Strains media and chemicals
The Escherichia coli Top 10 cells (Invitrogen) were grown in Luria–Bertani (LB) medium (10 g L-1 bactotryptone, 10 g L-1 NaCl, 5 g L-1 yeast extract), using 50 μg ml-1 ampicillin.
The heterologous expression was performed using as host the strain E. coli BL21 CodonPlus (DE3) RP (Novagen Ltd). The error-prone (ep) PCR (Polymerase Chain Reaction) mutagenesis was performed by the Gene Morph II Random Mutagenesis Kit (Agilent, La Jolla, California, USA). Restriction enzymes and the buffers were provided by New England Biolabs (Beverly, MA, USA). 4-Nitrophenyl β-D-glucopyranoside was obtained from Alfa Aesar (Kandel, Germany). Saccharification experiments were performed using the enzymatic commercial mixture used in Pennacchio et al. 2018 [14] and the enzymes were provided by Genencor (Rochester, New York, USA).
The enzymatic mixture MetZyme® SUNO TM BOOSTER 144 was supplied by MetGen Oy (15 U ml-1).
Gene sequencing
The new GH5 cellulases encoding genes from Streptomyces argenteolus AE58P, named Cel1 and Cel2, that in proteomics analyses showed peptide sequences matching with proteins with id 503810539 and 503918330, were synthetized by Polymerase chain reaction (PCR ) on S. argenteolus AE58P’s genomic DNA, using degenerate oligonucleotides as primers. Primers sequences were designed on the basis of the sequences of peptides identified in our previous work [25].
The degenerate oligonucleotides are reported below. 1 FW/1 REV for the amplification of the central region: (gtc ggc aty gtg ggc ctg ggc/gcc ytc sgg rta rtc ytc gaa aaa and gcg acc acc tty tgy ctg gay gay gtg/ rta sga ctg gtt cag sgg gtt for Cel1 and Cel2, respectively); 2 FW/2 REV for the amplification of the 5’ region (atg aay gay gay gtn ccn ggn ggt ctg gac/ ccc cac agy gar tat ccs gay ggc and gct ngc gca sgc scg gcc gtt cag/ cac rtc rtc cag rca raa ggt ggt cgc for Cel1 and Cel2, respectively); 3 FW/3 REV for the amplification of 3’ terminal region (ccc cac agy gar tat ccs gay ggc/ sgc ytt scg cca ytc rcc ccg ggt and aac ccs ctg aac cag tcs tay/ gct ngc gca sgc scg gcc gtt cag for Cel1 and Cel2 respectively).
The sequencing of the DNA fragments was performed by Eurofins Genomics Service (Milan, Italy).
Sequences of the genes coding for the new GH5 cellulases Cel1 and Cel2 from S. argenteolus AE58P, are available in the EMBL Data Library (MN845760 and MN845759 respectively).
Cloning and recombinant expression
Cel1 and Cel2 encoding genes were cloned into pBAD and pET22b (Novagen, Inc.) expression vectors, obtaining Cel1- pBAD, Cel2- pBAD, Cel1- pET22b, Cel2- pET22b and they were expressed in E. coli BL21- (DE3) RP (Novargen Ltd).
The cells were grown at 37°C in 0.05 L of LB (50 µg mL-1 ampicillin), until optical density (O.D.) 0.7. Different concentrations of Isopropil-β-D-1-tiogalattopiranoside, IPTG (0.01, 0.1 and 0.5 mM) and arabinose (0.002% and 0.02%), different incubation times (3 hours and overnight) and temperatures (20°C and 37°C) were tested for proteins expression.
The mutant cells were cultured at 37°C in 0.05 L of LB medium with 50 µg mL-1 ampicillin until optical density (O.D.) 0.7 and 0.02% arabinose was used as inducer of the protein expression over night at 20°C.
The cells were removed by centrifugation of the liquid cultures for 20 minutes at 4°C (6,000 rpm). The supernatants of the samples were used to measure the cellulase activity towards 4-Nitrophenyl β-D glucopyranoside (Sigma Aldrich, Milan, Italy).
The results correspond to the average of three experiments, everyone in triplicates.
Determination of optimal temperature and pH
The optimal temperature of the recombinant enzymes was evaluated using 4-Nitrophenyl β-D-glucopyranoside dissolved in 0.05 M Na citrate buffer at pH 5 as substrate, and performing the incubation for 10 minutes at 37°C, 45°C, 50°C, 55°C and 60°C.
The determination of optimal pH of cellulase activity was performed using the substrate 4-Nitrophenyl β-D-glucopyranoside, dissolved in 0.05 M citrate phosphate buffers, with pH values between 4.0 and 6.0 and performing the incubation (10 min) at 45°C and 55°C for Cel1 and Cel2, respectively.
