathway (Albermann gene can be used to increase penicillin titers in P. et al. 2013). For P. chrysogenum, it was shown that even in chrysogenum production strains (Cepeda-García et al. 2014). production strains, the additional overexpression of penDE However, not every genomic alteration introduced by classical can lead to elevated penicillin yields (Weber et al. 2012). strain improvement using random mutagenesis can be con- The acyl-coenzyme A: isopenicillin N acyltransferase nected to improved product formation. Production strains of encoded by penDE is involved in the last biosynthetic step T. reesei have lost a genomic fragment with a size of 85 kb of penicillin formation, which is the exchange of the sidechain compromising 29 genes (Seidl et al. 2008). But the loss of L-α-aminoadipic acid with phenylacetic acid or these fragments has no effect on cellulase production phenoxyacetic acid. However, the overexpression of penDE (Vitikainen et al. 2010). An alternative approach is the com- needs to be balanced since very high levels of the encoded parison of strains that are unable to synthesize a given product. enzyme lead to accumulation of 6-aminopenicillic acid in- The genome sequence of a cellulase-negative T. reesei strain stead of penicillin. In this case, the formation of 6- revealed an SNP in the xyr1 gene leading to a truncated and aminopenicillic acid by uncontrolled removal of the sidechain biologically inactive transcription factor (Lichius et al. 2015). of penicillin occurs probably due to an insufficient supply In P. chrysogenum, comparative genomics was used to an- with sidechain precursors. The supply of precursors is also a alyze strain improvement processes. In a comparison of the very promising field for strain improvement. The involved genomes of the wild-type strain NRRL1951 with two derived 6366 Appl Microbiol Biotechnol (2018) 102:6357–6372 production strains, a total of 558 SNPs were identified in cod- metabolism seems to be beneficial for cephalosporin C bio- ing regions leading to changes in the amino acid sequences of synthesis in this fungus. These conclusions are based on the encoded proteins (Salo et al. 2015). In this comparison, the knock-down and overexpression studies and provide the po- authors identified SNPs in 29% of the secondary metabolite tential to identify targets for strain improvement. cluster. Besides the deactivation of secondary metabolite clus- Besides understanding differences in secondary metabo- ters, no enrichment of functional categories of mutated genes lism, proteome studies are used to identify bottlenecks during in production strains was observed. This led to the hypothesis the secretion of recombinant proteins. The comparison of A. that these clusters were inactivated in favor of β-lactam bio- nidulans strains, showing different capacities of recombinant synthesis. An example of such inactivation is the sorbicillinoid protein production, disclosed alterations in proteins related to biosynthesis cluster, which is involved in the biosynthesis of a amino acid metabolism, ribosome biogenesis, translation, and yellow-colored pigment produced in wild-type strains. The endoplasmic reticulum and provides targets for rational strain formation of this class of pigments can hamper the downstream improvement (Zubieta et al. 2018). Strains used for the com- purification of secondary metabolites and therefore was also parisons mentioned above can be derived from long-term eliminated in other industrial filamentous fungi such as A. strain improvement programs, but also from forward genetic chrysogenum and T. reesei (Derntl et al. 2016). approaches. Using the latter approach together with whole ge- The genetic alterations in production strains in comparison nome sequencing enables the identification of the low-affinity to the wild type were a relevant aspect of strain improvement glucose transporter MstC from A. niger, whose deletion im- programs for a long time. So far, SNP analysis has not pro- proved secretion of heterologous proteins (Reilly et al. 2018). vided substantial evidence for the preferred mutagenesis of A different approach was used for Aspergillus species. genes from distinct functional categories. Here, a genus-wide comparative genomics approach was used An alternative approach to decipher genetic alterations in to identify genomic differences concerning primary and sec- production strains was recently successfully conducted. The ondary metabolism, stress response, biomass degradation, and comparative transcriptional analysis of the wild-type and pro- signal transduction (de Vries et al. 2017). In the case of organ- duction strains from the two antibiotic producers A. ic acid production, the authors identified a link between the chrysogenum and P. chrysogenum (Terfehr et al. 2017), re- number of isoenzymes and the phylogeny of the analyzed vealed that both only distantly related industrial fungi show fungi. Members from the section Nigri possess extra isoen- common transcriptional adaptions towards high-level β-lac- zymes relevant for citrate and gluconic acid production, which tam antibiotic biosynthesis. For