3.1 Di-CRISPR design for XR-XDH pathway
We tested Di-CRISPR at certain delta-targeting guide RNA to achieve multicopy chromosomal integration with high efficiency of reductase-xylitol dehydrogenase pathway in S. cerevisiae. Two xylose metabolic genes, XYL1 and XYL2, were introduced into δ-integration of yeast Y6 genomes simultaneously by using CRISPR/Cas9. As expected, multicopy genome integration of heterologous xylose metabolic pathway was observed to a remarkable increase with elevated DNA levels.
The highest copy number of XYL1 and XYL2 for δ-integration through CRISPR/Cas9 mediation was 2.8 and 6.3, respectiv, ,whereas it was 1.6 and 3.2 for conventional delta integration. It demonstrated that the new replicative CRISPR/Cas9-mediated targeted δ-integration was effective in genome integration. As a result, the transformants with optimized protein expression were subsequently selected by combining enzyme activity analysis. As shown in Fig. 1, the yeast strain Y6g/XR-XDH with higher copy-numbers than Y6δ/XR-XDH exhibited the higher enzyme activities, thus suggesting that the optimized regulation of xylose metabolic flux in S. cerevisiae is proportional to the efficiency enhancement of multi-copy chromosomal integration and multiplex genome engineering approach.
3.2 Construction of Cellulosome-producing CBP Reaction System
We designed an artificial hemicellulosome by mimicking the natural mechanism on the recombinant xylose-utilizing strain Y6g under the optimization of δ-integration CRISPR Cas9. Although some of functional cellulases are already genetically immobilized on S. cerevisiae for combinatorics and on-site enzyme loading, “chimeric hemicellulases and minihemicellulosome” have few been employed in “arming yeast” strategy for simultaneous hydrolysis and fermentation. As shown in Fig. 2, the displaying scaffoldin was functionally served as the integrating carrier for hemicellulase recruitment and was docked on Y6g cell surface via a-agglutinin anchor system. The set-up of scaffoldin can serve to enhance the quality of multi-enzymatic co-localization and also provide flexibility for adaptive assembly of chimeric components. Hence, the cellulosome-producing engineered microorganism enables us to achieve the synergistic reaction of CBP system on biomass accessibility by consolidating the simultaneous lignocellulose hydrolysis and available sugar co-fermentation. Moreover, considering the metabolic burden of host cell imparted by mini-cellulosome with the complex structur, ,we devise a CBP-enabling S. cerevisiae consortium, in which every engineered yeast strain could secrete or display different assembly component to be adaptive assembled on the surface of scaffoldin-displaying yeast cell.
3.3 Secreted Expression and Enzyme Assay
We constructed recombinant yeasts for expressing dockerin-fused xylanases (endoxylanase and xylosidase). The secreted expressions of full-length XynII-SdbA (29 kDa) and XylA-OlpA (92 kDa) were detected by Western-blot (Fig. 3). On the basis of docking of catalytic modules into the displaying scaffoldin, the functional enzyme activities were also determined. As shown in Table 3, two types of xylanases were examined in the cell culture supernatants of Y6g/XylA and Y6g/XynII, respectively. Each recombinant strain showed enzymatic activity of xylanase according to the xylooligosaccharides tested in the culture. No enzyme activity of control strain was detected.
Table 3
Enzyme activities of xylanases.
Strain | Substrate | Enzyme activity (U/ml) |
Y6 | 1% PASC | ND |
Y6g/XylA | 1% Xylan | 3.13 ± 0.29 |
Y6g/XynII | 0.8% pNPX | 3.46 ± 0.34 |
3.4 Functional Localization and Assembly of Artificial Cellulosome
With the successful expression of catalytic units, the capability of the artificial cellulosome to recruit chimeric enzyme components onto the displaying-scaffoldin via the specific binding interaction between cohesion and dockerin was further illustrated by immunofluorescence examination. The accessibility test of scaffoldin and dockerin-containing xylanase targeting with different epitope tags was performed. As shown in Fig. 4A column 3, the green immunofluorescent labeling of Y6g/ScafI was detected by using goat anti-mouse IgG (H + L) conjugated with Alexa Fluor 488. This result confirmed that the displaying scaffoldin has successfully been anchored on the host cell surface. In additionally, both recombinant yeast strains of Y6g/XylA and Y6g/XynII were marked with brightly shining red fluorescence by goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor 647 (Fig. 4A, column 2), indicating that the two types of dockerin containing xylanase were already integrated into the scaffoldin. Finally, the relative assembling levels of these recruited enzymes were quantitatively assessed via testing the corresponding epitope by flowcytometry. As shown in Fig. 4B (upper right quadrant), positive populations were detected for all recombinant strains, which suggested that whole compositions of minicellulosomal architecture were successfully displayed on the Y6g cell surface and achieved the desired results. However, compared to docking single cellulosomal component, it should be noted that co-docking the scaffoldin and chimeric enzymes continuously on the cellulosome structure resulted in an obviously decrease in the positive populations (Fig. 4B), which reflected a reduced assembly efficiency caused by steric hindrance. Therefore, it might be necessary for us to improve the minicellulosomal machinery and organization by designing scaffoldin linker between the protein modules as well as the construction of carbohydrate binding modules (CBMs) to target appended catalytic units to substrate.
