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Improving lignocellulolytic enzyme production with Penicillium: from strain screening to systems biology
Guodong Liu1, Yuqi Qin1,2, Zhonghai Li1 & Yinbo Qu*1,2
Many Penicillium species produce enzyme systems with good performances in lignocellulose degradation. In our laboratory, lignocellulolytic enzyme-producing Penicillium oxalicum (formerly classified as Penicillium decumbens) strains have been studied for more than 30 years. High cellulase-producing mutants have been obtained through random mutagenesis and genetic engineering, and the components in the enzyme systems have been elucidated using systems biology tools. The effects of different carbon sources on the production level of lignocellulolytic enzymes have been studied, and the related molecular mechanisms have been investigated. When compared with the widely used cellulase producer Trichoderma reesei, some unique features have been found in P. oxalicum, including higher b-glucosidase activity, higher numbers of lignocellulolytic enzyme gene, and different response of cellulase gene expression to some disaccharides. To boost the economic potential of the biorefineries using lignocellulosic biomass, P. oxalicum strains need to be further improved regarding the performance and production level of the enzyme systems.
Review
1State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, PR China 2National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, PR China *Author for correspondence: Tel.: +86 531 88365954; Fax: +86 531 88565610; E-mail: [email protected]
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One of the key technologies in biofuel production is the degradation of complex lignocellulosic biomass to monosaccharides. Lignocellulolytic enzyme systems, mainly produced by filamentous fungi in industry, are widely used in this process. To achieve efficient hydrolysis of lignocellulosics, high dosages of lignocellulolytic enzymes are needed due to the natural recalcitrance of the materials [1,2]. Thus, the high cost of lignocellulolytic enzymes is now a major barrier in the economically competitive production of biofuels from lignocellulosic materials [3]. Trichoderma reesei is now the main source of commercial lignocellulolytic enzymes [4]. Accordingly, T. reesei has become a paradigm for researches on lignocellulolytic enzymes and the regulatory mechanisms controlling their synthesis in the scientific community [5].
Compared with that of T. reesei, lignocellulolytic enzyme systems produced by many Penicillium species have generally better performances (higher cellulose conversion at equal protein loadings) in lignocellulose
hydrolysis [6–9]. Some Penicillium species (e.g., Penicillium funiculosum [10]) have been used for the production of commercial lignocellulolytic enzymes. The related reports about the production levels, hydrolysis efficiencies and industrial traits of crude lignocellulolytic enzyme systems from Penicillium strains have been reviewed [6]; however, knowledge on the composition of these enzyme systems and the regulation of their production is still poor compared with those in T. reesei and Aspergillus species, which significantly limits the progress of further strain improvement.
In our laboratory, a lignocellulolytic enzymeproducing Penicillium has been studied for more than 30 years. The fungus was first identified to be P. decumbens, and was recently reclassified to be P. oxalicum based on sequence ana lysis (Figure 1) [11,12]. Several carbon catabolite repression (CCR)resistant mutants with higher cellulase productivities were obtained, and have been used for industrialscale cellulase production and pilotscale cellulosic ethanol production in China.
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The wholegenome sequence of P. oxalicum was determined in 2009, which allowed the application of systems biology tools on it. In this review, we summarize the progress of our research on P. oxalicum strains. The studies include the development of lignocellulolytic enzyme overproducing mutants, biochemical characterization of enzyme components, exploration of the cellular network controlling enzyme synthesis and application of the enzymes in biorefinery. Results based on systems biology studies will be highlighted.
Screening & development of P. oxalicum strains for cellulase productionThe wildtype P. oxalicum isolate 114 was screened from decayed strawcovered soil in the suburb of Jining, east China, in 1979 using doublelayer agar plates [13]. Ballmilled holocellulose, instead of pure cellulose, was used as the carbon source in the screening plates, ensuring the screened isolates (forming hydrolytic halos) could produce complete cellulolytic and hemicellulolytic enzyme systems. Several singlespore isolates with similar cellulase production levels were purified from 114. One of the purified strains, 114–2, was kept in our laboratory as the wildtype strain and used in most subsequent studies (Figure 2). When 114–2 was cultivated in cellulosecontaining liquid medium, cellulase, xylanase, amylase and pectinase activities were detected.
Production of cellulase and hemicellulase by the wildtype P. oxalicum strain was severely repressed in the presence of excessive glucose or glycerol. After multiple rounds of mutagenesis of isolate 114 using ultraviolet irradiation and nitrosoguanidine, a mutant JU1 with the ability to produce cellulase in glucosecontaining medium was obtained (Figure 2) [13]. In
glucosefree medium (containing 2% holocellulose plus 0.5% wheat bran), JU1 also produced higher cellulase activity (3.1 units of filter paper activity [FPA] per ml) than 114–2 did (0.8 U ml1). Notably, JU1 showed quite different morphology from 114–2, including slower hyphal extension, thicker hyphae, loss of bluishgreen conidial pigment, lower amounts of conidia production and circumscribed pink colonies.
