BACKGROUND
efficient saccharification of cellulosic biomass to fermentable sugars is necessary for economical production of second generation biofuels.
several enzymes  act in concert to hydrolyze cellulose to glucose.
the industrially employed filamentous fungus trichoderma reesei has been evolved for cellulase production and is capable of secreting large quantities of cellulolytic enzymes at a relatively low cost  <cit> .
these include two exoglucanases, cel7a and cel6a , which represent 50% and 20% of the total cellulase content, respectively, four endoglucanases, cel7b , cel5a , cel12a , and cel45a  that represent approximately 15%, 10%, 1%, and <1% of the total cellulase content, respectively, and several lytic polysaccharide monooxygenases  <cit> .
lignocellulose hydrolysis at relatively high temperatures  may offer potential advantages, including reduced solution viscosity at high-solids loadings , lower risk of microbial contamination during saccharification, greater compatibility with high-temperature pretreatments, and potentially faster rates of hydrolysis  <cit> .
the short half-lives of t. reesei cellulases at temperatures above 50°c, together with very low expression levels of thermophilic cellulases   <cit> , motivates the development of thermostable t. reesei cellulases that can hydrolyze lignocellulose efficiently at temperatures beyond 50°c.
t. reesei endoglucanase i  is known to randomly cleave internal cellulosic bonds, thereby creating shorter cellulosic chains.
recent studies on optimizing the components of cellulase systems have underscored the importance of having tregi comprise a high fraction of the cellulase mixture  in order to efficiently hydrolyze pretreated lignocellulosic biomass  <cit> .
tregi has a bimodular structure with a 375-amino-acid  catalytic domain  attached to a 35-aa carbohydrate-binding module  via a 26-aa linker.
tregi has  <dig> disulfide bonds , 6 n-glycosylation sites in the cd , and a linker region rich in serine and threonine that are potential o-glycosylation sites.
it was selected for engineering enhanced thermostability because of its importance in cellulose hydrolysis and the availability of its crystal structure  <cit> .figure  <dig>
crystal structure of endoglucanase i from
trichoderma reesei
.
disulfide bonds are shown in blue.
n-glycosylation sites are shown in magenta.
mutagenesis sites are shown in red and are labeled as follows: a , b , c , d , e , f , and g .
mutations at site c and site e resulted in improved tregi enzyme variants.
pdb code 1eg <dig>
although protein engineering has been used to improve the thermostability of numerous cellulases  <cit> , there are no reports on improving the thermostability or increasing the optimal temperature of tregi.
in this study, we employed a b-factor guided approach  <cit>  to engineer thermostable variants of tregi using e. coli cell-free protein synthesis due its ease and throughput .
many thermostable mutants of tregi identified using cell-free protein synthesis were subsequently expressed in the fungal hosts s. cerevisiae, n. crassa, and t. reesei to better mimic the properties of the native tregi enzyme in terms of folding and glycosylation .
tregi variants, particularly g230a/d113s/d115t, were more stable compared to their recombinant wild-type versions when both the variant and wild-type were expressed in s. cerevisiae, n. crassa, or t. reesei, indicating that the positive influence of these mutations on stability translates across different expression hosts.
however, the s. cerevisiae-expressed enzymes were inferior in terms of activity and stability compared to the same enzymes expressed in t. reesei or n. crassa, which we attribute to the lack of cyclization of the n-terminal glutamine of treg <dig> expressed in our s. cerevisiae system.figure  <dig>
engineering thermostable endoglucanase i from
trichoderma reesei
.
RESULTS
b-factor guided mutagenesis
our efforts to improve the thermostability of tregi entailed a b-factor guided approach   <cit> .
the b-factor is a measure of the average deviation of a given residue from its mean position in the crystal structure.
residues having the highest average b-factors  correspond to the most flexible sites in the protein and provide targets for mutagenesis to improve thermostability.
this strategy is based on the premise that the thermostability of a mesophilic enzyme can be enhanced by increasing its rigidity at the most flexible sites in its structure.
examples of b-factor guided mutagenesis to increase thermostability include a bacillus subtilis lipase   <cit>  and a pseudomonas fluorescens esterase   <cit> , where Δt <dig> corresponds to the change in temperature at which the enzyme loses half its activity after an hour of incubation.
using the crystal structure of the tregi catalytic domain , we identified the  <dig> amino acids with the highest b-factors.
these amino acids were grouped in seven sites  comprising one or more amino acids , and were chosen for site-saturation mutagenesis.
amino acids with high b-factors that were in close proximity to the n- or c-terminus, disulfide bridges, or n-glycosylation sites in the tregi crystal structure were not selected because they could interfere with disulfide-bond formation or lead to false positives due to compensation for non-glycosylation in a bacterial expression system.
a modification of the cell-free protein synthesis protocol developed in our laboratory  <cit>  was used to express tregi in an active, soluble form .
the stability of the cell-free expressed tregi is comparable to tregi expressed in e. coli  <cit> , but is considerably reduced compared to the native enzyme expressed in t. reesei  <cit> .
although cell-free protein synthesis yielded an inferior tregi enzyme, we proceeded to use cell-free expression as a tool to rapidly screen mutants of tregi, with the assumption that the effect of mutations imparting thermostability will be conserved upon expression in fungal hosts such as t. reesei.
the protocol for screening and selection of mutant tregi enzymes is shown in .
