Self-cleavages of polyprotein with FMDV P2A in K. marxianus
Due to the chemical diversity in structure of hemicelluloses that are heterogeneous polysaccharides with both linear and branched molecules cross-linked to cellulose microfibrils, complete degradation requires multiple hemicellulases to act synergically . Aiming to facilely express multiple enzymes in ethanologenic K. marxianus for the hemicellulose degradation, we resorted to a 2A-mediated ribosomes skipping for co-translational cleavage of the polyprotein. The 2A-mediated cleavage is a common phenomenon in eukaryotic cells that it skips the synthesis of a glycyl–prolyl peptide bond at the C-terminus of 2A, releases the nascent protein, and resumes the downstream translation . While the efficiency of 2A self-cleavage is strongly related ot the sequence contexts of upstream and downstream ORFs in the polycistrons . Given that we tested the efficiency of FMDV P2A self-cleaving in K. marxianus by expression of three polycistronic genes IMX, IMPX, and IMPαX (Fig1a and b). The IMX gene consisted of a M330 coding sequence (INU1 signal peptide+mature protein coding sequence) and a C-terminal 6xHis-tagged Xyn-CDBFV mature protein coding sequence fused in-frame directly. In the IMPX gene, the P2A sequence was incorporated between M330 and Xyn-CDBFV without stop codon. The IMPαX gene had an extra α-factor signal sequence between P2A and Xyn-CDBFV. These three polycistronic genes were all cloned into the vector pUKDN132, in which their expressions were all driven by an INU1 promoter from K. marxianus.
After cultured in flasks, expressions of M330 and Xyn-CDBFV were detected by measuring the activities β-mannanase and β-xylanase in both supernatants and cell lysates of the IMX, IMPX, and IMPαX strains, which were transformed with the plasmids pUKDN132/IMX, pUKDN132/IMPX, and pUKDN132/IMPαX, respectively. Unexpectedly, as a control, we observed that the IMX strain produced high activities of both β-mannanase and β-xylanase in the supernatant, with approximately 24.03 U/ml and 155.26 U/ml respectively (Table 2), suggesting that these two genes fused directly did not impair their catalytic activities. This double-activities of the IMX strain provided a good reference to the P2A effect on the expression of downstream Xyn-CDBFV. The extracellular β-mannanase activities of IMPX and IMPαX strains were about 21.34 and 15.50 U/mL respectively, which were slightly lower than that of the IMX strain, whereas the intracellular activities of M330 in the two strains were higher, inferring that fusion of Xyn-CDBFV to the C-terminus of M330 with P2A slightly decreased the secretory expression of M330.
In our constructs, the efficiency of FMDV P2A self-cleavage was related to the production of Xyn-CDBFV. Enzymatic determinations demonstrated that the IMPαX strains secreted 136.17 U/mL β-xylanase into the supernatants, and retained 39.43 U/mL intracellularly. By contrast, the supernatant β-xylanase of IMPX strain was 42.07 U/mL, which was far less than the intracellular activity 87.59 U/mL. To confirm whether the β-xylanase activities of IMPX and IMPαX strains were the self-cleavaged Xyn-CDBFV by the 2A-mediated ribosomes skipping during translation, these samples were further analyzed by SDS-PAGE and Western blot. As shown in figure 1c and e, protein bands with approximate 57 kDa molecular weight in the supernatants of IMX strain were in accord with the theoretical prediction of the fusion protein IMX. In both supernatants of the IMPαX and IMPX strains, M330 and Xyn-CDBFV were secreted alone, while the secretory Xyn-CDBFV of IMPαX strain was much higher than that of the IMPX strain, suggesting that, in the presence of P2A and α-factor signal sequence, Xyn-CDBFV could be secreted to medium more efficiently. This result was in agreement with the previous literature . Furthermore, western blot assays for the His-tagged Xyn-CDBFV in the above samples were in compliance with the enzymatic assays and SDS-PAGE above (Fig 1 d and f). Thus, to extracellularly express two proteins via FMDV P2A self-cleavage, an extra signal sequence should be included at the N-terminus of downstream gene. The 2A-mediated ribosomal 'skipping' is an attractive alternative to the internal ribosomal entry site (IRES), first identified in the encephalomyocarditis virus, since it can express multiple cistrons at equimolar levels . However, in our results, we also found that this ribosomal 'skipping' in co-translation would decrease in apparent the total protein express levels, especially for the downstream gene.
