Huiling Wei,Mengyue Wu,Aili Fan*,Haijia Su*

Beijing Key Laboratory of Bioprocess,College of Life Science and Technology,Beijing University of Chemical Technology,North Third Ring Road 15,Chaoyang District,Beijing 100029,China

ABSTRACT Trichoderma is an ascomycete fungal genus widely distributed in the soils.Several species were selected,engineered and utilized for protein production for decades.The high extracellular secretion capability and eukaryotic post-translational modification machinery make Trichoderma spp.particularly interesting hosts.In this review,we summarized the recombinant proteins produced in Trichoderma since 2014,concerning their origins,hosts,promoters,terminators,signal peptides,yields and commonly used media.Meanwhile,strategies and merging trends in protein production and strain engineering are classified and summarized regarding codon optimization,promoter utilization,transcription factor regulation,post-translational modification and proteolytic degradation inhibition.With state-of-art biotechnologies and more available expression platforms, Trichoderma spp.could be more successful hosts to produce recombinant proteins as desired, i.e.better enzyme formula for efficient cellulose degradation or functional protein with high purity and yield.

Keywords:Trichoderma Heterologous expression Protein Catalysis Biotechnology

1.Introduction

Trichodermais an ascomycete fungal genus widely distributed in the soils and generally recognized as safe (GRAS) by U.S.Food and Drug Administration [1].It is famous for biocontrol species,such asT.harzianumandT.atrovirideas well as protein production hostT.reesei(teleomorph:Hypocrea jecorina) [2].NowadaysTrichodermaspp.,especiallyT.reeseistrains,are important workhorses to produce large quantities of diverse enzymes [3–6].T.reeseiQM6a,the parental strain of currentT.reeseimutants used in industry,was initially isolated from rotting U.S.Army equipment on the Solomon Islands during World War II and demonstrated significant ability to degrade cellulose,which was of great importance and potential to economically produce ethanolbased fuel from renewable lignocellulosic biomass.Driven by this idea,random mutagenesis of this strain in 1970s resulted inT.reeseiRUT-30,which was reported to strikingly produce 100 g·L-1of native proteins[5,7].Comparative genomic screening revealed that this unparallel ability of cellulases secretion partially resulted from truncatedcre1gene,frameshift mutation of glucosidase II αsubunit gene,higher endoplasmic reticulum (ER) content,upregulated unfolded protein response (UPR) and low proteases[8,9].Therefore,T.reeseiwas advantageous to produce recombinant proteins from different origins,including β-glucosidases(BGL),xylanase,lipase,mammalian obestatin,etc[10–15].In addition toT.reesei,T.atroviride,T.harzianum,T.virensandT.asperellumhave also been exploited as expression hosts.

Several excellent reviews have provided abundant information regarding protein production inTrichoderma,including improvement ofT.reeseiRUT-C30 as heterologous host [8,16],cellulases and cellobiohydrolases expression inTrichoderma[4,5],heterologous protein production inTrichoderma[2,3,17]and protein secretion in filamentous fungi [10].In this review,we summarized the recombinant proteins produced inTrichodermasince 2014,concerning their origins,hosts,promoters,terminators,signal peptides,yields and commonly used media (Table 1) and provided general information regarding the current strategies used to promote this system.

2.Recombinant protein produced in Trichoderma

Heterologous protein production inTrichodermawas commonly used to improve strain function.Since lignocellulosic biomass degradation involves a series of microorganisms and various enzymes in nature,most of the recombinant proteins produced inT.reeseiwere intended to optimize the enzyme cocktail for better degradation efficiency,such as the heterologous production of cellobiohydrolase Cel7A [19],BGL [18,25],lytic polysaccharidemonooxygenase TcAA9A [22],FAE-encoding genesAnfaeAandAnfaeB[21]and versatile peroxidase gene(vp1)[32](Table 1).Similarly,introduction of the mannase Man5A fromA.nigerinT.reeseiresulted in simultaneously production of cellulase and mannanase at high titers,leading to cost reduction for these two important enzyme additives in animal feeds (Table 1) [12].Furthermore,heterologous expression of an acetamidase gene (amdS) inT.harzianumT34 resulted in an “all-in-one”functional strain,which was able to protect and promote plant growth at the same time(Table 1) [31].Moreover,higher removal rate of phenolic compounds was achieved in treating industrial wastewater,when one laccase fromTrametes sanguineuswas produced inT.atroviride(Table 1) [28].