mutagenesis and screening of  <dig>  mutants at sites a-g generated ~ <dig> mutants with higher activity toward carboxymethyl cellulose  after pre-incubation at 50°c for 45 min than the wild-type tregi expressed using cell-free synthesis.
sequencing the  <dig> mutants that showed highest activities after the heat treatment revealed that all but six mutants had a mutation at site e .
alanine, arginine, serine, threonine, leucine, lysine, glutamic acid, glutamine, and methionine substitutions at this position all imparted improved thermostability.
in addition, we found mutations at site c  that gave rise to more stable variants of tregi.
amino acid changes at this site for these tregi mutants were d113s/d113t and d115l/d115g.
even though the d113l/d115g mutant had greater thermostability at 50°c, it was not pursued further because it had low specific activity relative to wild-type tregi expressed using cell-free synthesis.
since the single mutants  were not isolated during the screening process that covered >95% of all possible amino acid combinations at site c  <cit> , it is highly likely that mutations at both these sites need to be present simultaneously to afford increased stability.
activity measurements of round i variants with improved thermostability
the activities of some of these thermostable tregi mutants toward cmc, based on the amount of reducing sugar released before and after exposure to 50°c for various times, are summarized in figure  <dig>  combining mutations at g <dig> with mutations at sites d <dig> and d <dig> resulted in triple mutant enzymes  that were more active at 50°c than either of the engineered parents .
all the triple mutants showed higher specific activity at 50°c on cmc compared to the wild-type tregi expressed using cell-free synthesis at 50°c; mutants g230k/d113s/d115t and g230t/d113s/d115t exhibited ~ <dig> fold improvement in specific activity.figure  <dig>
activity of
t. reesei
endoglucanase i mutants at 50°c on cmc.
error bars represent one standard deviation.
tregi proteins were expressed using cell-free protein synthesis and were used without any purification.
the engineered tregi enzymes also showed higher activity on solid substrates: avicel and ionic-liquid pretreated avicel  .
all of the engineered enzymes showed ~2-fold higher activity for avicel hydrolysis  and ~5-fold improvement for il-avicel hydrolysis  at 50°c relative to the wild-type tregi expressed by cell-free synthesis.
many of these tregi mutants were active against both substrates at 55°c, conditions under which the wild-type tregi expressed by cell-free synthesis was essentially inactive.
characterization of trichoderma reesei endoglucanase i variants expressed in s. cerevisiae
considering the impact of proper folding and glycosylation on the stability, activity, and structure of proteins  <cit> , we sought to express tregi enzymes in a fungal host.
because recombinant expression of proteins in filamentous fungi is typically tedious, time-consuming, and not amenable to high-throughput, we used s. cerevisiae  <cit>  as an intermediate fungal expression host to evaluate the stability and activity of the engineered tregi triple mutants .
of all the triple mutants tested , only five of the tregi mutants were expressed in active and soluble forms .
the enzymes were secreted from yeast in a hyperglycosylated state  as observed previously  <cit> , and thus were treated with an endoglycosidase  that cleaves n-glycans, reducing the molecular weight to 54–56 kda , which nearly matches the molecular weight of the natively-expressed tregi enzyme   <cit> .
many of these tregi mutants exhibited higher hydrolytic activities on soluble  and solid substrates  compared to sc_tregi  at 50–65°c.
the mutant g230a/d113s/d115t sc_tregi  is >  <dig> -fold better than sc_tregi  at hydrolyzing avicel at 60°c and il-avicel at 65°c.
in addition, the activity of g230a/d113s/d115t sc_tregi at 60°c against avicel and il-avicel is similar to that of sc_tregi at 50°c on these substrates.
hence, b-factor guided engineering of tregi provided variants with hydrolytic activities on avicel and il-avicel at 60°c similar to the activity of sc_tregi toward these same substrates at 50°c.figure  <dig>
temperature activity profile of wild-type
t. reesei
endoglucanase i  and g230a/d113s/d115t mutant  on avicel , il-avicel , and mu-cellobiose  expressed in
s. cerevisiae
,
n. cra
ssa,
t. reesei
, and
s. cerevisiae
followed by n-terminal cyclization of the wild-type tregi with gutaminyl cyclase.
error bars represent one standard deviation.
assays were carried out with purified tregi proteins .
all of the engineered triple mutants expressed in s. cerevisiae exhibited higher apparent melting temperatures  than sc_tregi .
mutant g230a/d113s/d115t sc_tregi  showed a ~4°c increase in apparent melting temperature over sc_tregi.
no significant difference in half-life at 70°c was observed between sc_tregi and this mutant .
it is important to note that our initial efforts included expressing the tregi enzymes bearing a c-terminal his <dig> tag, which lowered their activity on avicel , but had little effect on their activity on il-avicel .
this result suggests abrogated binding to avicel of tregi enzymes bearing a c-terminal his <dig> tag.table  <dig>
biochemical characterization of tregi expressed in different hosts
expression host
enzyme
t
m
t
1/2
50°c
60°c
65°c
70°c
s. cerevisiae
s. cerevisiae
s. cerevisiae
n. crassa
n. crassa
t. reesei
t. reesei
tm is the apparent melting temperature for the enzyme, and t1/ <dig> is half-life in hours.