example, industrial strains correlates with the increased capacity of these strains to pro- from both fungi show an enrichment of differentially regulat- duce organic acid. ed genes associated with the supply of precursors and energy for β-lactam production. Furthermore, genes required for Optimizing endogenous metabolic pathways pathways and cellular functions that are not needed for anti- biotic production are downregulated. In addition, the effect of The identification of competing metabolic pathways can be a Velvet, a major regulator of secondary metabolism in filamen- valuable approach for strain improvement by genetic manip- tous fungi, was investigated in production strains of A. ulation. In this context, pathways that stay in direct competi- chrysogenum and P. chrysogenum. This global regulator con- tion because of using the same substrate and pathways that trols approximately 50% of all secondary metabolite clusters. degrade the desired product are of great interest. In addition to Most importantly, strain improvement and Velvet affect the competing pathways, transport-associated processes can also expression of a large set of genes in a similar manner in both be a bottleneck. Comparisons of strains with different charac- improved industrial fungi. Comparisons of production and teristics concerning these problems and basic research can wild-type strains can be also conducted at the protein level. provide hints for optimization. Using two-dimensional electrophoresis, it was possible to Because of the high secretion capacity, filamentous fungi identify protein level changes between a wild-type strain and are broadly used in industry to produce endogenous and het- two production strains with moderate and high penicillin bio- erologous products, particularly, A. oryzae, A. niger,and T. synthesis capacities (Jami et al. 2010). The two production reesei. However, these fungi also secrete a couple of proteases strains have undergone a global metabolic reorganization. which will degrade the products of interest and thus may di- They showed an overrepresentation of enzymes involved in minish the maximum product yield. Therefore, protease- penicillin precursor and energy metabolism, while proteins deficient Trichoderma and Aspergillus strains were generated involved in stress response, virulence, and the biosynthesis to circumvent this problem. Using A. oryzae strains that lack of other secondary metabolites were nearly lost. A similar two proteases, the heterologous production of human lyso- comparison with A. chrysogenum strains also identified com- zyme and bovine chymosin was further improved by deletion parable changes (Liu et al. 2015). Since thiamine biosynthesis of additional protease-encoding genes. A consequence was enzymes are overrepresented in the production strain, this the generation of a protease-deficient strain with an enhanced Appl Microbiol Biotechnol (2018) 102:6357–6372 6367 capability of heterologous protein production (Yoon et al. Protein degradation can also be a problem if the desired 2011). In a similar approach with T. reesei strains, the deletion heterologous product is usually catabolized by the host organ- of nine protease-encoding genes led to a doubled interferon ism. In order to produce galactaric acid using A. niger, a gene alpha-2b production (Landowski et al. 2016). These yields are coding for a uronate dehydrogenase of prokaryotic origin was comparable to those achieved with E. coli, where the product introduced into the genome. In addition to that, endogenous must be refolded from inclusion bodies. The T. reesei system pathways for galactaric acid degradation were identified using even outperforms the Pichia pastoris system, which was used RNA-seq data and inactivated using the CRISPR/Cas9 system frequently for interferon production. Another option to de- (Kuivanen et al. 2016). crease extracellular protease levels is the modification of the underlying gene regulation. The transcription factor PrtT from A. niger is applicable for this approach since it regulates the Product modification for optimized application expression of extracellular protease-encoding genes (Punt et al. 2008). The abovementioned strategies, developed for well- Products synthesized by filamentous fungi are used in a known production hosts, may be transferred to less- broad area of applications. This area ranges from prote- established production organisms. Genome sequencing of ases and lipases in washing agents in private households the thermophilic filamentous fungus Myceliophthora to the industrial scale use of enzymes, e.g., cellulases for thermophila and subsequent identification of protease- bioethanol production. Based on the diverse areas of ap- encoding genes were used to generate protease-deficient plication, the products must meet different criteria for dif- strains for cellulase production (Berka et al. 2011; Szabo et ferent physical and chemical parameters. While enzymes al. 2013). in washing agents often need ambient temperature optima, Misfolding is another problem in the production of heter- the vast majority of industrial used enzymes should be ologous