3.5 Xylan Hydrolysis and ethanol fermentation
The recombinant yeast Y6g/ScafI-XylA-XynII displaying the cellulosomal structure with two types of hemicellulase was tested to determine its ability to simultaneous hydrolysis and fermentation from birchwood xylan which contains >90% xylose residues. Figure 4 shows the time course of xylan utilization and ethanol production during consolidated bio-processing process. An amount of hemicellulosic substrate was ceaselessly consumed by cellulosome-producing yeast Y6g/ScafI-XylA-XynII within 96h with a slow but steady consumption rate of 0.006 g/l/h. Meanwhile, the decrease of the xylan concentration was consequently accompanied by the increase of ethanol titer. The highest ethanol concentration reached 0.61 g/l with 67.01% theoretical yield based on the consumed xylan. Throughout the whole simultaneous saccharification and fermentation, the released reducing sugar concentration was always below the detection limit, which demonstrated a desired pentose fermentability of xylose-utilizing recombinant S. cerevisiae strain associated with minicellulosome. However, as shown in Fig. 5, xylitol was produced unavoidably from xylose under the influence of redox imbalance in xylose reductase-xylitol dehydrogenase pathway, which was directly caused by different cofactor specificities of NAD+-dependent XDH and NADPH-preferring in recombinant strain. Nevertheless, the results demonstrated the functional activity of chimeric xylanases and the applicability of consolidated bioprocessing-enabling yeast strain in direct hemicellulose-to-ethanol conversion.
3.6 SSCF performance from steam-exploded P. purperum
We conducted a fed-batch operation mode for cellulosic ethanol production previously [17]. We followed the process to SSCF from steam-exploded P. purpereum in this work, involving the adaption of yeast culture and the pre-hydrolysis prior to inoculation. The functionality of CBP-microorganism with synergistic combination of endoxylanase and xylosidase in simultaneous saccharification holocellulose and co-fermentation ethanol were further studied. As presented in Fig. 6 and Table 4, as the initial carbon source for growth and metabolism of yeast recombinant strain, an amount of glucose was released after 6h-prehydrolysis of solid liquefactions. However, the subsequent concentration of glucose during the whole fermentation process maintained a considerable low level, suggesting the robust metabolic capacity and fermentation property of yeast strain. Produced xylose derived from hemicellulose hydrolysis was accumulated before 60h, it was not used until glucose was depleted due to catabolite repression. It’s worth noting that the amount of released xylose additionally affording by hemicellulosome-enabling yeast strain Y6g/ScafI-XylA-XynII, led to an increase of 14.81% and 8.33%, respectively, both in ethanol productivity and substrate utilization rate, when compared to control strain. Despite the accumulation of xylitol, the maximum ethanol concentration achieved 12.88 g/l with the maximal cellulose conversion rate of 91.21% and hemicellulose conversion rate of 86.41% after 96 h (Table 4).
As the accessory enzyme for hydrolysis, as well as the supplement of commercial cellulases, xylanase could alter the macromolecular structure of lignocellulosic material and thus improve cellulose enzymatic hydrolysis by increasing access of cellulases to substrate. A xylan-degrading yeast strain by co-displaying hemicellulases on the surface of xylose-utilizing host cell has been reported previously but it did not involve the scaffoldin and minicellulosome [18]. While in recent years, increasing attentions have been focused on the heterologous expression of engineering xylanolytic mini-hemicellulosome for CBP whole-cell biocatalyst. Sun et al. successfully used the artificial cellulosome strategy to design the structures of cellulosomal modules consisting of a scaffoldin and three xylanases but did not evaluate the lignocellulolytic potential and co-fermentation property for natural lignocellulosic biomass [10]. In this work, the holocellulose-to-ethanol conversion from pretreated perennial C4 grass was achieved on the basis of incorporating the functional mini-hemicellulosome into the xylose-utilizing yeast strain under the optimization of δ-integration CRISPR Cas9. The structure of the hemicellulosome and its responsible function in SSCF bioreaction system of pretreated lignocellulose were further evaluated with the addition of commercial cellulase.