JU1 was then adapted repeatedly on spent ammonium sulfite liquor (SASL; one kind of pulp mill effluent) gradient agar plates to improve its tolerance to this toxic liquor [14]. The experimental evolution generated a SASLtolerant mutant JUA10. JUA10 grew faster than JU1 in SASL, synthetic highsulfate medium and even nontoxic glucose medium [15]. The time of maximum cellulase production by JUA10 was 20 h earlier than that of JU1 in SASLwaste fiber medium [14]. Through genome shuffling, three fusants with more than twofold higher cellulase (FPA) productivities were obtained from JUA10 [16]. In addition, several other cellulase highproducing mutants, such as JUA10–1 [17], JU140–12 [18] and JUA10T [19], were also derived from JUA10 by further mutagenesis and screening (Figure 2).
Rational strain engineering of P. oxalicum was performed based on the clear genetic background provided by genome sequencing. The assembled genome size of wildtype strain 114–2 was 30.19 Mb, which was predicted to encode 10,021 proteins [20]. For mutant JUA10T, the genome size was 30.69 Mb, with 10,473 proteincoding genes predicted [19]. To our knowledge, these are the first genome sequences of industrial cellulaseproducing Penicillium species. Homologs of most proteins known to affect cellulase production in fungi were annotated in P. oxalicum, some of which were targeted for the purpose of strain improvement. The gene encoding the ortholog of Neurospora crassa NCU05137 (a secreted protein whose deletion led to increased cellulase expression [21]) in P. oxalicum, PDE_01641, was deleted in both 114–2 and JUA10T [22]. Deletion of the PDE_01641 gene increased cellulase (FPA) production and cell growth on cellulose in 114–2, and resulted in a 36% increase in cellulase production at 48 h of fermentation in JUA10T. Deletion of gene bgl2 (encoding major intracellular bglucosidase) improved the final cellulase activity (after 144 h of fermentation in the medium containing 1% cellulose plus 1% wheat bran) to 0.88 U ml1, which was 3.3fold that of 114–2 [23]. Combined manipulations of some crucial (positive or negative) transcription regulators resulted in an up to tenfold elevation of cellulase production in 114–2 [Li Z, Qu Y,
Unpublished Data]. The engineering strategies are being implemented in industrial hyperproducing mutants to further improve production levels.
Key terms
Lignocellulolytic enzyme system: Mixture of many kinds of enzymes acting together for the degradation of complex lignocellulosics. Cellulases, hemicellulases and ligninases are the main components.
Penicillium: Genus of fungi belonging to the division Ascomycota, order Eurotiales, class Eurotiomycetes, family Trichocomaceae. Penicillium species are widely distributed in the natural environment, and have been used in the production of drugs, food and enzymes in industry.
Carbon catabolite repression: A regulatory system ensuring that micro organisms use rapidly metabolized carbon sources (e.g., glucose) first. When these carbon sources are present, the transcription of genes encoding enzymes for the utilization of alternative carbon sources (e.g., cellulose) are repressed.
Systems biology: Study aimed at under standing how all the components in biological systems (cell, tissue, organism, and so on) function together. Genome-wide experimental techniques are widely used for systems biology studies.
Holocellulose: Total polysaccharides (cellulose and hemicellulose) of wood or other lignocellulosic materials. Holo cellulose can be prepared from natural lignocellulosic materials by removing extractives and lignin using chemical reagents (e.g., acidified sodium chlorite).
Filter paper activity: Widely used, standard representation of cellulase activity. Filter paper (a blend of amorphous and crystalline cellulose) is hydrolyzed and the amount of reducing sugars released is determined for activity calculation.
Genome shuffling: Technique recom-bining multi-parental genome sequences to concentrate beneficial mutations in one genome for strain improvement.
Homolog: Genes or proteins of shared ancestry. Homologs that originated by vertical descent from a common ancestor are called orthologs.
Nonhomologous endjoining pathway: A cellular process ligating dsDNA ends without the need for sequence homology.
Processivity: For cellulases, processivity is the ability of a cellulase to continuously hydrolyze one cellulose chain without dissociating from it. Cellobiohydrolase I from Trichoderma reesei is a typical processive cellulase.