To determine the optimal temperature and optimal pH of cellulase activity for the best mutants, 4-Nitrophenyl β-D-glucopyranoside was dissolved in 0.05 M phosphate buffer at different pH (4-6), performing the incubation for 10 minutes at 37°C, 45°C, 50, 55°C and 60°C.
The results correspond to the average of three experiments, everyone in triplicates.
Effect of temperature and pH on the enzyme activity
To determine the thermoresistance of the recombinant enzymes, 4-Nitrophenyl β-D-glucopyranoside was dissolved in 0.1 M Na acetate buffer at pH 5, performing the assay at 37°C, 45°C, 50°C, 55°C and 60°C for 72 h.
The determination of pH resistance of cellulase activity was performed assaying the enzymatic activity with the substrate 4-Nitrophenyl β-D-glucopyranoside, dissolved in 50 mM citrate phosphate buffers, with pH values between 3.0 and 7.0, performing the assay by incubation for 10 min at 45°C and 55°C for Cel1 and Cel2, respectively, and monitoring the maintenance of enzymatic activity for 72 hours.
To determine the thermoresistance of the best clones, 4-Nitrophenyl β-D-glucopyranoside was dissolved in 0.1 M Na- acetate buffer at the optimal pH of wild type Cel2 (pH 5), and at the optimal pH of each clone and the incubation was performed at the optimal temperature of each clone and of wild type Cel2 (55°C), for 72 hours, collecting the samples at different times.
The results correspond to the average of three experiments, everyone in triplicates.
Evaluation of substrate specificity
Substrate specificity of Cel1 and Cel2 was measured towards 4 substrates (4-Nitrophenyl β-D glucopyranoside pNPG, azo-CMC, azo-Avicel, filter paper). The substrate pNPG (Sigma Aldrich, Milan, Italy) was dissolved in 0.1 M sodium acetate buffer at pH 5 in a final concentration of 20 mM and it was incubated, with the culture supernatants of E. coli recombinantly expressing Cel1 and Cel2, at 45°C and 55°C, respectively, for 10 minutes. After adding 0.5 M Na2CO3, the absorbance was read at 405 nm. One unit of glycoside hydrolase activity was the measure of enzyme needed to release 1 µmol min-1 of p-nitrophenol.
The substrate AZO-CMC (Megazyme, Ireland) was dissolved in 0.05 M Na- citrate buffer pH 5 to obtain a concentration of 2% w/v and the incubation (50° C) was performed with the culture supernatants of E. coli, expressing Cel1 and Cel2, for 5 minutes. After adding the precipitating agent and centrifuging at 12,000 rpm for 10 minutes, the absorbance was read at 590 nm for each sample.
A slurry of the substrate Azo-Avicel in 0.1 M Na- acetate buffer (pH 4.5) was incubated for 1 hour in a final concentration of 2% w/v, with the culture supernatants of E. coli, expressing Cel1 and Cel2 at 40°C. The reaction was stopped by 2% trizma base solution (pH ~ 8.5). The absorbance was measured at 590 nm, after the centrifugation of the samples for 10 min (3,000 rpm).
The activity assay towards Filter Paper (no. 1), purchased from Whatman (Chiltern, UK) was performed as in [14].
The results correspond to the average of three experiments, everyone in triplicates.
Determination of Vmax and KM
The kinetics experiments for Cel1 and Cel2 were performed using the substrate 4-Nitrophenyl β-D-glucopyranoside (pNPG) (1 mM, 10 mM, 20 mM, 30 mM and 50 mM), dissolved in 0.1 M Na- acetate buffer (pH 5). The culture supernatants of E. coli, expressing Cel1 and Cel2, were incubated with the substrate at their optimal temperature (45° C and 55° C, respectively), for 15 minutes and the absorbance values were measured by taking the readings at 405 nm every minute and, from these, it was possible to calculate the kinetic constants.
The values of Michaelis–Menten constants (KM and Vmax) of Cel1 and Cel2 were identified using GraphPad software.
The results correspond to the average of three experiments, everyone in triplicates.
Feedstock and feedstock combinations
Biomass substrates used as raw materials are spent mushroom compost substrate (SMS) and wheat (WS). Spent mushroom compost (solid residue generated after growing mushrooms) and wheat straw were the same materials used in Beckers et al. (2019) [28].
For the development of the assays, feedstock combinations were prepared consisting of a mixture of SMS and WS in a wet weight ratio of 40% and 60% (20% and 80% in dry basis), respectively.