- = not determined.
errors are reported in parentheses and represent one standard deviation for measurements.
apparent melting temperature data for sc_tregi  and tr_tregi are shown in .
apparent melting temperature data are not shown for other tregi enzymes.
half-life measurements for t. reesei- and n. crassa-expressed enzymes are shown in , respectively.
half-life measurements for s. cerevisiae-expressed tregi enzymes are not shown.
characterization of trichoderma reesei endoglucanase i variants expressed in the filamentous fungi n. crassa and t. reesei
sc_tregi showed a much lower apparent melting temperature  and temperature optimum  compared to the native tregi   <cit> , which could be due to non-optimal folding and/or aberrant glycosylation of tregi when expressed in s. cerevisiae.
in order to investigate these possibilities, and to compare the properties  of the engineered tregi enzymes to the native tregi, we pursued tregi expression in t. reesei.
thus the wild-type tregi and the best mutant  were expressed in t. reesei qm <dig>  in addition, our interest in evaluating alternative filamentous fungal systems for cellulase expression prompted us to express tregi enzymes  in the filamentous fungus n. crassa, which is closely related to t. reesei.
tregi enzymes  expressed in t. reesei and n. crassa were found to have identical temperature optima on mu-cellobiose , and apparent melting temperatures , which in the case of wild-type enzyme  is also identical to the values reported previously for the native tregi.
furthermore, little difference was observed between their activities on solid substrates avicel  and il-avicel , thus validating n. crassa as a suitable surrogate of t. reesei for tregi production.
g230a/d113s/d115t tr_tregi showed similar or higher activity on solid substrates  than tr_tregi when assayed at 50–70°c ; specifically, a ~2-fold increase in activity was observed at 65°c.
in addition, g230a/d113s/d115t tr_tregi showed similar hydrolytic activity on solid substrates at 60°c as the tr_tregi at 50°c.
as expected, similar improvements were also observed for n. crassa-expressed tregi variants.
hence, b-factor guided engineering of tregi provided variants with hydrolytic activities on avicel and il-avicel at 60°c similar to the activity of tr_tregi toward these same substrates at 50°c.
g230a/d113s/d115t tr_tregi , g230a/d113s/d115t nc_tregi , and g230e/d113s/d115t nc_tregi  showed a 3°c increase in apparent melting temperature  compared to tr_tregi or nc_tregi .
in order to assess whether the thermostable mutants also show improved half-lives over the wild-type tregi, half-lives of the thermostable mutant g230a/d113s/d115t tregi and the wild-type tregi  were measured at 50°c, 60°c, 65°c and 70°c .
although the half-life for tregi enzymes at 50°c was too long  to distinguish any gains made at this temperature , the g230a/d113s/d115t tregi variant showed a >2-fold increase in half-life at 60°c compared to the corresponding recombinant  wild-type tregi .
the improved half-life  of the g230a/d113s/d115t tr_tregi variant at 60°c results in improved or comparable performance of this mutant in biomass saccharification carried out at 60°c compared to the performance of tr_tregi at 50°c .
we also expect that the increased stability and half-life of the g230a/d113s/d115t tr_tregi at temperatures such as 60°c will correlate with improved half-life at lower temperatures  compared to tr_tregi.
the half-lives of both these enzymes were significantly shorter   at higher temperatures .
it is surprising that little or no difference was found between the half-lives of g230a/d11s/d115t tregi and the wild-type enzyme  at either 65°c or 70°c , which may reflect a shift in the inactivation mechanism with temperature mediated by the different enzyme structures and/or glycosylation patterns as observed previously with other β-sandwich or multi-domain proteins  <cit> .
impact of n-terminal pyroglutamate on activity and stability of t. reesei endoglucanase i
tregi and g230a/d113s/d115t tregi expressed in both t. reesei and n. crassa were found to have much higher tm values  than their s. cerevisiae-expressed counterparts .
in addition, they were up to  <dig> -fold more active on avicel  at 50–65°c and up to  <dig> -fold more active on il-avicel  at 50–65°c compared to their counterparts expressed in s. cerevisiae.
given the similarity of molecular weights of the tregi glycoforms expressed in s. cerevisiae and n. crassa , the lower stability and activity of s. cerevisiae tregi could potentially be due to non-optimal folding  and/or incorrect disulfide formation upon expression in s. cerevisiae.
in this connection, we investigated the role of n-terminal pyroglutamate observed in the t. reesei egi crystal structure.
the available crystal structure of t. reesei egi   <cit>  reveals a buried n-terminal pyroglutamate in the natively-expressed enzyme resulting from the cyclization of the n-terminal glutamine.
we hypothesized that the lack of this n-terminal glutamine cyclization  in s. cerevisiae-expressed enzyme would most likely result in non-optimal protein structure and contribute to the observed decreased stability and activity compared to the t. reesei-expressed enzyme.
glutaminyl cyclases  are known to catalyze the irreversible lactamization of a n-terminal glutamine to a hydrophobic pyroglutamate in many proteins and peptides  <cit> .
using a human glutaminyl cyclase enzyme  <cit>  we cyclized the n-terminal glutamine residue of sc_tregi to a pyroglutamate, as evidenced by a blocked n-terminal sequence, typical of proteins/peptides with n-terminal pyroglutamate  <cit> .
treating sc_tregi with the human glutaminyl cyclase substantially increased its activity on solid substrates compared to the corresponding enzyme without glutaminyl-cyclase treatment , and shifted its temperature optimum from 50°c to 60°c on soluble substrates .
the activity of the glutaminyl-cyclase treated sc_tregi on avicel and il-avicel is similar if not identical to its counterpart expressed in t. reesei or n. crassa .