Table 1 Examples of recombinant protein produced in Trichoderma sp

Besides,Trichodermasystem demonstrated unique advantages in many aspects in recombinant protein production.For example,compared toE.coli,P.pastorisandA.niger,Corynascus thermophiluscellobiose dehydrogenase (CDH) produced byT.reeseiwas most similar to the original concerning glycosylation,cofactor loading and catalytic constants (Table 1) [20].When produced inT.atroviride,the basidiomycete Dye-decolorizing peroxidase,Pleos Dyp1,exhibited better recovery and higher activity than those obtained inE.coli(Table 1) [29].Trichodermaare endowed with high extracellular secretion capability and eukaryotic posttranslational modification (PTM),making them particularly interesting hosts for recombinant protein production and beneficial complementary to other systems [3].A growing number of investigations have deepened our understanding ofTrichodermasystem in target protein production,secretion and proteolysis and shed light on improvement of this system.

3.Processes and strategies for heterologous protein production in Trichoderma

Protein production is a complicated and strictly controlled process.With state-of-art techniques,such as omics and bioinformatic analysis,CRISPR/Cas system and high-throughput screening method,Trichodermaexpression system was better understood and significantly improved to increase target gene expression,protein stability,activity and yield.We summarized the progress of protein production inTrichodermaconcerning codon optimization of target gene,promoter utilization,new expression system construction,transcription factor regulation,PTM and proteolysis inhibition.An overview of these processes is presented in Fig.1.For the protein secretion and secretion promotion in filamentous fungi,please refer to the excellent reviews by Saloheimo and Pakula[33]and Sun and Su [10].

3.1.Codon optimization of gene of interest

Codon optimization is one of the most commonly used strategies for heterologous expression of protein,which could greatly increase the stability of mRNA and translation efficiency[12,19,22,23,30].During codon optimization,codon bias,GC content,free energies and secondary structures of the RNA are usually taken into account with the aid of bioinformatic tools [11,12,23].Nowadays,this process can be conveniently outsourced to gene synthesis company,which largely reduces the time-consumption.

3.2.Promoter utilization

Promoters are defined as the DNA sequence upstream of the transcription start site and controll the transcription of genes.Another important factor for efficient protein production inTrichodermais the usage of promoter.The potentcbh1promoter of CBH1 used to be the“default”promoter for heterologous protein production inT.reesei[2].Nowadays,more promoters are available and utilized in this system (Table 1),such as inducible promoterscbh2,egl1,egl2,xyn1andxyn2,as well as constitutive promoterscdna1,pdc,eno,pki,gpdA,pccg6,etc.[28,29,31,35].With the development in genome mining and transcriptomics,new promoters with different expression strength were identified,which could be independent from cellulase production.For instance,the copperrepressible promoterPtcu1was identified and overexpression of the regulator XYR1 by this promoter accomplished a full cellulase production inT.reeseiunder catabolite repression condition with no need for inducing sugars for enzyme production [36].The promoter toolbox for recombinant gene expression inT.reeseiwas excellently reviewed by Fitz et al in 2018 concerning the new developments in the field of promoters as well as their advantages and disadvantages in applications [37].