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Strains with specific genotypes were also developed to facilitate the engineering and functional genomics study in P. oxalicum. A strain Dpku70, in which the nonhomologous endjoining pathway was disrupted, was constructed to improve genetargeting efficiency [24]. The frequency of homologous recombination in Dpku70 reached 100%, much higher than that in the parent wildtype 114–2 (20–90%, depending on genes). A pyrGauxotrophic mutant M12 was isolated through the spontaneous mutagenesis of 114–2 [25]. The pyrG marker can be selected bidirectionally (either presence or absence), thus allowing marker recycling for multiple gene manipulations [26].
Characterization of the lignocellulolytic enzyme system of P. oxalicumSome enzymatic properties of P. oxalicum enzyme systems were studied using crude enzymes. The enzyme system of mutant JU1 had optimum temperatures of 40, 55, 60 and 70°C, respectively, when absorbent cotton (hydrolyzed for 24 h), filter paper (hydrolyzed for 1 h), sodium carboxymethyl cellulose (hydrolyzed for 15 min) and salicin (hydrolyzed for 30 min) were used as the substrates for hydrolase activity assays [27]. The optimum pH of the enzyme system on filter paper was 4.8. These results are similar to those of enzyme systems from many other mesophilic fungi [28–30]. For the effect of metal ions, cellulase activity of the enzyme system of mutant JUA101 was found to be inhibited by Fe3+ [31]. To get a better understanding of the lignocellulolytic enzyme
system of P. oxalicum, the composition of the enzyme mixtures and properties of single enzymes were studied.
� Studies of single-enzyme components prior to genome sequencingCellobiohydrolase (CBH; exoacting cellulase), endo1,4bglucanase (endoglucanase [EG]) and bglucosidase (BGL; cellobiase) are the three major types of enzymes in cellulose hydrolysis [32]. When the enzyme systems of P. oxalicum were separated by polyacrylamide gel electrophoresis and then stained for enzyme activities, one to two bands for CBH activities and one band for BGL activity were detected, while more than ten bands were detected for EG activities [33]. Through a combination of several different column chromatographic procedures, six CBHs, eight EGs and one BGL were purified from strain JU1 [34]. The purified enzymes differed in molecular weight, isoelectric point, carbohydrate content, substrate specificity, specific activity, kinetic parameters, product profile and randomness (processivity) of action. The major cellobiohydrolase, CBHI (later renamed Cel7A2), was purified from strain JUA10 and studied for the relationship between glycosylation type and enzymatic properties [35] (for details, see the subsection ‘Posttranslational modification of cellulases’). Another cellobiohydrolase purified from JUA10, PdCel6A, was very stable at acidic pH, and thus could efficiently improve the production of ethanol from lignocellulosics under acidic conditions [36]. Using degenerate primers, one CBH gene cbh1 (later renamed cel7A-1) [37] and
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P. oxalicum 114-2 (KF152942)
P. oxalicum JU-A10-T (KB908899)
P. oxalicum NRRL 790 (AF034455)
P. oxalicum CCF:2052 (HE651146)
P. oxalicum CCF:2315 (HE651152)
P. oxalicum NRRL 787 (AF033438) TYPE
P. decumbens NRRL 741 (AF033453) TYPE
P. chrysogenum NRRL 807 (AF033465) TYPE
Figure 1. Maximum-likelihood phylogenetic tree of Penicillium strains. DNA sequences including internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2 and partial 28S ribosomal RNA gene were aligned using ClustalX 2.0 [11], and then the phylogenetic tree was generated by MEGA 5.10 [12] using the bootstrap maximum likelihood method with 1000 replicates. GenBank accession numbers are shown in parentheses. Partial nucleotide sequences of the assemblies (2493–3639 for 114–2 and 1064521–1065666 for JU-A10-T) were used for alignment. The type strains of Penicillium oxalicum, Penicillium decumbens and Penicillium chrysogenum are marked in the figure. Nucleotide sequence identities between the sequence from strain 114–2 and those of type strains are shown (numbers on the right sides of dotted lines). The identification of 114–2 and JU-A10-T as P. oxalicum was also supported by aligning the coding sequences of calmodulin, b-tubulin and RNA polymerase II subunit 2 genes with those from identified P. oxalicum strains, respectively (nucleotide sequence identities are 98–100%).
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three EG genes cel7B, cel5A and cel45A [38,39] were cloned from P. oxalicum. The three EGs were heterologously expressed and characterized in yeasts. The high hydrolytic activity of recombinant Cel7B [39] and the thermophilic property of recombinant Cel45A [38] were detected.