the increased stability of sc_tregi from n-terminal glutamine cyclization was evaluated by measurements of apparent melting temperatures .
glutaminyl-cyclase treated sc_tregi  showed an ~8°c increase in apparent melting temperature compared to the corresponding uncyclized version .
the apparent melting temperature of sc_tregi upon cyclization  was identical to that of tr_egi .
the large increase in tm upon n-terminal glutamine cyclization indicates that the lower stability of the s. cerevisiae enzymes is due to the uncyclized n-terminal glutamine.
in addition, it is worth noting that the differences in glycosylation between sc_tregi and nc_tregi  are irrelevant to the activity and stability differences between these enzymes in comparison to the cyclization of the n-terminal glutamine.
recently our group also demonstrated a similar increase in stability and activity upon cyclization of n-terminal glutamine to pyroglutamate in s. cerevisiae-expressed talaromyces emersonii cel7a  <cit> .
the ubiquitous presence of the n-terminal pyroglutamate in gh <dig> enzymes , along with its role in imparting high stability/activity for these enzymes, highlights the importance of n-terminal glutamine cyclization for achieving high-efficiency cellulases.
impact of mutagenesis and expression host on activity and stability of t. reesei endoglucanase i
combinations of tregi mutations at amino acids g <dig>  and d113/d <dig> identified using the b-factor method resulted in improved tregi enzymes irrespective of the expression host for the enzyme.
in particular, tregi mutant g230a/d113s/d115t showed improved activity on solid substrates as well as improved thermostability for each expression host when compared to wild-type tregi enzyme expressed in the same host.
these mutations may rigidify the mobile portions of the tregi enzyme, and thus enhance its stability.
in particular, it is likely that the mutation at g <dig>  which is located on a solvent exposed loop, imparts improved stability by reducing the entropy  of the  unfolded tregi, a well-known mechanism for improving protein stability by replacing conformationally flexible residues such as glycine  with more rigid residues such as alanine  <cit> .
removal of unfavorable electrostatic interactions involving negative charges on d113/d <dig> in the wild-type enzyme could also lead to increased stability  <cit> .
a small difference in mass was observed between nc_tregi and g230a/d113s/d115t nc_tregi  and between sc_tregi and g230a/d113s/d115t sc_tregi , after accounting for the mass difference resulting from the g230a/d113s/d115t mutation.
this shows that there is little or no difference in the extent of glycosylation  between the wild-type tregi and the mutant g230a/d113s/d115t tregi when they are expressed in the same fungal host.
thus, the improved stability of g230a/d113s/d115t tregi compared to wild-type tregi when expressed in either n. crassa or s. cerevisiae is due to the g230a/d113s/d115t mutation and not due to changes in glycosylation.
the specific activity of tregi enzymes  on solid substrates was dramatically altered upon expression in different hosts  with t. reesei or n. crassa producing the most active enzymes on avicel and il-avicel .
glycosylation of tregi enzymes renders them more thermostable compared to their cell-free expressed counterparts, which is consistent with previous reports showing the importance of glycosylation in protein thermostability  <cit> .
the cyclization of n-terminal glutamine to a pyroglutamate in s. cerevisiae expressed tregi resulted in a tregi enzyme with near identical properties to the natively expressed enzyme.
CONCLUSIONS
using b-factor guided mutagenesis, t.reesei endoglucanase i variants were developed that were up to 2-fold more active on solid cellulosic substrates at 65°c than the recombinant wild-type enzyme expressed in the same hosts, and exhibited the same activity at 60°c as the recombinant wild-type enzyme at 50°c.
the most stable tregi mutant g230a/d113s/d115t showed a ~3°c increase in tm, as well as more than 2-fold improved half-life at 60°c over the native tregi.
amino acid changes at g <dig> coupled with the d113s/d115t double mutation in tregi gave rise to variants with improved thermostability and activity at 50–70°c, irrespective of their expression host.
however, the stability and activity of the tregi enzymes varied significantly when expressed using e. coli cell extract, s. cerevisiae, n. crassa, or t. reesei.
tregi variants expressed in n. crassa or t. reesei yielded the most active and stable tregi enzymes and s. cerevisiae yielded inferior tregi enzymes.
cyclization of n-terminal glutamine to a pyroglutamate in s. cerevisiae-expressed tregi improved its properties to closely match those of the native enzyme, highlighting the importance of this ubiquitous modification in cel7b enzymes.
in this study we show that a non-glycosylating host can be used to screen and discover thermostability imparting mutations in fungal enzymes that result in improved enzymes irrespective of the protein production host.
the choice of host for protein engineering of cellulases may not be as critical as the protein production host, which plays a major role in determining the properties of the expressed enzymes.