Fig.1.An overview of recombinant protein production in Trichoderma and strategies used to promote these processes.TF:transcription factor.Recombinant proteins are synthesized in the endoplasmic reticulum(ER),travel to the Golgi for further post-translational modification and then were secreted into the culture.With the aid of omics and bioinformatic analysis,protein production in Trichoderma are better understood concerning target gene expression,translation,post-translational modification,secretion and proteolysis.The strategies used to promote these processes aimed to increase target gene transcription and translation,provide suitable post-translational modification,enhance secretion and inhibit protein degradation,which can be examined and optimized with high efficiency via highly efficient genetic editing toolbox and highthroughput screening method.

Regarding their utilization,promoter usage can significantly affect the protein yield [22].Expression of target gene simultaneously under multiple promoters was found to enhance protein yield [2].Besides,during expression of the mannanase fromA.niger,promoter engineering by replacing eight binding sites of the transcription repressor ACE1 to those of the activators ACE2 and Xyr1 largely improved the transcription efficiency by 3.6-and 5.0-fold,respectively,leading to increased protein yield [38].Furthermore,different promoters can be used to adjust enzyme formula produced byT.reesei.Wang et al constructed a promoter collection includingcbh1,cbh2,egl1,egl2,xyn1andxyn2forT.reeseiRUT-C30.Multiple heterologous genes were then expressed under different promoters,by which means the cellulolytic profile ofT.reeseiwas adjusted for different utilizations [39].In addition,promoter usage can remarkably improve heterologous protein purity.As we all know,Trichodermaspp.are prolific protein producers.9 characterized cellulases and 15 characterized hemicellulases were produced byT.reeseiand at least 42 genes were predicted to encode cellulolytic or hemicellulolytic enzymes[40].The abundant native proteins made it difficult for recombinant protein purification.Luckily,constitutive promoters provide one possibility to produce target protein with high purity.With constitutiveenopromoters and glucose-containing medium,Cel7A was produced inHypocrea jecorinawith repressed native cellulase production,which greatly simplified its purification [41].With more available promoters in hand,recombinant proteins with high yield,better formula or high purity could be achieved inTrichodermasystem,which facilitates the development of a wider range of applications.

3.3.Development in expression system

For protein production inTrichoderma,various expression strategies are developed.The first and most commonly used strategies is to fuse target gene to a native highly secreted gene (“Carrier”),such ascbh1,which has been reported to stabilize the recombinant mRNA,facilitate translocation in the secretory pathway and avoid protein degradation [42].The protein expression cassettes usually consist of promoter,signal peptide,potential carrier protein,target gene and terminator.The most popular signal peptide used inT.reeseiis the 17-aa CBH1 signal peptide [11,19–21](Table 1).Native signal peptides from eukaryotic origins also worked in some cases,such as in CDH,laccase and Pleos-dyp1 production [28,29](Table 1).As the cost of gene synthesis getting cheaper and affordable,the expression cassette containing codon optimized gene,promoter,terminator and signal peptide sequence as well as protein purification tags can be designed and synthesized in one step,which could significantly reduce the timeconsumption for expression cassette construction.

Another strategy is to replace native highly secreted gene with gene of interest.Previous studies suggested that deletion of gene coding for a major native protein could “make room”for production and secretion of other proteins [2].Using multiplexed CRISPR/Cas9 and a synthetic expression system (Fig.2A),cellulase genes inT.reesei,i.e.cbh1,egl1andegl2,were replaced by lipase B gene ofCandida antarctica(calB) under regulation of a synthetic transcription factor.The resulted strain produced high amounts of highly pure CalB in cellulase repressing glucose medium(Fig.2B) [43].