Thermochemical Pretreatment
Biomasses were treated at CENER facilities of Biorefinery and Bioenergy Centre (BIO2C), located in Aoiz (Spain). Several thermochemical pretreatment experiments were performed on SMS/WS mixtures with different ratios, considering the recalcitrance of this biomass and allowing to fractionate the biomass into different components in order to obtain valuable bio-based products. The thermochemical pretreatment assays were carried out in the reactor provided by Advancebio Systems LLC, USA as cited by Beckers et al. (2019) [28]. Due to the stickiness behaviour of SMS it was not possible to feed the horizontal continuous reactor automatically, but it worked successfully when SMS was mixed with WS in a 40 to 60 ratio on wet basis. The samples used for this particular study were treated either at 170ºC and 183ºC with a fixed residence time (10 min). Considering that for an optimum performance of the TC pretreatment reactor an initial moisture content of 50% in the feedstock is required, part of this required water can be supplied through SMS addition, reducing the water plant necessities. SMS and WS feedstocks were mixed based on dry matter content and adjusting the moisture up to 50% by adding the catalyst (NaOH 50%) using 2% of dry weight on the total solid content. This was followed by a manual mixing to reach the homogenisation and before feeding the reactor, the mixture was overnight at room temperature. Indeed, these conditions were part of a broader experimental design for thermochemical fractionation using the feedstock selected (data not shown).
Pretreated samples, generated under the conditions tested, were taken and after collecting a sample for compositional analysis, were subjected to a manual filter press for solid and liquid fraction separation. Solid fraction was store at 4ºC in zipped plastic bags until being used for enzymatic hydrolysis.
Compositional analysis
Moisture content in SMS was calculated as in Beckers et al. (2019) [28], following the CEN/TS 14774-3:2010 procedure. Ash content was determined as mentioned in Beckers et al. (2019) [28]following the UNE-EN ISO18122:2015. Elemental analysis of Nitrogen determination is carried out following the UNE-EN ISO 16948:2015 and using a conversion factor into protein of 5.25.
Pretreated materials (slurries) were separated to insoluble and soluble fractions, and cellulose, hemicellulose, and lignin contents were quantified from the insoluble fraction by the NREL procedures nº 42618 (A. Sluiter, Laboratory Analytical Procedures, (2011). www.nrel.gov/biomass/pdfs/42618.pdf) and nº 42627 (A. Sluiter, Laboratory Analytical Procedures, (2008). www.nrel.gov/biomass/pdfs/42627.pdf) respectively. Oligomeric sugars were hydrolysed by adding 72% H2SO4 (w/w), followed by autoclaving at 121ºC for 1 h. Samples were stored at -20ºC until analysis.
Soluble sugar analysis was performed by high-performance liquid chromatography (HPLC), quantifying the content of monosaccharides (d-glucose, d-xylose-fructose-galactose, l-arabinose), disaccharides (cellobiose, maltose) and trisaccharides (maltotriose) as reported in Beckers et al. (2019) [28].
Enzymatic hydrolysis
Saccharification experiments was performed using pretreated spent mushroom substrate supplemented with wheat straw on a rotary shaker (ThermoMixer C, Eppendorf, Milan, Italy).
The culture supernatants of E. coli recombinantly expressing Cel1 and Cel2 and culture supernatants of Streptomyces argenteolus AE58P [25] were incubated with 10% (w/v) of dried pretreated biomass in 2.5 mL of 0.05 M Na- citrate buffer (pH 5). The enzymatic commercial cocktail, used as benchmark, was composed by Accelerase1500, Accelerase BG, and Accelerase XY provided by Genencor and were prepared at the amounts expressed as units per gram of pretreated biomass: 5.4, 145 and 4000, respectively [14].
The saccharification yields of Cel1 and Cel2 were tested, replacing Accelerase 1500 with the same amount, in terms of units, of culture supernatants of E. coli expressing Cel1 and Cel2 and culture supernatant of S. argenteolus AE58P, performing the incubation at 50°C (600 rpm). The samples were collected daily until 72 hours and centrifuged at 4°C for 30 min (6,000 rpm). The glucose and xylose released were quantified, analysing the supernatants by high-performance liquid chromatography (HPLC; Dionex, Sunnyvale, CA, USA) as in Ventorino et al. (2016) [25]. Moreover, the saccharification experiments with SMS/WS (40:60) were also carried out using the culture supernatants of E. coli recombinantly expressing Cel2 and epCel2 mutants added to the enzymatic mixture MetZyme® SUNO TM BOOSTER 144 supplied by MetGen Oy. MetZyme® SUNO TM BOOSTER 144 is a high performance booster cocktail custom designed to significantly enhance the cellulose hydrolysis efficiency of cellulases. Three different conditions were tested for two clones and wild type enzyme, to select the best one and apply it to the other mutants, as reported below:
•Condition 1- The enzymatic mixture SUNO TM BOOSTER 144 and the cellulase enzyme (Cel2 wild type or epCel2 mutants) were added to the biomass in the same volume/volume ratio (50:50 V/V- 1.5 U and 0.35 U, respectively).