BGL hydrolyzing cellobiose to glucose is important for the release of product inhibition in cellulose hydrolysis [40]. Like many other Penicillium species [6], P. oxalicum produces enzyme systems with higher BGL activity than that of T. reesei, which thereby have better performances in simultaneous saccharification and fermentation of acidpretreated corncobs to ethanol [41]. pBGL1 is the primary BGL in the enzyme system of P. oxalicum [42]. Either supplementation of purified pBGL1 to the enzyme mixture [42]
or integration of pbgl1 gene into the genome [43] could significantly enhance the saccharifying ability of the enzyme system from T. reesei.
� Global understanding using genomic & proteomic approachesIntegrated genomic and proteomic ana lysis provides a rapid and comprehensive understanding on the composition of the enzyme system of P. oxalicum [20]. Eighteen genes encoding cellulolytic enzymes, including three CBHs, 11 EGs and four polysaccharide monooxygenases (PMOs; previously known as glycoside hydrolase [GH] family 61 EGs [44]) were predicted in P. oxalicum (Figure 3) [45]. Some of the enzymes were subsequently heterologously expressed in yeast and characterized for their properties. One GH family 5 EG (Cel5C) containing an unusual CBM_X2 domain showed activities on both cellulose and xyloglucan [46]. Three of the four PMOs showed synergism with commercial cellulase from T. reesei in cellulose hydrolysis [Qu Y, Unpublished Data]. For other degrading enzymes, 11 BGLs, 51 hemicellulases, 25 pectinases and 11 amylolytic enzymes were predicted [20]. The higher number of hemicellulases and cellulose binding domain (CBM1)containing proteins in P. oxalicum was highlighted in the comparison with those in T. reesei [20]. Particularly, the number of CBM1containing proteins (n = 23) is the highest among all the sofar sequenced Aspergillus and Penicillium species (three to 22 in the remaining species).
The actual compositions of enzyme systems were further elucidated by proteomic analyses of the secretomes. Cellulases, hemicellulases, amylases and proteases were found to be the major components [19,20]. When compared with those of T. reesei [47,48], some unique features of the composition of P. oxalicum enzyme systems are noted:
� Two GH family 7 CBHs, one with and the other without CBM1 (Figure 3), are present in the secretome of P. oxalicum [19]. The later type of CBH is genetically absent in T. reesei [49];
� In addition to Cel7B (ortholog of T. reesei Cel7B/EG I), Cel5B, but not Cel5A (ortholog of T. reesei Cel5A/EG II), is another major EG in P. oxalicum [19]. It was noted that a close homolog of Cel5B was also reported to be a major EG in Penicillium brasilianum [50], but its ortholog is absent in T. reesei [20];
� A PMO (Cel61A) accounts for approximately 6% of the total secretome of 114–2 and approximately 14% of that of JUA10T [19], much higher than the proportions of PMOs in enzyme preparations produced by T. reesei (<1%) [47,48]. The role of this PMO in lignocellulose hydrolysis by P. oxalicum is being studied in our group;
� P. oxalicum and T. reesei each have some unique hemicellulases in their secretomes, such as feruloyl esterase and endob1,4galactanase in P. oxalicum [20], and GH74 family xyloglucanase and bxylosidase in T. reesei [47]. The information provided a rational basis for further improvement of the enzyme systems.
� Post-translational modification of cellulasesPost-translational modifications such as glycosylation are widely detected for secreted proteins from fungi [51]. Some cellulases purified from strain JU1 had very similar amino acid compositions but different molecular weights, indicating that they might be encoded by the same gene but underwent diverse posttranslational modifications [34]. On the 2D electrophoresis map of cultured supernatant, 106 secreted protein spots were assigned into a total of 38 protein models [20], which clearly confirmed the hypothesis of posttranslation modification. The map also showed that most isoforms of one protein had similar molecular weights but differed greatly in isoelectric points.
The diversity of Nglycosylation on CBHI (Cel7A2) from P. oxalicum was studied in detail [35]. Four glycoforms of CBHI with identical amino acid sequences but different Nglycan structures were purified from JUA10. The four proteins had different specific activities, optimum temperatures and optimum pHs. In particular, one of the glycoforms, CBHIA (carrying
Key terms
Product inhibition: Inhibition of the activity of an enzyme by its product.
Simultaneous saccharification and fermentation: Method for biomass conversion in which hydrolytic enzymes and fermenting microorganisms (e.g., yeast) are simultaneously added into the reactor for substrate utilization.
Glycoside hydrolase: Type of enzyme hydrolyzing glycosidic bonds. Glycoside hydrolases are classified into more than 100 families in the Carbohydrate-Active Enzyme database based on amino acid sequence similarities.
Cellulose binding domain: Amino acid sequence folding independently and showing cellulose-binding activity. Cellulose binding domains are usually found in cellulases and hemicellulases.
Post-translational modification: Processing of polypeptides after translation. The modifications include folding, cutting and adding chemical groups (e.g., phosphate and carbohydrates).