Fig.2.(A)Scheme of the CRISPR/Cas9-mediated multiplexed gene deletion in T.reesei.Donor DNA were constructed,each containing the pyr4 selection marker gene flanked by～1000 bp of DNA region homologous to 5′-upstream and 3′-downstream regions of cellulase gene.Two gRNAs were designed to introduce double stranded break at the 5′-and 3′-ends of each targeted coding sequence.A single-transformation was performed combining 6 in vitro pre-assembled Cas9 ribonucleocomplexes,three donor DNAs and T.reesei protoplasts.Simultaneous deletion of the three cellulase genes,cbh2,egl1 and egl2,was achieved in about 12%of the resulting colonies.(B)Scheme of the synthetic expression system for CBHI-calB production.Bm3R1-VP16:synthetic transcription factor (sTF).A low and constitutive sTF expression was driven by hfb2 core promoter (T.reesei origin).The sTF recognizes the binding sequences (8 × BS) in an engineered promoter of the cbh1-calB gene.The sTF binding sites are followed by the An201 core promoter (A.niger origin),forming the synthetic promoter.The sTF expression cassette was integrated in a single copy into the cbh2 locus and the CBHI-calB expression cassette was integrated as three copies in the cbh1, egl1 and egl2 loci [43].

Besides,Zhang et al constructed a new efficient expression system to co-express two heterologous genes inT.reeseiRUT-C30 by replacement ofcbh1andcbh2genes in the genome[44].Moreover,a versatile 2A peptide-based bicistronic protein expression platform was developed inT.reeseifor co-expression of target genes[45].Cel7A(“gene of interest”) and eGFP (“Marker protein”) were simultaneously expressed in a 1:1 ratio from a single transcript using the 2A peptide approach.Since the expression level ofcel7Awas directly correlated to that of eGFP,the recombinant protein production can be monitored with fluorescent marker.Furthermore,the 2A peptide approach can be expanded for production of multiple proteins simultaneously by introducing additional 2A peptide sequences between target genes.However,this platform is only suitable for target genes,when addition of amino acids to the gene products’ C terminus does not affect their function [45].

Along with the development of synthetic biology,a design–bu ild–test–learn (DBTL) cycle was invented for construction ofTrichodermamutants for protein production,which used to be timeconsuming and took about 2 months.To increase the efficiency,a GFP-fusion coupled fluorescence-activated cell sorting(FACS)platform was developed to analyze and sortT.reeseiharboring GFPfused protein using flow cytometry and speed up the DBTL cycle,with no need for spore separation and transformant screening.The total costed time for the DBTL cycle was significantly reduced by approx.75% to 2 weeks (Fig.3) [46,47].The developments in expression system and screening processes not only facilitated but also significantly sped up the construction of recombinant protein production inT.reesei,making this system more efficient and convenient.

3.4.Transcription factor regulation

Transcription factor (TF) regulation has emerged as a useful approach for protein production inTrichoderma.More and more TFs related to cellulolytic enzymes or protease production are characterized,including transcription activators,such as ACE2[48,49],Hap2/3/5 [50],ACE3 [40],VEL1 [51],Str1 [52],CRZ1 [53],VIB-1 [54]and Are1 [55],as well as transcription repressors,such as ACE1 [38,56,57],Pac1 [58,59],Rce1 [60],LAE1 [9,61]and Ctf1[62](Table 2).The effects of these TFs on cellulolytic enzyme or protease production were summarized in Table 2.

In addition to regulation of native enzymes,TFs can facilitate target enzyme production with high purity.A versatileT.reeseistrain was developed by inducing a mutation(A824V)in XYR1 regulator and a bicomponent carbon source strategy.The mutated strain constantly produced high-purity xylanase with an almost cellulase-free phenotype in glucose-based medium.By easily replacing the xylanase gene in the genome,it could be used as a convenient expression platform for recombinant protein production with high purity [24].