• Condition 2- 0.54 U of the SUNO TM BOOSTER 144 and 0.54 U of the cellulase enzyme (Cel2 wild type or epCel2 mutants) were added to the biomass.
• Condition 3- 1.08 U of the SUNO TM BOOSTER 144 and 1.08 U of cellulase enzyme (Cel2 wild type or epCel2 mutants) were added to the biomass.
The experiments for the hydrolysis of pretreated SMS/WS were performed incubating the enzymatic cocktails with 10% (w/v) of the dried biomass in 2.5 mL of 0.5 M succinic acid, at the optimal pH and temperature and of each enzyme on a rotary shaker (500 rpm). The samples were collected daily and until 72 hours and centrifuged at 4 °C for 30 min (6,000 rpm). The monosaccharides obtained was quantified analysing the supernatants, by HPLC [25]and the yields of the hydrolysed was expressed in g L-1.
The results correspond to the average of three experiments, everyone in triplicates.
Construction of Cel2 error-prone PCR library
pBAD-Cel2 construct was used as target sequence for epPCR and the random mutations were introduced by the Gene Morph II Random Mutagenesis Kit. PCR reactions were performed using the mix and program reported in Cecchini et al. (2018) [29]. About 20 epPCR were performed to obtain 48 µg total of Cel2 cDNA, for the construction of the 30,000 mutants library. The DNA regions obtained were purified by the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), the digestion with the same restrictions enzymes used for Cel2 was performed and the cloning into a pBAD vector was carried out. The mixture obtained after the ligation was employed for E. coli TOP10 cells transformation, plated into LB agar medium with ampicillin (50 µg mL-1) and grown at 37°C overnight. Liquid medium LB with 50 μg mL-1 ampicillin was added to the plates surface in order to collect the colonies with a scraper. After the cells centrifugation for 10 minutes at 4°C (6,000 rpm), the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was used for plasmid DNA preparation to transform E. coli BL21 (DE3) RP cells for the mutants expression.
The cells were incubated overnight at 37°C on LB agar (50 µg mL-1 ampicillin), to select the error-prone Cel2 (epCel2) mutants.
Screening of epCel2 mutants library
The Cel2 mutants were inoculated in LB medium (200 µL in 96-well plates), by the robot QPIX 450 (Molecular Devices, LLC, California, USA). The mutants were incubated at 37°C overnight and they were screened on solid medium in order to discriminate transformants expressing extracellular cellulase activity from those that do not express it. Therefore, each clone of the library was transferred on 22 × 22 cm Q-Tray bioassay plates containing LB Agar, ampicillin (50 µg mL-1), the substrate 1% w/v carboxymethylcellulose (CMC) and the inducer 0.002% w/v arabinose using a QPix colony picker with 96-well gridding head (Molecular Devices, Sunnyvale, California).
Several trials of the solid medium assay were performed, incubating the plates at 37°C for 2, 4, 6, 8 hours and overnight. In order to identify the transformants able to degrade CMC, the plates were dyed using a Congo red (0.1% w/v) solution for 15 minutes, followed by 3 washing with 3 M sodium chloride (NaCl). The trials of the solid medium assay were performed three times.
The mutants, forming clear halo around the colony, were selected as positive and they were screened in liquid medium, in order to detect the clones more active than Cel2 towards 4-Nitrophenyl β-D-glucopyranoside.
To define the best conditions for the enzymes production trials of recombinant expression of Cel2 were performed in microscale, using different concentration of the inducer arabinose (0.002%, 0.02%, 0.2%) and two different time for the adding to the growth medium (at the beginning of the growth, at 0.7 OD). The experiments were repeated three times, and data were reported as means ± standard deviations.
After the mutants incubation at 37°C overnight, 200 µL of the cultures were diluted by LB medium (800 µL in 96 deep-well microplates) with ampicillin (50 µg mL-1) , using the robot BioMek NXP (Beckman Coulter, California, USA) and the incubation was performed at 37°C. When the cultures reached 0.7 OD, arabinose (0.002% w/v) was used to induce the protein production at 37°C, overnight.
After centrifugation at 3,000 rpm for 30 minutes at 4°C, the activity of the clones was analysed towards the chromogenic substrate 4-Nitrophenyl β-D-glucopyranoside, dissolved in 0.1 M Na- acetate buffer at pH 5. The enzymes were incubated with the substrate at 55°C (Cel2 optimal temperature) for 10 minutes and the absorbance was measured at 405 nm by the Multi Detection SystemGloMax® Discover System (Promega, Wisconsin, USA).