Бурение грунтовых зондов, установка энергетических колодцев

As we discussed earlier, common yeast S. cerevisiae has many advantages as a producer of lignocellulosic ethanol, such as faster sugar consumption, higher ethanol yield, and higher resistance to ethanol and fermentation inhibitors present in pretreated lignocel — lulosic materials [106,107]. In addition to this, S. cerevisiae is ame­nable to genetic manipulation and is generally regarded as safe (GRAS) due to its long association with the food and beverage industries. Therefore, a number of researchers have focused their efforts in the heterologous expression of cellulase genes with yeast hosts [108,109] in order to produce genetically engineered yeasts for CBP. A genetically engineered yeast cell acts as a host cell for

Figure 8.5 Genetically engineered yeast cell acting as a host cell for cellulase genes with promoters which secretes the cellulases endoglucanase (EG), exoglucanases including cellobiohydrolase (CBH) and cellodextrinase, and jS-glucosidase (BGL), and its operation in consolidated bioprocessing. (Reprinted with permission from reference [110]; copyright 2012 Elsevier).

cellulase genes with promoters which secretes the cellulases and its operation in consolidated bioprocessing is illustrated in Figure 8.5. There are several examples of expression of cellulases and hemi- cellulases by Saccharomyces cerevisiae in recent literature, especially after 2008, and some of the selected examples and their references are shown in Table 8.7.

Multiple enzymatic activities are required to hydrolyze cellulose into soluble sugars, as described in Chapter 6. These include endo — glucanase (EG), exoglucanases including cellobiohydrolase (CBH) and cellodextrinase, and S-glucosidase (BGL). Endoglucanase produces nicks in the cellulose polymer, exposing reducing and non-reducing ends for cellobiohydrolase, which liberates cellooligo — saccharides, cellobiose and glucose. In the last step of saccharifica­tion, S-glucosidase cleaves the cellooligosaccharides and cellobiose to liberate glucose. Given that cellobiose and cellooligosaccharide are potent inhibitors of cellulose hydrolysis, S-glucosidase action has been shown to be one of the major rate-limiting steps in the hydrolysis of cellulose. Therefore, S-glucosidase genes with a fun­gal origin such as BGL1 from Saccharomycopsis fibuligera, BGL1 from

A. aculeatus, bglA from Aspergillus kawachii, bglB from Candida wick — erhamii, bgl from Trichoderma reesei, and BGL1 from Endomyces fibu — liger, have been heterologously expressed in S. cerevisiae [117-120]. Cellobiose fermentation ability of industrial Saccharomyces strains carrying S. fibuligera BGL1 depends on their ability to accumulate BGL1 but also on their genetic background [121].

In one of the early examples, Den Haan et al. demonstrated the construction of a yeast strain capable of growing on and one-step conversion of amorphous cellulose to ethanol [122]. This report represents a significant progress towards realization of one-step processing of cellulosic biomass in a consolidated bioprocessing configuration. In 2007, Den Haan et al. claimed this was the first report of a recombinant strain of S. cerevisiae growing on pure cel­lulose. In this study, they expressed two cellulase encoding genes, an endoglucanase of Trichoderma reesei (EGI) and the ^-glucosidase of Saccharomycopsis fibuligera (BGL1) combination in Saccharomyces cerevisiae. The resulting strain was able to grow on phosphoric acid-swollen cellulose (PASC) through simultaneous production of sufficient extracellular endoglucanase and ^-glucosidase activ­ity. Anaerobic growth (0.03 h-1) up to 0.27 g l-1 DCW was observed on medium containing 10 g l-1 phosphoric acid-swollen cellulose as the sole carbohydrate source with concomitant ethanol production of up to 1.0 g l-1 [122].

In another example, Jeon and coworkers reported the direct cellulosic alcohol fermentation using recombinant Saccharomyces cerevisiae engineered for the production of Clostridium cellulovo — rans endoglucanase and Saccharomycopsis fibuligera ^-glucosidase [116,123]. In this study, Saccharomyces cerevisiae was engineered for simultaneous saccharification and fermentation of cellulose by the overexpression of the endoglucanase D (EngD) from Clostridium cellulovorans and the ^-glucosidase (Bgl1) from Saccharomycopsis fibuligera. To promote secretion of the two enzymes, the genes were fused to the secretion signal of the S. cerevisiae a mating factor gene. The recombinant yeast developed could produce ethanol through simultaneous production of sufficient extracellular endoglucanase and ^-glucosidase. When direct ethanol fermentation from 20 g L-1 ^-glucan as a substrate was performed with these recombinant strains, the ethanol concentration reached 9.15 g L-1 after 50 h of fermentation. Furthermore, Jeon and coworkers reported the con­version ratio of ethanol from b-glucan as 80.3% of the theoretical ethanol concentration produced from 20 g L-1 b-glucan [123].

Recently, genes encoding T. reesei endoglucanase II (EGII) and cel — lobiohydrolyase II (CBHII) and A. aculeatus BGL1 were integrated into the chromosome of a wine yeast strain with a single vector car­rying a gene responsible for resistance to the antibiotic G418 [115]. The resultant S. cerevisiae strain produced ethanol from pretreated corn stover cellulose without addition of exogenously-produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fer­mented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v [115].

Yamada and coworkers constructed a diploid Saccharomyces cere — visiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw [124]. In this study they found that the engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was pre­cultured in molasses medium (381.4 mU/g wet cell) and displayed approximately six-fold higher phosphoric acid-swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours [124].

Комментарии запрещены.