Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for L-(؉)-lactate production. We constructed a P. stipitis strain that expressed the L-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to ϳ15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.

number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.The filamentous fungus Trichoderma reesei is known as an efficient producer of cellulases. The cellulolytic system of T. reesei is composed of two cellobiohydrolases (CBHI and CBHII) and at least five endoglucanases (EGI, EGII, EGIII, EGIV, and EGV) (19,20). Lack of EGII (originally called EGIII [18]) production reduces the endoglucanase activity in the culture supernatant by as much as 55%, whereas lack of EGI reduces it by only 25% (21). Thus, EGII is proposed to account for most of the endoglucanase activity produced by T. reesei (21). T. reesei EGI represents 5 to 10% of the secreted protein (16). The production of EGI has been improved in T. reesei by placing the egl1 gene under the control of the strong promoter of the Trichoderma CBHI (cbh1) gene and by increasing the copy number of the egl1 gene (8).Cellulases are used widely in the textile industry in treatments of cellulose-containing textile materials during their manufacture and finishing (5). The most well-known application is the use of cellulases in biostoning. Biostoning of fabric means the use of cellulases in place of, or in addition to, the use of pumice stones for the treatment of denim fabric to impart a stonewashed effect. Heikinheimo et al. (7) showed that T. reesei-purified cellulase EGII was the most effective at removing color from denim, producing a good stonewashing effect with the lowest hydrolysis level. Endoglucanases are important also for degradation of ␤-glucan in feed. Degradation of ␤-glucan lowers the viscosity of the intestinal contents and this improves the quality of the feed (3).In this study we have constructed T. reesei strains that produce elevated amounts of endoglucanase activity. The aim of our work was to construct different tailored high endoglucanase activity-producing strains for specific applications. We have improved the production of the EGII enzyme in T. reesei and we have constructed a T. reesei strain that produces high amounts of EGI and -II without any cellobiohydrolases. Cellulase preparations derived from these T. reesei overproduction strains were tested on the biostoning application. MATERIALS AND METHODSMicrobial strains and plasmi...

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BackgroundPolylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding l-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway.ResultsEach LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5.ConclusionsWe developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional ethanol pathway, at neutral and low pH, generated a comprehensive picture of lactic acid production in this yeast. The findings are applicable in generation other lactic acid producing yeast, thus providing a significant contribution to the field of biotechnical production of lactic acid.