Surface-Engineered Yeast Strains for the CBP
Another approach for producing cellulolytic yeast strain is displaying various types of functional proteins on microbial cell surfaces without loss of their activity. This can be achieved by yeast cell surface engineering, where functional proteins are genetically fused to an anchor protein such as a-agglutinin, a-agglutinin and Flo1p, and expressed on the yeast cell surface [125]. Display of S. fibulig — era BGL1 on the S. cerevisiae cell surface by fusing the mature protein and a-agglutinin anchoring moieties enabled the recombinant strain to grow on cellobiose at almost the same rate as on glucose under anaerobic conditions [120]. The application of surface-engineered yeast cell with endoglucanase (EG), exoglucanases including cellobiohydrolase (CBH), cellodextrinase, and ^-glucosidase (BGL) fused on to yeast cell surface in consolidated bioprocessing is shown in Figure 8.6.
A number of examples of using cell surface engineering to express cellulases and their applications in direct conversion of cellulose and lignocellulosic materials have appeared in recent literature. Guo and coworkers reported a recombinant S. cerevisiae expressing cell-wall associated BGL1 from S. fibuligera utilized 5.2 g/L cellobiose and produced 2.3 g/L ethanol in 48 h, while a comparable S. cerevisiae secreting BGL1 into the culture broth used
3.6 g/L cellobiose and produced 1.5 g/L ethanol over the same period [126]. In another example, Fujita and coworkers produced ethanol from pure cellulose such as phosphoric acid-swollen cellulose as well as from biomass such as barley straw without the addition of cellulases using recombinant S. cerevisiae strains displaying T. reesei EGII and CBHII and A. aculeatus BGL1 on the cell surface. A yeast strain codisplaying endoglucanase II and cellobiohydro — lase II showed significantly higher hydrolytic activity with amorphous phosphoric acid-swollen cellulose than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of ^-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose. The yield of ethanol produced from the PASC consumed was 0.45 g/g, which corresponds to 88.5% of the theoretical yield [127, 128].
Additionally, Matano and coworkers reported the enhancement of cellulase activities on a recombinant yeast cell surface displaying T. reesei EGII, CBHII and A. aculeatus BGL1by additionally integrating EGII and CBHII genes into the recombinant strain [129].
As a result, high-titer ethanol (43.1 g/L) was produced from high — solid (200 g-dry weight/L) rice straw by performing 2 h liquefaction and subsequent 72 h fermentation in the presence of 10 FPU/g biomass added cellulase. The yield of ethanol produced from the cellulosic material by the recombinant strain reached 89% of the theoretical yield, which was 1.4-fold higher than the wild-type strain. Consequently, cell surface engineering successfully reduced the amount of commercial enzyme required for the fermentation of cellulose. Notably, the recombinant strain was able to hydrolyze a portion of the cellulosic material that was not hydrolyzed by commercial cellulase [129].
Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes on the cell surface was reported by Yanase and coworkers [130]. In this experiment EGII from T. reesei and BGL1 from A. aculeatus were codisplayed on the cell surface of the recombinant K. marxianus, which produced 20.4 g/L ethanol from 53.4 g/L cellobiose at 45°C. The recombinant strains successfully converted cellulose ^-glucan at 48°C with a yield of 4.24 g/L from 10 g/L within 12 h, without any cellulase addition, while the amount of ethanol produced at 30°C was only 0.930 g/L [130]. Ethanol yields as high as 0.48 g per gram of b-glucan consumed could be achieved by this technique. This result indicates that high — temperature cellulose fermentation to ethanol is efficiently accomplished using this recombinant thermotolerant Kluyveromyces marxianus strain displaying thermostable cellulolytic enzymes on its cell surface. Although many approaches have attempted to utilize cellulose at elevated temperatures, Kluyveromyces marxianus, generally regarded as safe (GRAS) microorganism, would be specifically suitable for this purpose because of its ability to grow at higher temperature with a shorter doubling time and on a wide variety of carbon sources. Therefore, further developments in the surface engineering of Kluyveromyces marxianus would make a significant contribution to the improvement of CBP for bioethanol production.
Displaying the cellulases on the yeast cell surface has certain advantages as well as disadvantages, some of the main advantages include:
1. Close proximity of multiple cellulases on the cell surface enables synergistic hydrolysis of cellulose, which leads to increased sugar availability for ethanol production [127,129].
2. Glucose liberated from cellulose is concurrently taken up on the yeast cell surface so that the glucose concentration is maintained at low levels, which reduces both the risk of contamination by other glucose-dependent organisms and product inhibition by cellulases [127].
3. Since the steady-state concentration of glucose in the medium can be maintained near zero, glucose repression, which prevents the uptake, catabolism or both of non-glucose sugar, is alleviated to facilitate consumption of xylose [131].
4. Reutilization of the yeast cells enables reuse of the enzymes displayed on their cell surface without reproduction of the yeast cells, which would reduce the cost of yeast propagation as well as enzyme addition [132,133].
5. Cellulolytic enzymes are genetically self-immobilized on the yeast cell surface so that the activities of the enzymes are retained as long as the yeast continues to grow, while the activity of enzymes secreted into the medium is poorly maintained over a long reaction period [134].
However, despite all these advantages, surface immobilization of cellulases on yeasts is still an immature technology and the main disadvantage is that ethanol yields are still very low and most of the studies so far have been done on simple model compounds or pure cellulose or xylan. Significant biotechnological advances are needed in engineering recombinant yeast that can display a vast array of cellu — lases required to handle very complex lignocellulosic biomass forms.