 efficient saccharification of cellulosic biomass to fermentable sugars is necessary for economical production of second generation biofuels. several enzymes act in concert to hydrolyze cellulose to glucose. the industrially employed filamentous fungus trichoderma reesei has been evolved for cellulase production and is capable of secreting large quantities of cellulolytic enzymes at a relatively low cost. these include two exoglucanases, cel7a and cel6a, which represent 50% and 20% of the total cellulase content, respectively, four endoglucanases, cel7b, cel5a, cel12a, and cel45a that represent approximately 15%, 10%, 1%, and <1% of the total cellulase content, respectively, and several lytic polysaccharide monooxygenases. lignocellulose hydrolysis at relatively high temperatures may offer potential advantages, including reduced solution viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature pretreatments, and potentially faster rates of hydrolysis. the short half-lives of t. reesei cellulases at temperatures above 50°c, together with very low expression levels of thermophilic cellulases, motivates the development of thermostable t. reesei cellulases that can hydrolyze lignocellulose efficiently at temperatures beyond 50°c. t. reesei endoglucanase i is known to randomly cleave internal cellulosic bonds, thereby creating shorter cellulosic chains. recent studies on optimizing the components of cellulase systems have underscored the importance of having tregi comprise a high fraction of the cellulase mixture in order to efficiently hydrolyze pretreated lignocellulosic biomass. tregi has a bimodular structure with a 375-amino-acid catalytic domain attached to a 35-aa carbohydrate-binding module via a 26-aa linker. tregi has disulfide bonds, 6 n-glycosylation sites in the cd, and a linker region rich in serine and threonine that are potential o-glycosylation sites. it was selected for engineering enhanced thermostability because of its importance in cellulose hydrolysis and the availability of its crystal structure.figure crystal structure of endoglucanase i from trichoderma reesei. disulfide bonds are shown in blue. n-glycosylation sites are shown in magenta. mutagenesis sites are shown in red and are labeled as follows: a, b, c, d, e, f, and g. mutations at site c and site e resulted in improved tregi enzyme variants. pdb code 1eg although protein engineering has been used to improve the thermostability of numerous cellulases, there are no reports on improving the thermostability or increasing the optimal temperature of tregi. in this study, we employed a b-factor guided approach to engineer thermostable variants of tregi using e. coli cell-free protein synthesis due its ease and throughput. many thermostable mutants of tregi identified using cell-free protein synthesis were subsequently expressed in the fungal hosts s. cerevisiae, n. crassa, and t. reesei to better mimic the properties of the native tregi enzyme in terms of folding and glycosylation. tregi variants, particularly g230a/d113s/d115t, were more stable compared to their recombinant wild-type versions when both the variant and wild-type were expressed in s. cerevisiae, n. crassa, or t. reesei, indicating that the positive influence of these mutations on stability translates across different expression hosts. however, the s. cerevisiae-expressed enzymes were inferior in terms of activity and stability compared to the same enzymes expressed in t. reesei or n. crassa, which we attribute to the lack of cyclization of the n-terminal glutamine of treg expressed in our s. cerevisiae system.figure engineering thermostable endoglucanase i from trichoderma reesei.  b-factor guided mutagenesis our efforts to improve the thermostability of tregi entailed a b-factor guided approach. the b-factor is a measure of the average deviation of a given residue from its mean position in the crystal structure. residues having the highest average b-factors correspond to the most flexible sites in the protein and provide targets for mutagenesis to improve thermostability. this strategy is based on the premise that the thermostability of a mesophilic enzyme can be enhanced by increasing its rigidity at the most flexible sites in its structure. examples of b-factor guided mutagenesis to increase thermostability include a bacillus subtilis lipase and a pseudomonas fluorescens esterase, where Δt corresponds to the change in temperature at which the enzyme loses half its activity after an hour of incubation. using the crystal structure of the tregi catalytic domain, we identified the amino acids with the highest b-factors. these amino acids were grouped in seven sites comprising one or more amino acids, and were chosen for site-saturation mutagenesis. amino acids with high b-factors that were in close proximity to the n- or c-terminus, disulfide bridges, or n-glycosylation sites in the tregi crystal structure were not selected because they could interfere with disulfide-bond formation or lead to false positives due to compensation for non-glycosylation in a bacterial expression system. a modification of the cell-free protein synthesis protocol developed in our laboratory was used to express tregi in an active, soluble form. the stability of the cell-free expressed tregi is comparable to tregi expressed in e. coli, but is considerably reduced compared to the native enzyme expressed in t. reesei. although cell-free protein synthesis yielded an inferior tregi enzyme, we proceeded to use cell-free expression as a tool to rapidly screen mutants of tregi, with the assumption that the effect of mutations imparting thermostability will be conserved upon expression in fungal hosts such as t. reesei. the protocol for screening and selection of mutant tregi enzymes is shown in. mutagenesis and screening of mutants at sites a-g generated ~ mutants with higher activity toward carboxymethyl cellulose after pre-incubation at 50°c for 45 min than the wild-type tregi expressed using cell-free synthesis. sequencing the mutants that showed highest activities after the heat treatment revealed that all but six mutants had a mutation at site e. alanine, arginine, serine, threonine, leucine, lysine, glutamic acid, glutamine, and methionine substitutions at this position all imparted improved thermostability. in addition, we found mutations at site c that gave rise to more stable variants of tregi. amino acid changes at this site for these tregi mutants were d113s/d113t and d115l/d115g. even though the d113l/d115g mutant had greater thermostability at 50°c, it was not pursued