proteins. Based on transcriptomic data, it was shown thermotolerant (Struvay and Feller 2012). High tempera- that the biosynthesis of heterologous proteins activates the tures offer advantages, e.g., faster conversation rates, a unfolded protein response (UPR) in A. niger (Guillemette et reduced risk of contamination, and an increased mass al. 2007;Kwon et al. 2012). The activation of UPR leads to transfer (Grigoriev et al. 2011). However, even heat labil- proteolysis, which can reduce the product yield substantially. ity can also be beneficial because these enzymes can be Using the transcriptomic data, it was possible to identify core easily inactivated by a moderate increase in temperature. genes involved in this pathway as promising targets for strain Another crucial parameter for the activity and stability of improvement. The amount of secreted heterologous proteins enzymes is the pH optimum. For instance, acid-stable cel- was increased by deletion of the doaA gene, encoding an lulases are highly required for bioethanol production, be- essential factor for ubiquitin-mediated proteolysis. This was cause substrates are often treated with acids prior to the further optimized by overexpression of the sttC gene, enzymatic processes. Functional analysis of genomes and encoding a protein involved in the glycosylation of secreted even metagenomes from extremophiles can help to iden- products (Jacobs et al. 2009). tify enzymes that meet these criteria, even if the original Autophagy is a highly conserved intracellular pathway host cannot be used for production. that degrades non-functional or not needed cellular compo- Phytases, phosphohydrolases that catalyze the hydrolysis nents. This process may affect high yield expression of pro- of phytic acid, releasing bioavailable phosphorus, were opti- teins and is thus a promising target for strain improvement mized based on various homologous phytase-encoding genes. programs. In chymosin-producing A. oryzae strains, the in- The construction of a consensus phytase gene led to an en- activation of autophagy leads to a threefold increase in prod- zyme with improved activity and thermostability (Lehmann et uct formation (Yoon et al. 2013). Besides heterologous pro- al. 2000; Tomschy et al. 2000). In T. reesei, the temperature tein production, secondary metabolite production can also be optimum of endoglucanase was increased by 16 °C in a sim- affected by autophagy. Autophagy-deficient P. chrysogenum ilar approach (Trudeau et al. 2014). Besides optimizing en- strains have an increased amount of penicillin biosynthesis zymes to fit certain criteria, one can also directly choose en- enzymes and thus synthesize more penicillin (Bartoszewska zymes from extremophiles. The comparative genomic analy- et al. 2011). Similarly, the lack of Atg1, which is essential sis of the two thermophilic biomass-degrading fungi for autophagosome formation, led to increased cephalospo- Myceliophthora thermophila and Thielavia terrestris rin C production rates in A. chrysogenum strains (Wang et disclosed a wealth of thermostable enzymes for hydrolysis al. 2014). However, the modification of autophagy does not of all major polysaccharides occurring in biomass (Berka et necessarily lead to increased product yields. The deletion of al. 2011). Via the heterologous expression of a thermostable Acatg11 results in autophagy deficiency and a reduced ceph- β-glucosidase of Neosartorya fischeri, a closely related spe- alosporin C titer in the corresponding A. chrysogenum strain cies to A. fumigatus, the cellulytic activity of T. reesei was (Liu et al. 2017a). improved (Xue et al. 2016). 6368 Appl Microbiol Biotechnol (2018) 102:6357–6372 Another alternative strategy is the use of metagenome li- References braries. Using a library derived from deep-sea sediments, a cold-active lipase was identified with an optimal activity at Albermann S, Linnemannstons P, Tudzynski B (2013) Strategies for strain improvement in Fusarium fujikuroi: overexpression and lo- 25 °C (Zhiwei et al. 2015). 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Albright JC, Goering AW, Thomas PM, Kelleher NL, Keller NP, Wu CC (2015) Fungal artificial chromosomes for mining of the Ethical approval This article does not contain any studies with human fungal secondary metabolome. BMC Genomics 16:343. https:// participants or animals performed by any of the authors. doi.org/10.1186/s12864-015-1561-x Bulakhov AG, Volkov PV, Rozhkova AM, Gusakov AV, Nemashkalov Open Access This article is distributed under the terms of the Creative VA, Satrutdinov AD, Sinitsyn AP (2017) Using an inducible pro- Commons Attribution 4.0 International License (http:// moter of a gene encoding Penicillium verruculosum glucoamylase creativecommons.org/licenses/by/4.0/), which permits unrestricted use, for production of enzyme preparations with enhanced cellulase per- distribution, and reproduction in any medium, provided you give appro- formance. 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Applied Microbiology and Biotechnology – Springer Journals
Published: Jun 2, 2018
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