Coexpression of hemicellulolytic enzymes with FMDV P2A
Hemicelluloses act as one important factor contributing to the recalcitrance of lignocelluloses, and they, even in low quantities, can prevent cellulases to degrade the cellulose efficiently . Cellulase supplemented with endoxylanase promoted the hydrolysis of steam-exploded feed stocks, released more glucose, accumulated higher content of xylobiose and xylooligosaccharides [43, 44]. Content of xylose, however, was not significantly elevated, which may be due to the insufficient β-xylosidase in most cellulase enzymes produced by filamentous fungi Trichoderma reesei [5, 45]. We reasoned that an ethanologenic strain co-expressed multiple hemicellulases, especially β-xylanase and β-xylosidase, would eliminate the accumulation of xylooligosaccharides and produce more fermentable xylose. A β-xylosidase RuXyn1 that has high capability of converting intermediate xylo-oligosaccharides into xylose was used to co-express with β-xylanase in K. marxianus . The RuXyn1 coding sequence was fused to Xyn-CDBFV with a P2A and an α-factor signal sequence (Fig. 2a), and the resulting IXPαR was expressed in K. marxianus under the unique INU1 promoter. The IXPαR strain transformed with the pUKDN132/IXPαR produced 59.01 and 0.05 U/ml of extracellular β-xylanase and β-xylosidase in flask cultures respectively (Fig 2 b-d).
Supplements of β-mannanase facilitated the total enzymatic hydrolysis of lignocellulose feedstock and brewery’s by-product, such as beech sawdust, spruce, Douglas fir wood and chips spent grain [46-49]. Given critical roles of β-mannanase, β-xylanase and β-xylosidase in the hydrolysis of lignocellulose, we tested the feasibility of P2A for coordinately expressing these three selected enzymes in one ORF. The polycistronic gene IMPαXPαR that compacted M330, Xyn-CDBFV and RuXyn1 into one ORF, each with a signal sequence (Fig. 2a). Consistent with that of the IMPαX and IXPαR strains, all activities of these three enzymes were detectable in the crude culture supernatant of the IMPαXPαR strain, which is obtained by transformation of the pUKDN132/IMPαXPαR plasmid. The β-mannanase, β-xylanase and β-xylosidase activities were 18.90, 61.00, and 0.07 U/mL, respectively (Fig. 2c-e). As expected, figure 2b showed three protein bands in culture supernatants of the IMPαXPαR strain that were corresponding to the molecular weights of M330, Xyn-CDBFV and RuXyn1, confirming that FMDV P2A is applicable for secretory co-expression of multiple enzymes in K. marxianus.
Preparation of hemicellulolase mixtures by recombinant K. marxianus strains
We have previously developed a high-cell density fed-batch fermentation for single hemicellulolytic enzyme production in K. marxianus . In this study, we evaluated the productions of multiple enzymes in fed-batch fermentation for both the IXPαR and IMPαXPαR strains. K. marxianus is Crabtree negative yeast that does not perform aerobic alcoholic fermentation, and but can respire even in high glucose concentrations [50, 51]. However, high concentration of glucose could adversely cause respiratory repression and turn to alcoholic fermentation especially in high-cell density, probably due to the insufficient oxygen supply. Similar to S. cerevisiae, a Crabtree positive yeast that predominantly produces ethanol in high glucose even in sufficient oxygen levels, it is practicable to guide K. marxianus to utilize glucose for respiratory metabolism and convert carbon resources into cell biomass, as glucose can be fed slowly to maintain a concentration below the threshold value in fed-batch fermentation [52, 53]. Additionally, ethanol fermentation could affect cell growth in K. marxianus, and thus decreases expressions of enzymes. To circumvent this, we controlled the dissolved O2 above 10% by limiting the fed rate of glucose during fermentation. Cell densities of both strains reached more than 450 (OD600nm) after 48 h (Fig. 3a). Productions of secretory proteins synchronized with the cell growths, and all enzymes were dramatically accumulated during the stages from 16 h to 48 h (Fig. 3b-d). After 72 h, the IXPαR strain secreted 1664.2 U/ml of l β-xylanase and 0.90 U/ml β-xylosidase, which were about 28 and 18 folds that of in the flask cultures respectively. SDS-PAGE showed that the IXαR strain secreted two different protein bands that represented mature forms of Xyn-CDBFV and RuXyn1 respectively. The IMPαXPαR strain produced 2210.5 U/ml of β-xylanase and 1.25 U/ml of β-xylosidase, slightly higher than that of the IXαR strain. As well, this strain also produced 159.8 U/ml of β-mannanase concurrently, and all enzymes were secreted extracellularly as their mature forms (Fig. 3e and f).