In generally, it is practical benefit in integrating catalytic units of xylanase into the multi-enzymatic formation of CBP system for enhancing biomass accessibility and cellulosic ethanol co-fermentation of pentose and hexose. On the other hand, besides the potential enzyme proximity synergy, the enhanced hydrolysis rate of holocellulose observed for the functional minicellulosome might be due to an overall effect caused by yeast cell surface-display system, secretion efficiency of exogenous enzyme, minicellulosomal machinery, and so on. Further studies are therefore required to improve the elaborated structural organization of designer hemicellulosome for the ideal consolidated bioprocessing based on simultaneous holo-cellulose saccharification and fermentation.
Table 4
Summaries of SSCF performance of engineered yeast strain.
Engineered strains | Maximum ethanol concentration (g/l) | Theoretical ethanol yielda (%) | Ethanol productivity (mg/l/h) | Ehanol yield (g/g) | Substrate consumption rate (g/l/h)d | Cellulose conversion rateb (%) | Hemicellulose conversion ratec (%) |
Y6g/XylA-XynII | 12.88 | 84.87 | 0.13 | 0.43 | 0.31 | 91.21 | 55.25 |
Y6g | 11.47 | 86.92 | 0.12 | 0.44 | 0.27 | 86.41 | - |
a\(\text{E}\text{t}\text{h}\text{a}\text{n}\text{o}\text{l} \text{y}\text{i}\text{e}\text{l}\text{d}\left(\text{\%}\right)=\frac{\text{p}\text{r}\text{o}\text{d}\text{u}\text{c}\text{e}\text{d} \text{e}\text{t}\text{h}\text{n}\text{a}\text{o}\text{l} \left(\text{g}\right)}{\text{c}\text{o}\text{n}\text{s}\text{u}\text{m}\text{e}\text{d} \text{g}\text{l}\text{u}\text{c}\text{o}\text{s}\text{e} \left(\text{g}\right)\times 0.51+\text{c}\text{o}\text{n}\text{s}\text{u}\text{m}\text{e}\text{d} \text{x}\text{y}\text{l}\text{o}\text{s}\text{e}\left(\text{g}\right)\times 0.51}\times 100\text{\%}\) |
b \(\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e} \text{c}\text{o}\text{n}\text{v}\text{e}\text{r}\text{s}\text{i}\text{o}\text{n} \text{y}\text{i}\text{e}\text{l}\text{d}\left(\text{\%}\right)=\frac{{\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{s}}\left(\text{g}\right)-{\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{R}}\left(\text{g}\right)}{{\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{s}}\left(\text{g}\right)}\times 100\text{\%}\)\(({\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{S}}:\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e} \text{f}\text{r}\text{o}\text{m} \text{t}\text{o}\text{t}\text{a}\text{l} \text{s}\text{u}\text{b}\text{s}\text{t}\text{r}\text{a}\text{t}\text{e}; {\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{R}}:\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e} \text{f}\text{r}\text{o}\text{m} \text{f}\text{e}\text{r}\text{m}\text{e}\text{n}\text{t}\text{a}\text{t}\text{i}\text{o}\text{n} \text{r}\text{e}\text{s}\text{i}\text{d}\text{u}\text{e}\text{s})\)
c \(\text{H}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e} \text{c}\text{o}\text{n}\text{v}\text{e}\text{r}\text{s}\text{i}\text{o}\text{n} \text{y}\text{i}\text{e}\text{l}\text{d}\left(\text{\%}\right)=\frac{{\text{H}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{s}}\left(\text{g}\right)-{\text{H}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{R}}\left(\text{g}\right)}{{\text{H}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{s}}\left(\text{g}\right)}\times 100\text{\%}\)\(({\text{H}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{S}}:\text{h}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e} \text{f}\text{r}\text{o}\text{m} \text{t}\text{o}\text{t}\text{a}\text{l} \text{s}\text{u}\text{b}\text{s}\text{t}\text{r}\text{a}\text{t}\text{e}; {\text{H}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}}_{\text{R}}:\text{h}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e} \text{f}\text{r}\text{o}\text{m} \text{f}\text{e}\text{r}\text{m}\text{e}\text{n}\text{t}\text{a}\text{t}\text{i}\text{o}\text{n} \text{r}\text{e}\text{s}\text{i}\text{d}\text{u}\text{e}\text{s})\)
d\(\text{S}\text{u}\text{b}\text{s}\text{t}\text{r}\text{a}\text{t}\text{e} \text{c}\text{o}\text{n}\text{s}\text{u}\text{m}\text{p}\text{t}\text{i}\text{o}\text{n} \text{r}\text{a}\text{t}\text{e} (\text{g}/\text{l}/\text{h})=\frac{\text{C}\text{o}\text{n}\text{s}\text{u}\text{m}\text{e}\text{d} \text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}\times 1.1 \left(\text{g}/\text{l}\right)-\text{C}\text{o}\text{n}\text{s}\text{u}\text{m}\text{e}\text{d} \text{h}\text{e}\text{m}\text{i}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}\times 1.136\left(\text{g}/\text{l}\right)}{96\left(\text{h}\right)}\)