The sequences encoding the genes for endoglucanase II and cellobiohydrolase II from the fungus Trichoderma reesei QM9414 were successfully cloned and expressed in Yarrowia lipolytica under the control of the POX2 or TEF promoters, and using either the native or preproLip2 secretion signals. The expression level of both recombinant enzymes was compared with that obtained using Pichia pastoris, under the control of the AOX1 promoter to evaluate the utility of Y. lipolytica as a host strain for recombinant EGII and CBHII production. Extracellular endoglucanase activity was similar between TEF-preoproLip2-eglII expressed in Y. lipolytica and P. pastoris induced by 0.5 % (v/v) methanol, but when recombinant protein expression in P. pastoris was induced with 3 % (v/v) methanol, the activity was increased by about sevenfold. In contrast, the expression level of cellobiohydrolase from the TEF-preproLip2-cbhII cassette was higher in Y. lipolytica than in P. pastoris. Transformed Y. lipolytica produced up to 15 mg/l endoglucanase and 50 mg/l cellobiohydrolase, with the specific activity of both proteins being greater than their homologs produced by P. pastoris. Partial characterization of recombinant endoglucanase II and cellobiohydrolase II expressed in both yeasts revealed their optimum pH and temperature, and their pH and temperature stabilities were identical and hyperglycosylation had little effect on their enzymatic activity and properties.

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0–9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s−1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.

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Biotransformation of fatty acids from renewable wastewater as feedstock to value-added chemicals is a fascinating commercial opportunity. α,ω-dicarboxylic acids (DCAs) are building blocks in many industries, such as polymers, cosmetic intermediates, and pharmaceuticals, and can be obtained by chemical synthesis under extreme conditions. However, biological synthesis can replace the traditional chemical synthesis using cytochrome P450 enzymes to oxidize fatty acids to DCAs. Saccharomyces cerevisiae BY(2R)/pYeDP60-CYP52A17SS (BCM), a transgenic strain expressing the galactose-inducible CYP52A17SS cytochrome P450 enzyme, was able to grow in a coconut milk factory wastewater (CCW) medium and produced 12-hydroxydodecanoic acid (HDDA) and 1,12-dodecanedioic acid (DDA). The supplementation of CCW with 10 g/L yeast extract and 20 g/L peptone (YPCCW) markedly increased the yeast growth rate and the yields of 12-HDDA and 1,12-DDA, with the highest levels of approximately 60 and 38 µg/L, respectively, obtained at 30 °C and pH 5. The incubation temperature and medium pH strongly influenced the yeast growth and 1,12-DDA yield, with the highest 1,12-DDA formation at 30 °C and pH 5–5.5. Hence, the S. cerevisiae BCM strain can potentially be used for producing value-added products from CCW.

Effect of commercial cellulose enzymes was investigated by batch enzymatic hydrolysis at 15.0% (w/v) solid. It was found that the best commercial cellulose enzyme was Cellic ® CTec comparing to Accellerase 1000 TM and Accelerase 1500 TM . The Cellic ® CTec gave the highest reducing sugar concentration and rice straw conversion. Moreover, when the hydrolysate obtained from hydrolysis using Cellic ® CTec was fermented by Saccharomyces cerevisiae TISTR 5596, it would give the highest ethanol. In this study, the Cellic ® CTec was used for fed-batch prehydrolysis prior to ethanol production by simultaneous saccharification and fermentation (SSF) way at 20% (w/v) solid loading. It could produce 35.76 g/L or 4.6% (v/v) of ethanol concentration and 83.67 L/ton dry matter (DM) of yield.

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The suitability of crude glycerol (75% (v/v) glycerol content) from a biodiesel production plant, and soybean meal and rice bran hydrolysates as replacements in the culture medium for commercially available glycerol, peptone and yeast extract, respectively, to form a low cost culture medium for the growth of, and recombinant endoglucanase II production by, Pichia pastoris was evaluated. The use of 1% (v/v) crude glycerol as a carbon source, and a mixture of 0.175% (v/v) soybean meal and 0.15% (v/v) rice bran hydrolysate in the presence of 0.5% (w/v) ammonium sulfate was found to be a suitable medium in the presence of 3% (v/v) methanol as the inducer. Under these conditions, the highest level of extracellular endoglucanase II activity (61.5 U/mL) was found after 120 h culture at 30°C and pH 6.0.