Enzymatic hydrolyses of pretreated corncobs
Hemicellulases supplementation to commercial cellulases enhanced the enzymatic hydrolyses of lignocellulose significantly [54, 55]. Using prepared hemicellulase cocktails, we next evaluated their performances on the promotion of lignocellulose hydrolyses. We chosed corncob as the feedstock for enzymatic hydrolyses because it is one of the most abundant inedible agricultural residues and consists of a relatively high content of hemicellulose (∼40%) . Enzymatic hydrolyses were conducted with 10 % (w/v) corncobs pretreated by aqueous dilute acid, and 5 FPU of Cellic® CTec2 cellulase per gram solids. After 96 h, about 300 mM soluble sugars were released from the pretreated corncobs. To test the β-xylanase Xyn CDBFV and β-xylosidase RuXy1 performances on the enzymatic hydrolyses, 300 μl of supernatant collected from the IXPαR strain fed-batch culture at 48 h, equal to 531.29 U β-xylanase and 0.22 U β-xylosidase, was supplemented to the Cellic® CTec2 cellulase. In accord with previous literatures on pine kraft pulp and softwood [47, 57], supplementations of xylanolytic enzymes to the Cellic® CTec2 cellulase improved the enzymatic hydrolysis of corncobs. At each sampling point, addition of the IXPαR strain culture supernatant generated higher contents of soluble sugars. After 96 h of hydrolysis, the release of soluble sugars increased by 15.7% (Fig 4a). Similarly, the amounts of monomeric glucose and xylose increased to 8.32 and 61.39 g/L respectively, which were 11.2% and 11.1% higher than that of Cellic® CTec2 cellulase alone (Fig 4b and c).
The role of β-mannanase M330 for the corncob hydrolysis in combination of β-xylanase and β-xylosidase was also evaluated. The culture supernatant of IMPαXPαR strain containing 49.50 U β-mannanase, 485.70 U β-xylanase, and 0.28 U β-xylosidase was supplemented to the Cellic® CTec2 cellulase. As shown in Figure 4a, the supplementary β-mannanase increased the amount of total soluble sugars over time. At 96 h, about 12.1% more soluble sugars were obtained comparing to that of the IXPαR strain, and the glucose and xylose contents were increased to 65.48 and 8.45 g/L (Fig 4b and c), which were 11.9% and 11.4% higher than that of xylanolytic enzymes respectively, showing that β-mannanase could facilitate a more extensive break-down of corncobs. This promotion may be ascribed to the deep hydrolysis glucomannan by the endoglucanase TrCel5A presented in Cellic® CTec2 .
HSFs of ethanol from pretreated corncobs
Besides the application in expression of heterologous protein, the K. marxianus strain used in this study can produce ethanol from multiple substrates, including glucose, xylose, lactose, and inulin, with a maximum ethanol concentration more than 100 g/L [58, 59]. The hemicellulolase activities of IMPαXPαR strain would be conducive to ethanol production from pretreated lignocellulosic biomass. Subsequently, we investigated the potential of recombinant IMPαXPαR strain as a CBP strain to produce ethanol from pretreated corncobs with a high solids content, as it is more economic for cellulosic ethanol processes to generate high sugar titers necessary for high ethanol production [60, 61]. HSFs were performed by pre-hydrolyzing pretreated corncobs with 10 FPU commercial cellulase per gram solids for 72 h before inoculated with the IMPαXPαR or FIM-1 (control) strain. As shown in Figure 5a, glucose in HSFs with IMPαXPαR strain decreased more rapidly than with FIM-1 through the first 96 h. After 216 h, glucose concentrations were 5.2 and 8.7 g/L in HSFs with the IMPαXPαR and FIM-1strains respectively. The glucose consumption rates in our K. marxianus strains were in apparent lower than other K. marxianus strains in simultaneous saccharification and fermentation of switchgrass [4, 62, 63], but the reason for this is not clear. In HSFs with both IMPαXPαR and FIM-1 strain, xylose concentrations increased within the first 120 h, and then decreased slightly when prolonging the fermentation time (Fig. 5b). Glucose could strongly repress xylose utilization in simultaneous fermentation of them with K. marxianus , presumably, responsible for the xylose accumulation during the preceding stage of HSF. Different with the above in vitro saccharification of pretreated corncobs, no significant increase of xylose was found in the HSFs with IMPαXPαR strain comparing to that of FIM-1 strain. The reason may be due to lower glucose concentrations in the HSFs with IMPαXPαR strain that might promote its xylose assimilation.
Ethanol production profiles indicated the IMPαXPαR strain had higher efficiency of ethanol conversion rate, and produced more ethanol during fermentation. At 144h, 20.8 g/L of ethanol was obtained in HSFs with IMPαXPαR strain, which is about 34.2% more than that of FIM-1 (Fig. 5c). After prolonging fermentation time to 216 h, ethanol in HSFs with the IMPαXPαR strain increased to 23.9 g/L, while it was 21.3 g/L in that of FIM-1 strain. Considering the high glucose consumption rate in HSFs with IMPαXPαR strain, we used the sugar-ethanol conversion rates based on their initial glucose contends to present the contribution of hemicellulases produced for ethanol production. As shown in Figure 5d, the ethanol conversion rates of IMPαXPαR strain at 144, 168, 192, and 216 h were 0.54, 0.55, 0.55, and 0.55, respectively, which were higher than the theoretical ethanol conversion rate 0.51 from glucose, indicating additional amounts of glucose were produced during fermentation. While, at the same points, ethanol conversion rates for the control strain were 0.51, 0.51, 0.51, and 0.52 respectively. Consequently, we confirmed that the hemicellulolytic enzymes produced by IMPαXPαR strain improved the hydrolysis and ethanol conversion in HSFs of pretreated corncobs.