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[Man]3 [GlcNAc]
2 + GlcNAc at
Asn137), showed a signif icant synergism on cellulose degradation with commercial enzyme systems. CBHIA had no hydrolytic activity on cellulose and pnitrophenylbdcellobioside (a commonly used substrate for CBH assays), but could decrease the hydrogen bond intensity and crystalline degree of cotton fibers. It is reasonable to suppose that the properties of other enzyme components are also affected by diverse glycosylations.
The cellular machine for lignocellulolytic enzyme production in P. oxalicumSome sequenced filamentous fungi, such as Aspergillus niger, Aspergillus oryzae and Penicillium chrysogenum, have comparable or even higher numbers of lignocellulolytic enzymeencoding genes than P. oxalicum [20]; however, they are not good producers of lignocellulolytic enzymes. This indicates that P. oxalicum holds more efficient transcription, translation or secretion machines for the production of lignocellulolytic enzymes.
Generally, lignocellulolytic enzymes in filamentous fungi are produced at high levels only in the presence of inducers (e.g., cellulose) [52]. The same feature was observed in P. oxalicum. The effects of different carbon sources on lignocellulolytic enzyme production, and the involved regulatory proteins or pathways (Figure 4), have been investigated in P. oxalicum.
� Effects of different carbon sourcesWhen P. oxalicum was grown in medium with glycerol or glucose as the sole carbon source, low activities of cellulase and xylanase were detected, suggesting that some enzyme components are expressed at basal levels [20,33,53]. When lignocellulosic substrates (e.g., cellulose, xylan, xylose and wheat bran) were used as carbon sources, significantly higher lignocellulolytic enzyme activities were detected. The repressing effect of glucose and inductive effect of cellulose on the expression of cellulases and xylanases were verified at genetranscriptional levels [54]. Lactose, which is used for cellulase induction in T. reesei in industry [55], also induces the transcription of cellulase genes in P. oxalicum. However, the inductive effect on cellulase gene transcription is not sustained and no improved
cellulase production was detected. It is assumed that glucose produced by lactose hydrolysis repressed cellulase gene expression quickly [54]. Another strong inducer of cellulase in T. reesei, sophorose (b1,2glucobiose) [56], cannot induce the production of cellulases in P. oxalicum [57]. It is also noted that the wildtype strain 114–2 and mutant JUA10 respond to specific sugars differently [54]. Lactose does not induce cellulase gene transcription and even cannot support cell growth in JUA10. By contrast, lsorbose induces cellulase gene transcription in JUA10 but not in 114–2. The mechanisms for these differences are as yet unknown.
� How the cell senses celluloseThe inductive effect of insoluble cellulose on cellulase expression is generally believed to be mediated by cellodextrins (cellulose hydrolytic product) [58,59]. Supplementation of a small amount of exogenous cellodextrins (0.05%) to 2% cellulose medium did lead to improved cellulase production in P. oxalicum 114–2 [60]. However, endogenous cellodextrins are hardly detected when cellulose is used as the inducer. This might be due to the gradual release and rapid catabolism (by BGLs) of cellodextrins [58]. BGLdeficient mutants were constructed to study the role of cellodextrins in celluloseinduced cellulase expression in P. oxalicum [23].
M12
140-12JU-A10-TJU-A10-1
JU-A10 GS2-15
JU1
JN15
UV11
114 114-2Isolation
Mutagenesis
Mutagenesis
Mutagenesis
Mutagenesis
Adaption in SASL Genome shuffling
M12
∆pku70
Figure 2. Genealogy of Penicillium oxalicum strains. Strains shown in light gray have been lost over the years. SASL: Spent ammonium sulfite liquor.
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Key terms
Transcription factor: Protein activating or inhibiting the transcription of DNA to RNA through binding to specific DNA sequences.
Inverse metabolic engineering: Concept in metabolic engineering first identifying the genetic basis of a desired phenotype and then introducing the genotype into another host.
Submerged fermentation: Microorganisms are cultured in liquid medium in a large tank to make useful products. The culture is constantly mixed by stirring and supply of sterile air is needed.