However,the effects of TFs regulation are not as simple as listed in Table 2.Several studies revealed intricate interactions among a variety of TFs [60].For example,deletion ofvel1completely impaired the cellulase regulator XYR1 [51].CRZ1 was found to compete for the occupancy ofxyr1promoter with transcription repressor ACE1 [53].Comparative transcriptomic analysis demonstrated that deletion ofctf1up-regulated the expression ofvib-1,but down-regulatedrce1,which consequently up-regulatedxyr1andace3[62].Furthermore,comparative genome analysis of twoT.reeseihyper-cellulolytic mutants SS-II and RUT-30 revealed that full-lengthcre1existed in SS-II and resulted in faster growth rate and more efficient biomass degradation than RUT-C30 with a truncatedcre1.The truncatedcre1was believed to contribute to higher cellulase production in RUT-30.Hence,a positive attribute in oneTrichodermastrain might not always work well in otherTrichodermahosts due to different genetic backgrounds [9].Moreover,the pH-related TF Pac1 was reported to repress not only cellulase,but also protease Trasp production[58,59].Similarly,by deletion ofpacCinT.harzianum,proteaseproAwas significantly up-regulated(log2Fold Change 3.59,unpublished result).However,constitutive expression of thepacCgene undergpdApromoter inT.harzianumwas supposed to inhibitproAexpression,but resulted in a significantly up-regulated expression ofproA(log2Fold Change 4.39),which was even higher than thepacCdeletion strain(unpublished result).Therefore,TF regulation to increase protein production or decrease protease concentration should be carried out with caution.

Fig.3.Schematic illustration of the GFP-fusion coupling FACS platform accelerating the heterologous gene expression process in T.reesei,modified from [46].Gfp gene was fused to target gene as indicator for spore separation through the FACS platform.Desired strains with gene expressed at expected levels can be rapidly obtained from a large candidate pool through cell sorting,as well as an additional confirmation of hyphae fluorescence on 96-well plate cultures.The time and numbers of transformants are marked in each procedure.

Table 2 Effects of transcription factor regulation in Trichoderma spp

As the metabolism networks ofTrichodermaare highly complicated,the effects of TFs on protein production remained largely elusive.Further elucidation of the regulation network is of great importance for engineeringTrichodermahosts with robust target protein production.The development of system biology might be helpful to reveal the complex metabolism network and contribute to further promotion ofTrichodermasystem.

3.5.Post-translational modification

Glycosylation is one of the most important and common modifications to proteins,which could affect protein structure,localization,secretion and enzymatic characteristics [65–70].Several glycosylation types were characterized with distinct protein–sugar linkages in both Eukarya and Bacteria (Table 3) [71].Clear sugar differences were observed between Eukarya and Bacteria,which significantly affected heterologous protein.Compared toN-orO-glycosylation,C-,S-orP-glycosylation are less common.Therefore,onlyN-andO-glycosylation were discussed in this review.

On one hand,O-andN-glycans were found to play distinct roles in cellulase activity and stability regarding substrate bind and proteolysis resistance [72–74].By experimental and computational techniques,rational design of N-glycans can be used to enhance protein stability[75].On the other hand,eukaryotic PTM is a great advantage ofTrichodermasystem.With minor engineering,Trichodermamutants withO-mannosyltransferase deletion or recombinant glycoproteins of mammalian-type production were constructed and used for recombinant antibody [76]and other human-like glycoproteins production [76,77],which largely expanded the application ofTrichodermasystem.

In addition to glycosylation,protein phosphorylation plays a vital role in signaling transduction in the cell.Rodriguez-Iglesias&Schmoll [78]observed that six protein phosphatases demon-strated positive or negative regulations for cellulase production,which raised more questions and was interesting to be further explored.