further because it had low specific activity relative to wild-type tregi expressed using cell-free synthesis. since the single mutants were not isolated during the screening process that covered >95% of all possible amino acid combinations at site c, it is highly likely that mutations at both these sites need to be present simultaneously to afford increased stability. activity measurements of round i variants with improved thermostability the activities of some of these thermostable tregi mutants toward cmc, based on the amount of reducing sugar released before and after exposure to 50°c for various times, are summarized in figure  combining mutations at g with mutations at sites d and d resulted in triple mutant enzymes that were more active at 50°c than either of the engineered parents. all the triple mutants showed higher specific activity at 50°c on cmc compared to the wild-type tregi expressed using cell-free synthesis at 50°c; mutants g230k/d113s/d115t and g230t/d113s/d115t exhibited ~ fold improvement in specific activity.figure activity of t. reesei endoglucanase i mutants at 50°c on cmc. error bars represent one standard deviation. tregi proteins were expressed using cell-free protein synthesis and were used without any purification. the engineered tregi enzymes also showed higher activity on solid substrates: avicel and ionic-liquid pretreated avicel. all of the engineered enzymes showed ~2-fold higher activity for avicel hydrolysis and ~5-fold improvement for il-avicel hydrolysis at 50°c relative to the wild-type tregi expressed by cell-free synthesis. many of these tregi mutants were active against both substrates at 55°c, conditions under which the wild-type tregi expressed by cell-free synthesis was essentially inactive. characterization of trichoderma reesei endoglucanase i variants expressed in s. cerevisiae considering the impact of proper folding and glycosylation on the stability, activity, and structure of proteins, we sought to express tregi enzymes in a fungal host. because recombinant expression of proteins in filamentous fungi is typically tedious, time-consuming, and not amenable to high-throughput, we used s. cerevisiae as an intermediate fungal expression host to evaluate the stability and activity of the engineered tregi triple mutants. of all the triple mutants tested, only five of the tregi mutants were expressed in active and soluble forms. the enzymes were secreted from yeast in a hyperglycosylated state as observed previously, and thus were treated with an endoglycosidase that cleaves n-glycans, reducing the molecular weight to 54–56 kda, which nearly matches the molecular weight of the natively-expressed tregi enzyme. many of these tregi mutants exhibited higher hydrolytic activities on soluble and solid substrates compared to sc_tregi at 50–65°c. the mutant g230a/d113s/d115t sc_tregi is >  -fold better than sc_tregi at hydrolyzing avicel at 60°c and il-avicel at 65°c. in addition, the activity of g230a/d113s/d115t sc_tregi at 60°c against avicel and il-avicel is similar to that of sc_tregi at 50°c on these substrates. hence, b-factor guided engineering of tregi provided variants with hydrolytic activities on avicel and il-avicel at 60°c similar to the activity of sc_tregi toward these same substrates at 50°c.figure temperature activity profile of wild-type t. reesei endoglucanase i and g230a/d113s/d115t mutant on avicel, il-avicel, and mu-cellobiose expressed in s. cerevisiae, n. cra ssa, t. reesei, and s. cerevisiae followed by n-terminal cyclization of the wild-type tregi with gutaminyl cyclase. error bars represent one standard deviation. assays were carried out with purified tregi proteins. all of the engineered triple mutants expressed in s. cerevisiae exhibited higher apparent melting temperatures than sc_tregi. mutant g230a/d113s/d115t sc_tregi showed a ~4°c increase in apparent melting temperature over sc_tregi. no significant difference in half-life at 70°c was observed between sc_tregi and this mutant. it is important to note that our initial efforts included expressing the tregi enzymes bearing a c-terminal his tag, which lowered their activity on avicel, but had little effect on their activity on il-avicel. this result suggests abrogated binding to avicel of tregi enzymes bearing a c-terminal his tag.table biochemical characterization of tregi expressed in different hosts expression host enzyme t m t 1/2 50°c 60°c 65°c 70°c s. cerevisiae s. cerevisiae s. cerevisiae n. crassa n. crassa t. reesei t. reesei tm is the apparent melting temperature for the enzyme, and t1/ is half-life in hours. - = not determined. errors are reported in parentheses and represent one standard deviation for measurements. apparent melting temperature data for sc_tregi and tr_tregi are shown in. apparent melting temperature data are not shown for other tregi enzymes. half-life measurements for t. reesei- and n. crassa-expressed enzymes are shown in, respectively. half-life measurements for s. cerevisiae-expressed tregi enzymes are not shown. characterization of trichoderma reesei endoglucanase i variants expressed in the filamentous fungi n. crassa and t. reesei sc_tregi showed a much lower apparent melting temperature and temperature optimum compared to the native tregi, which could be due to non-optimal folding and/or aberrant glycosylation of tregi when expressed in s. cerevisiae. in order to investigate these possibilities, and to compare the properties of the engineered tregi enzymes to the native tregi, we pursued tregi expression in t. reesei. thus the wild-type tregi and the best mutant were expressed in t. reesei qm in addition, our interest in evaluating alternative filamentous fungal systems for cellulase expression prompted us to express tregi enzymes in the filamentous fungus n. crassa, which is closely related to t. reesei. tregi enzymes expressed in t. reesei and n. crassa were found to have identical temperature optima on mu-cellobiose, and apparent melting temperatures, which in the case of wild-type enzyme is also identical to the values reported previously for the native tregi. furthermore, little difference was observed between their activities on solid substrates avicel and il-avicel, thus validating n. crassa as a suitable surrogate of t. reesei for tregi production. g230a/d113s/d115t tr_tregi showed similar or higher activity on solid substrates than tr_tregi when assayed at 50–70°c; specifically, a ~2-fold increase in activity was observed at 65°c. in addition, g230a/d113s/d115t tr_tregi showed similar hydrolytic activity on solid substrates at 60°c