Deletion of the major extracellular BGL gene bgl1 resulted in remarkably higher cellodextrins in the culture broth, but did not enhance the expression of cellulases. Deletion of the major intracellular BGL gene bgl2 accumulated cellobiose in the cytoplasm along with significantly increased expression of cellulases. In the bgl2disrupting mutant, exogenous cellobiose alone can induce the transcription of cellulase genes. These results indicated that cellobiose released from cellulose is an inducer for cellulase gene expression in P. oxalicum after
being transported into the cell. This assumption was further verified by that in cellobiose transporterdeficient
mutant, celluloseinduced cellulase expression was dramatically reduced [Li J, Liu G, Li Z, Qin Y, Qu Y, Unpublished
Data].A subsequent question is how the cellobiose signal
is transduced after entering the cell. Sophorose and gentiobiose (b1,6glucobiose) are supposed to be important in cellobioseinduced cellulase expression in T. reesei and Penicillium purpurogenum, respectively, because they strongly induce cellulase gene expression and can be produced from cellobiose through transglycosylation by intracellular enzymes [61,62]. In P. oxalicum mutants with both bgl1 and bgl2 deleted, cellobiose, but not sophorose or gentiobiose, induced cellulase expression [61,62]. The result clearly suggests different mechanisms for cellulase induction among fungal species. The proteinsensing and transducing cellobiose signal has not been identified in fungi so far.
CBHs
EGs
PMOs
PDE_05445 (Cel7A-1)
PDE_07945 (Cel7A-2)
PDE_07124 (Cel6A)
PDE_07929 (Cel6B)
PDE_00507 (Cel5A)
PDE_09226 (Cel5B)
PDE_09969 (Cel5C)
PDE_05193 (Cel5D)
PDE_06439 (Cel12A)
PDE_03711
PDE_09267
PDE_02886
PDE_09014
PDE_00698
PDE_01261
PDE_06768
PDE_07928 (Cel45A)
PDE_05633 (Cel61A)
S
S
S
S
C
C
C
S C
S C
S C
S C
S C
S C
Glyco_hydro_7
Glyco_hydro_7
Glyco_hydro_12
Glyco_hydro_12
Glyco_hydro_12
Glyco_hydro_45
Glyco_hydro_61
Glyco_hydro_61
Glyco_hydro_61
Glyco_hydro_61
Glyco_hydro_7
Glyco_hydro_6
Cellulase (GH5)
Cellulase (GH5)
Cellulase (GH5)
Cellulase (GH5)
Cellulase (GH5)
Catalytic domainCBM_1
Cellulase (GH5) CBM_X2
Figure 3. The 18 cellulolytic enzymes predicted in Penicillium oxalicum. Domain compositions of the enzymes are shown according to the results of Pfam ana lysis [45]. C: Enzymes characterized for enzymatic properties; CBH: Cellobiohydrolase; CBM_1: Family 1 carbohydrate binding module (formerly known as fungal cellulose-binding domain); EG: Endoglucanase; PMO: Polysaccharide mono-oxygenase; S: Enzymes detected in experimental secretomic analyses.
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Some classical signaling pathways (e.g., G protein signaling) were reported to affect cellulase expression in T. reesei, although they did not directly transduce the cellulose/cellodextrin signal [63,64]. One heterotrimeric G protein a subunit coupled to cyclic adenosine monophosphate signaling, PGA3, negatively regulates cellulase gene transcription in P. oxalicum through a carbon sourceindependent manner [Hu Y, Liu G, Li Z,
Qin Y, Qu Y, Unpublished Data]. The regulatory role only occurs in the early stage of cultivation but does not affect cellulase production levels, suggesting it is not a dominant regulator in cellulose signaling.
� Transcriptional regulationRegulation of lignocellulolytic enzyme production is mainly accomplished by controlling the transcriptional levels of genes encoding these enzymes [52]. As reported in other fungi, transcriptions of most cellulases and hemicellulases in P. oxalicum are simultaneously induced or repressed (i.e., coregulated) by carbon sources [54]. Orthologs of several transcription factors previously known to be involved in this regulation were annotated in P. oxalicum [20], some of which were functionally studied. As expected, the CCR factor CreA [19] and deubiquitinating enzyme CreB that stabilizes CreA [65] negatively regulates cellulase production in P. oxalicum under both repressing and inducing conditions. The regulatory roles of transcription factors ACEI (negative regulator of cellulase expression [66]), ClrB (activator of cellulase and xylanase expression [67]) and XlnR (activator of xylanase and cellulase expression [68]) were also confirmed in P. oxalicum [Li Z,Qu Y, Unpublished Data].
To get a systematic understanding of the transcription factors regulating cellulase expression, a singlegene deletion strain set covering more than 400 transcription factors was constructed in P. oxalicum. The abovementioned strain, Dpku70 (Figure 2) [24], was used as the parent strain to facilitate the efficiency of targeted gene deletion. Some transcription factors, whose functions were not reported previously, were shown to strongly affect cellulase production. It appears that we are still far away from a complete understanding of the mechanisms controlling lignocellulolytic enzyme expression in fungi.