Table 3 Protein glycosylation in Eukarya and Bacteria

3.6.Proteolytic degradation inhibition

In addition to high protein secretion capacity,an idealTrichodermahost is supposed to have low protease production.However,bioinformatic analysis ofT.reeseigenome revealed the existence of 383 putative protease encoding genes,which could be a major hindrance to high levels of protein production [28].Therefore,lowprotease-producing strains have been developed via random mutagenesis,e.g.T.reeseiRUT-C30,as well as by the targeted protease gene deletion to inhibit the degradation of heterologous proteins in culture[79,80].For example,by deletion of the alkaline protease Spw in RUT-C30,the heterologous endoglucanase EGV fromHumicola insolensdemonstrated higher yield and better stability than that produced in parental strain[81].Inactivation of the main protease genetraspinT.reeseiQM9414 improved simultaneously the CMCase,avicelase and xylanase activities[59].For sensitive therapeutic proteins,13 major proteases related to degradation of therapeutic antibodies were identified and seven most problematic proteases were sequentially removed in oneT.reeseistrain,which became a better expression host for antibody,IFNα-2b and insulin like growth factor.Further deletion of two more proteases (slp7andamp2) provided the first study for interferon production inT.reesei[27,82].With the development of biotechniques,comparative secretomics provided one powerful tool to efficiently identify target protease.By this means,three target proteases were identified and further deleted in one-step genetic transformation.The mutated strain demonstrated a 78% decrease in protease activity and a 6-fold increase in cellulase activity[83].This study provided an efficient approach for precise host modification by state-of-art genetic engineering combined with bioinformatic and “omics”analysis.

3.7.Miscellaneous

The composition of cultivation medium is able to affect target protein production as well [23],such as the carbon nitrogen ratio(C:N) of medium.Higher nitrogen levels promoted growth and production of the recombinant Obe-HFBI up to a C:N ratio of 1:1.125,where high growth rate had a negative effect on protein production [15].Besides,the low dissolved oxygen supply in the highly viscous medium ofT.reeseiwas observed to hamper cellulase production during long cultivation period.By integratingVitreoscillahemoglobin (VHb) gene into the genome ofT.reesei,the mutated strain secreted 2.2-fold higher total protein than the parental strain,which indicated that VHb had a beneficial effect on total protein secretion in submerged fermentation[84].Moreover,co-culture appeared to be a promising strategy.The cellulase activity was significantly affected during co-culture ofPhanerochaetechrysosporiumandTrichoderma virideunder different inoculum time,inoculum size,solid–liquid ratio and temperature [85].In addition,light and its intensity could influence protein production,whose effects were mainly on the secretome ofT.reesei[86].

4.Conclusions

Researches onTrichoderma,especiallyT.reesei,were initially driven by their abundant enzymes and potential industrial application for biofuel production.Afterwards,the utilization ofTrichodermahosts expanded gradually to other functional proteins,such as lipase,peroxidase and even antibody and interferon.Due to high protein secretion ability and eukaryotic PTM machinery,Trichodermahosts provided an additional system for recombinant protein production with complementary features toE.coli,yeast andAspergillus.Although lots of studies were successfully carried out to improve protein production and reduce the time consumption inTrichoderma,the mechanism and regulation of recombinant protein production are still fragmented due to the limited understanding of the complicatedTrichodermametabolism.Fortunately,the development of system biology,bioinformatics,omics-based biotechnologies and genetic manipulation tools such as CRISPR/Cas9 system shed light on a much better understanding ofTrichodermahosts concerning the recombinant protein regulation,secretion,PTM and proteolysis,which could contribute greatly to rational design and efficient engineering this hyperproducing cell factory.Meanwhile,DBLT platform could increase the engineering and screening efficiency.Hence,it is expected that the process of strain improvement would speed up and theTrichodermahosts would be more efficient and convenient enzyme producers not only for various lignocellulosic enzymes,but also for a broader range of other functional proteins.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to Prof.Shu-Ming Li from Philipps-Universit?t Marburg for critical reading of this manuscript.This work was supported by the National Key R&D Program of China (No.2018YFA0902200],the National Natural Science Foundation of China (Nos.21838001,21525625,31961133018 and 81803409),and the Fundamental Research Funds for the Central Universities(Nos.buctrc201810 and XK1802-8).We sincerely acknowledge the Mix-Up project,which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 870294.