as the tr_tregi at 50°c. as expected, similar improvements were also observed for n. crassa-expressed tregi variants. hence, b-factor guided engineering of tregi provided variants with hydrolytic activities on avicel and il-avicel at 60°c similar to the activity of tr_tregi toward these same substrates at 50°c. g230a/d113s/d115t tr_tregi, g230a/d113s/d115t nc_tregi, and g230e/d113s/d115t nc_tregi showed a 3°c increase in apparent melting temperature compared to tr_tregi or nc_tregi. in order to assess whether the thermostable mutants also show improved half-lives over the wild-type tregi, half-lives of the thermostable mutant g230a/d113s/d115t tregi and the wild-type tregi were measured at 50°c, 60°c, 65°c and 70°c. although the half-life for tregi enzymes at 50°c was too long to distinguish any gains made at this temperature, the g230a/d113s/d115t tregi variant showed a >2-fold increase in half-life at 60°c compared to the corresponding recombinant wild-type tregi. the improved half-life of the g230a/d113s/d115t tr_tregi variant at 60°c results in improved or comparable performance of this mutant in biomass saccharification carried out at 60°c compared to the performance of tr_tregi at 50°c. we also expect that the increased stability and half-life of the g230a/d113s/d115t tr_tregi at temperatures such as 60°c will correlate with improved half-life at lower temperatures compared to tr_tregi. the half-lives of both these enzymes were significantly shorter at higher temperatures. it is surprising that little or no difference was found between the half-lives of g230a/d11s/d115t tregi and the wild-type enzyme at either 65°c or 70°c, which may reflect a shift in the inactivation mechanism with temperature mediated by the different enzyme structures and/or glycosylation patterns as observed previously with other β-sandwich or multi-domain proteins. impact of n-terminal pyroglutamate on activity and stability of t. reesei endoglucanase i tregi and g230a/d113s/d115t tregi expressed in both t. reesei and n. crassa were found to have much higher tm values than their s. cerevisiae-expressed counterparts. in addition, they were up to -fold more active on avicel at 50–65°c and up to -fold more active on il-avicel at 50–65°c compared to their counterparts expressed in s. cerevisiae. given the similarity of molecular weights of the tregi glycoforms expressed in s. cerevisiae and n. crassa, the lower stability and activity of s. cerevisiae tregi could potentially be due to non-optimal folding and/or incorrect disulfide formation upon expression in s. cerevisiae. in this connection, we investigated the role of n-terminal pyroglutamate observed in the t. reesei egi crystal structure. the available crystal structure of t. reesei egi reveals a buried n-terminal pyroglutamate in the natively-expressed enzyme resulting from the cyclization of the n-terminal glutamine. we hypothesized that the lack of this n-terminal glutamine cyclization in s. cerevisiae-expressed enzyme would most likely result in non-optimal protein structure and contribute to the observed decreased stability and activity compared to the t. reesei-expressed enzyme. glutaminyl cyclases are known to catalyze the irreversible lactamization of a n-terminal glutamine to a hydrophobic pyroglutamate in many proteins and peptides. using a human glutaminyl cyclase enzyme we cyclized the n-terminal glutamine residue of sc_tregi to a pyroglutamate, as evidenced by a blocked n-terminal sequence, typical of proteins/peptides with n-terminal pyroglutamate. treating sc_tregi with the human glutaminyl cyclase substantially increased its activity on solid substrates compared to the corresponding enzyme without glutaminyl-cyclase treatment, and shifted its temperature optimum from 50°c to 60°c on soluble substrates. the activity of the glutaminyl-cyclase treated sc_tregi on avicel and il-avicel is similar if not identical to its counterpart expressed in t. reesei or n. crassa. the increased stability of sc_tregi from n-terminal glutamine cyclization was evaluated by measurements of apparent melting temperatures. glutaminyl-cyclase treated sc_tregi showed an ~8°c increase in apparent melting temperature compared to the corresponding uncyclized version. the apparent melting temperature of sc_tregi upon cyclization was identical to that of tr_egi. the large increase in tm upon n-terminal glutamine cyclization indicates that the lower stability of the s. cerevisiae enzymes is due to the uncyclized n-terminal glutamine. in addition, it is worth noting that the differences in glycosylation between sc_tregi and nc_tregi are irrelevant to the activity and stability differences between these enzymes in comparison to the cyclization of the n-terminal glutamine. recently our group also demonstrated a similar increase in stability and activity upon cyclization of n-terminal glutamine to pyroglutamate in s. cerevisiae-expressed talaromyces emersonii cel7a. the ubiquitous presence of the n-terminal pyroglutamate in gh enzymes, along with its role in imparting high stability/activity for these enzymes, highlights the importance of n-terminal glutamine cyclization for achieving high-efficiency cellulases. impact of mutagenesis and expression host on activity and stability of t. reesei endoglucanase i combinations of tregi mutations at amino acids g and d113/d identified using the b-factor method resulted in improved tregi enzymes irrespective of the expression host for the enzyme. in particular, tregi mutant g230a/d113s/d115t showed improved activity on solid substrates as well as improved thermostability for each expression host when compared to wild-type tregi enzyme expressed in the same host. these mutations may rigidify the mobile portions of the tregi enzyme, and thus enhance its stability. in particular, it is likely that the mutation at g which is located on a solvent exposed loop, imparts improved stability by reducing the entropy of the unfolded tregi, a well-known mechanism for improving protein stability by replacing conformationally flexible residues such as glycine with more rigid residues such as alanine. removal of unfavorable electrostatic interactions involving negative charges on d113/d in the wild-type enzyme could also lead to increased stability. a small difference in mass was observed between nc_tregi and g230a/d113s/d115t nc_tregi and between sc_tregi and g230a/d113s/d115t sc_tregi, after accounting for the mass difference resulting from the g230a/d113s/d115t mutation. this shows that there is little or no difference in the extent of glycosylation between the wild-type tregi and the mutant g230a/d113s/d115t tregi when they are expressed in the same fungal host. thus, the improved stability of g230a/d113s/d115t tregi compared to wild-type tregi when expressed in either n. crassa or s. cerevisiae is due to the g230a/d113s/d115t mutation and not due to changes in glycosylation. the specific activity of tregi enzymes on solid substrates was dramatically altered upon expression in different hosts with t. reesei or n. crassa producing the most active enzymes on avicel and il-avicel. glycosylation of tregi enzymes renders them more thermostable compared to their cell-free expressed counterparts, which is consistent with previous reports showing the importance of glycosylation in protein thermostability. the cyclization of n-terminal glutamine to a pyroglutamate in s. cerevisiae expressed tregi resulted in a tregi enzyme with near identical properties to the natively expressed enzyme.  