� Lessons from the comparison of wild-type & mutant strainsThe cellulase hyperproducing mutants obtained through longterm random mutagenesis are good materials for the study of inverse metabolic engineering [69]. The genome sequences of mutant JUA10T (Figure 2) and wildtype 114–2 were compared. A large number of sequence variations (average 1.4 singlenucleotide variations per kilobase pair) were found between the two strains, making it difficult to identify the critical
variations accounting for the different production levels. The secretomes and transcriptomes of the two strains were then compared. Integrative ana lysis of these results showed significantly downregulated expression of amylases and proteases in addition to upregulated expression of cellulases and hemicellulases in JUA10T. The result led us to investigate the function of the wellknown amylase transcription activator AmyR (dramatically downregulated in JUA10T) in cellulase expression [70]. Interestingly, gene deletion suggested that AmyR plays a negative role in cellulase expression, even when starch was absent in the medium [19]. In addition, a frameshift mutation in CreA was proved to underlie the CCRresistant expression of cellulases in JUA10. Mutations in the genepromoter region of cellobiohydrolase Cel7A1 was proved to contribute to its exceptional overpresentation in the secretome of JUA10T [19].
Genomewide transcription ana lysis also provided some other clues about the hyperproducing phenotype in JUA10T (Figure 4). First, more genes in JUA10T are expressed at low levels in comparison to those in 114–2 [19]. That is to say, the cell factory of JUA10T concentrates on fewer biological processes. Second, the expression levels of genes for the pentose phosphate pathway (providing NADPH and precursors for amino acids biosynthesis), lysine and cysteine synthesis, ribosome and protein folding are upregulated in JUA10T, all facilitating the highlevel synthesis of lignocellulolytic enzymes. Third, amino acid degradation and most secondary metabolisms are reduced in JUA10T, which may increase the fluxes and energy used for enzyme synthesis. The results provide implications for future rational design of strains for higher production of lignocellulolytic enzymes.
Application of P. oxalicum enzyme systems in biorefinery
� Low-cost production of lignocellulolytic enzymesOne of the approaches for improving the economics of cellulosic biofuels production is to lower the cost of cellulase production. Submerged fermentation conditions were optimized for cellulase production by P. oxalicum. The optimal temperature and pH of cellulase production by strain JU1 is 28°C and 3–4, respectively [27]. Wheat bran, an agricultural residue mainly consisting of xylan, starch, crude protein and cellulose, has a remarkably stimulative effect on cellulase production by P. oxalicum [53,60]. Industrial wastes such as SASL, paper mill waste fiber, corncob residues (from xylooligosaccharide production) and acidic wastewater (from monosodium glutamate process) were used to substitute for part or all of the cultivation medium to
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further lower the cost of cellulase production [14,16,18]. Fedbatch fermentation was used to improve the volumetric cellulase activity in submerged fermentation [14]. Also, cellulase production by P. oxalicum in solid-state fermentation was studied, primarily focusing on the control of air flow [71–73].
As a combined result of strain improvement and process optimization, an extracellular protein concentration of 16.9 g l1 and cellulase activity of 15.4
filter paper unit (FPU) ml1 at 96 h of fermentation (i.e., productivity of 160 FPU l1 h1) was obtained for P. oxalicum mutant JUA10–1. The mutant has been used for industrial production of cellulase preparations for feed, food and the textile industry in China since 1996 [74]. Also, the
P. oxalicum enzyme systems were used in research on pulping for high xylanase activity [75] and extraction of plant flavonoids for high transglycosylation activity [76].
� Biorefinery of agricultural residues using P. oxalicum enzymesWe have developed a technology route for the biorefinery of nonwood lignocelluloses with pulp, ethanol and singlecell protein as the main products [77]. P. oxalicum was used for cellulase production in this process using wastes from pulping as the culture medium. Biorefinery of corncob was proposed based on the previously wellestablished production of xylose, xylitol, xylooligosaccharides and furfural, with ethanol and lignin as the coproducts [77]. In this process, corncob residues (containing mainly cellulose and lignin [17]) and wheat bran were used as the main carbon sources for the onsite
Lignocellulose
Cellulases Hemicellulases
Amylases, and so on
BGLs
Cellodextrins Glucose Pentoses
BGLsCellodextrins Glucose
Glycolysis
PPP
NADPH
Amino acidsynthesis
Citrate cycle
Secondary metabolism
Amino acidcatabolism
Amino acids
Protein synthesis
Protein folding
ClrB, XlnR, and so on
AmyR CreA
Nucleus Cytoplasm
(hemi-)cel
amy
others
GTsCDTs
?
PTs
Figure 4. Summary of proteins and cellular processes involved in cellulase production in Penicillium oxalicum. Black arrows indicate mass flows and gray arrows indicate regulatory interactions. Genes, proteins or pathways of remarkable expression changes in JU-A10-T compared with those in 114–2 are marked (triangle for upregulation and inverted triangle for downregulation). BGL: b-glucosidase; CDT: Cellodextrin transporter; GT: Glucose transporter; PPP: Pentose phosphate pathway; PT: Pentose transporter.
Key term
Solid-state fermentation: Microorganisms are cultured on solid medium (with reduced water activity) to make products. Solid-state fermentation has the advantage of producing some chemicals and enzymes.
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production of lignocellulolytic enzymes by P. oxalicum. The crude enzymes can be directly used for simultaneous saccharification and fermentation of corncob residues to ethanol without removing fungal cells. Thus, the cost of enzymes is further reduced. Using a fedbatch strategy, the concentration of ethanol produced from delignined corncob residues reached 57.2 g L1 at a lowenzyme dosage (9.3 FPU g1 substrate) after 141.5 h of fermentation [17]. At present, a corncobbased cellulosic ethanol project in a scale of 50,000 tonne per year has been built at Yucheng, China [101].
The negative role of lignin in the hydrolysis of complex lignocellulosic materials by P. oxalicum enzymes was found [17]. When delignined corncob residues were used as the substrate, significantly higher concentration of ethanol was produced in comparison to that using corncob residues as the substrate. Electrophoresis ana lysis indicated that the major cellulase CBHI was irreversibly adsorbed to the lignin, which might reduce its efficiency in cellulose hydrolysis. CBHIs from Penicillium pulvillorum and T. reesei were recently shown to be differently adsorbed to lignin [7], indicating the possibility to reduce the adsorption of CBHI to lignin by protein engineering in the future.
Future perspectiveAlthough many strategies have been employed, the performance and production level of lignocellulolytic enzyme systems produced by P. oxalicum still need
to be further improved to benefit the economy. For example, supplementation of exogenous BGL could further improve the performance of P. oxalicum enzyme systems in cellulosic ethanol production [78], despite its relatively higher BGL activity (compared with that of T. reesei). Some other components (e.g., CBHI, which binds to lignin) in the enzyme system also need to be optimized for the hydrolysis of different lignocellulolytic materials. On the other hand, the enzyme production level in P. oxalicum is still lower than those reported in T. reesei and some other fungi (up to 100 g l1 [49,79]). Based on the results from systems biology studies, the production level in P. oxalicum is expected to be further elevated. To achieve this goal, understanding of the related cellular processes (e.g., signal transduction, transcription regulation and protein secretion) is being enhanced and highly efficient genetic manipulation tools (e.g., multiplegene targeting) are being developed.
Financial & competing interests disclosureThis work was supported by grants from the National Basic Research Program of China (grant no. 2011CB707403) and the National Natural Sciences Foundation of China (grant no. 31030001). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Executive summary
Screening and development of Penicillium oxalicum strains for cellulase production � The wild-type P. oxalicum isolate 114 was screened from decayed straw-covered soil using double-layer agar plates containing ball-milled
holocellulose. � A series of carbon catabolite repression-resistant mutants with higher cellulase production levels were obtained through random
mutagenesis. � Genetic engineering of P. oxalicum was performed based on genomic ana lysis.
Characterization of the lignocellulolytic enzyme system of P. oxalicum � Some enzyme components were studied prior to genome sequencing through protein purification and gene cloning. � Integrated genomic and proteomic ana lysis showed that the enzyme system of P. oxalicum contained more diverse components than that
of Trichoderma reesei. � Post-translational modification widely occurs on lignocellulolytic enzymes and affects their activities.
The cellular machine for lignocellulolytic enzyme production in P. oxalicum � Glucose represses, while cellulose induces, the expression of lignocellulolytic enzymes. Lactose and sophorose cannot induce high-level
cellulase production in P. oxalicum. � Intracellular cellodextrins are important in the induction of lignocellulolytic enzyme expression by cellulose. � In addition to several well-documented regulators, novel transcription factors are found to be involved in the transcriptional regulation of
lignocellulolytic enzymes. � Comparative ana lysis of wild-type and mutant strains identified critical factors (including transcription factors CreA and AmyR) affecting
cellulase production level.Application of P. oxalicum enzyme systems in biorefinery
� Fermentation conditions were optimized and low-cost media were designed for the production of lignocellulolytic enzymes. � P. oxalicum enzymes were used in the biorefinery of corncob in China.
Future perspective � The hydrolysis performance and production level of P. oxalicum enzyme systems still need to be improved in the future.
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� Website101 Green Prospects Asia. Shandong longlive
biotechnology delivers first batch of fuel ethanol from corncobs. www.greenprospectsasia.com/content/shandonglonglivebiotechnologydeliversfirstbatchfuelethanolcorncobs