using b-factor guided mutagenesis, t.reesei endoglucanase i variants were developed that were up to 2-fold more active on solid cellulosic substrates at 65°c than the recombinant wild-type enzyme expressed in the same hosts, and exhibited the same activity at 60°c as the recombinant wild-type enzyme at 50°c. the most stable tregi mutant g230a/d113s/d115t showed a ~3°c increase in tm, as well as more than 2-fold improved half-life at 60°c over the native tregi. amino acid changes at g coupled with the d113s/d115t double mutation in tregi gave rise to variants with improved thermostability and activity at 50–70°c, irrespective of their expression host. however, the stability and activity of the tregi enzymes varied significantly when expressed using e. coli cell extract, s. cerevisiae, n. crassa, or t. reesei. tregi variants expressed in n. crassa or t. reesei yielded the most active and stable tregi enzymes and s. cerevisiae yielded inferior tregi enzymes. cyclization of n-terminal glutamine to a pyroglutamate in s. cerevisiae-expressed tregi improved its properties to closely match those of the native enzyme, highlighting the importance of this ubiquitous modification in cel7b enzymes. in this study we show that a non-glycosylating host can be used to screen and discover thermostability imparting mutations in fungal enzymes that result in improved enzymes irrespective of the protein production host. the choice of host for protein engineering of cellulases may not be as critical as the protein production host, which plays a major role in determining the properties of the expressed enzymes. 
trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. these potential advantages motivate efforts to engineer t. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°c. lignocellulose hydrolysis at temperatures above the optimum temperature of t. reesei cellulases could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. trichoderma reeseiendoglucanase ib-factorcellulasethermostabilitycell-free synthesissaccharomyces cerevisiaeneurospora crassapyroglutamateglutaminyl cylcaseissue-copyright-statement© the author 2015 furthermore, g230a/d113s/d115t tr_tregi showed 2-fold improved activity compared to tr_tregi at 65°c on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°c as tr_tregi was at 50°c. structure-guided evolution of t. reesei egi was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. the activities and stabilities of the recombinant tregi enzymes followed similar trends but differed significantly in magnitude depending on the expression host. production of tregi enzymes in four hosts highlighted the impact of the expression host and the role of n-terminal pyroglutamate formation on the activity and stability of tregi enzymes. n-terminal pyroglutamate formation in tregi expressed in s. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the t. reesei or n. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in gh enzymes. a b-factor guided approach for improving thermostability was used to engineer variants of endoglucanase i from t. reesei that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type tregi at temperatures ranging from 50–70°c. when expressed in t. reesei, tregi variant g230a/d113s/d115t had a higher apparent melting temperature and improved half-life at 60°c than the recombinant wild-type tregi. compared to n.crassa-expressed tregi, s. cerevisiae-expressed tregi showed inferior activity and stability, which was attributed to the lack of cyclization of the n-terminal glutamine in sc_tregi and not to differences in glycosylation.
 trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. lignocellulose hydrolysis at temperatures above the optimum temperature of t. reesei cellulases could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. these potential advantages motivate efforts to engineer t. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°c.  a b-factor guided approach for improving thermostability was used to engineer variants of endoglucanase i from t. reesei that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type tregi at temperatures ranging from 50–70°c. when expressed in t. reesei, tregi variant g230a/d113s/d115t had a higher apparent melting temperature and improved half-life at 60°c than the recombinant wild-type tregi. furthermore, g230a/d113s/d115t tr_tregi showed 2-fold improved activity compared to tr_tregi at 65°c on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°c as tr_tregi was at 50°c. the activities and stabilities of the recombinant tregi enzymes followed similar trends but differed significantly in magnitude depending on the expression host. compared to n.crassa-expressed tregi, s. cerevisiae-expressed tregi showed inferior activity and stability, which was attributed to the lack of cyclization of the n-terminal glutamine in sc_tregi and not to differences in glycosylation. n-terminal pyroglutamate formation in tregi expressed in s. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the t. reesei or n. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in gh enzymes.  structure-guided evolution of t. reesei egi was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. production of tregi enzymes in four hosts highlighted the impact of the expression host and the role of n-terminal pyroglutamate formation on the activity and stability of tregi enzymes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords trichoderma reeseiendoglucanase ib-factorcellulasethermostabilitycell-free synthesissaccharomyces cerevisiaeneurospora crassapyroglutamateglutaminyl cylcaseissue-copyright-statement© the author 2015 