Takushi Hatano

Overexpression and secretion of cellulolytic enzymes by δ-sequence-mediated multicopy integration of heterologous DNA sequences into the chromosomes of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae transformants which secrete high levels of cellulolytic enzymes, with chromosome-integrated multicopies of heterologous DNA sequences encoding the cellulolytic enzymes were constructed. An expression construct of β-glucosidase and carboxymethyl cellulase directed by the GAP promoter was integrated into the chromosomes of the haploid S. cerevisiae using the δ sequence-mediated integration system. Southern blot analysis of the chromosomes prepared from various integrants and separated by pulse-field gel electrophoresis demonstrated that the integration occurred mainly in a particular chromosome and the copy number of the integration was variable. The amount of enzymes secreted by the transformants correlated with the copy number of integration. For each enzyme, the highest activity was about 1.4-fold that produced by the transformant harboring the same expression cassette on a YEp-type plasmid. The δ-integrated exogenous DNA was mitotically stable in rich medium. A haploid double transformant which coexpresses and secretes β-glucosidase and carboxymethyl cellulase was further constructed by genetic crossing of the haploid transformant that produces a high level of the enzyme, followed by meiotic segregation of the resulting diploid strain. The haploid double transformant, but neither of the single transformant, could grow on a plate containing carboxymethyl cellulose as a sole carbon source. It is suggested that the δ-sequence-mediated integration system is a very useful means for the genetic engineering of yeast, especially when overproduction and secretion of multiple heterologous enzymes are desired.

Production of ketoalkanes from fatty acid esters by a fungus, Trichoderma

A fungal strain, Trichoderma sp. SM-30, isolated from soil as a kerosene-using strain, metabolized fatty acid esters and extracellularly accumulated ketoalkanes having a one-carbon-atom shorter chain than those of the substrate fatty acids. The molecular species of ketoalkane formed from tricaprin was only one and identified to be methylheptylketone, a methylalkylketone (MAK). When triacylglyceride was used as carbon source, the microorganism selectively converted 6–14 carbons fatty acids to the corresponding-chain-sized MAKs. The maximal yield of MAK was approximately 36% (wt. of formed MAK/wt. of consumed substrate) in a culture containing tricaprin as the sole carbon source. Among six type-culture strains of Trichoderma, three strains had the ability to accumulate MAK in the medium.

Screening of methylalkylketone-accumulating fungi from type culture strains

Abstract Fungal strains having the ability to accumulate methylalkylketones (MAKs) in culture medium containing tricaprin (synthetic triglyceride) or palm-kernel oil (natural triglycerides) were selected from type culture strains. Twenty two strains which were high accumulators were selected and concentrated taxonomically into the filamentous fungi of Deuteromycotina and Ascomycotina . The selected strains were divided into the following two types in terms of their MAK formation characteristics. The first type shows a high accumulation of methylheptylketone from tricaprin but a low accumulation of MAKs from palm-kernel oil, and the second type shows a high accumulation of MAKs from both tricaprin and palm-kernel oil. Fusarium, Trichoderma , and their related genera are grouped into the first type, and Penicillium, Aspergillus, Cladosporium , and their related genera are in the second type. The molecular sizes of the MAKs accumulated were short and middle carbon-lengths; carbon numbers 5–13. Specificity in the bioconversion of fatty acids to MAKs is as follows: one-carbon-atom-shorter MAK molecules (carbon number n-1) from fatty acids (carbon number n), constituents of triglycerides, are exclusively formed. However, some strains formed unknown product(s) other than MAKs. During time course experiments for MAK accumulation by P. decumbens IFO 7091, the degradation of accumulated MAK was discovered.

Extracellular formation of triglycerides from glucose by a mutant strain of Trichosporon

Abstract A strain, L-12, which secretes triglycerides (TG) was selected from low cell-density mutants of yeast Trichosporon sp. The strain was superior to the wild type strain in the extracellular production of TG from glucose; the production is over 75% of total TG accumulated.

Extracellular production of palmitoleic triglycerides by a yeast, Trichosporon

Abstract A yeast belonging to Trichosporon which produces triglycerides extracellularly was isolated. This strain accumulated palmitoleic triglycerides from ethyl palmitate used as a sole carbon source. To increase the level of extracellular palmitoleic triglycerides, mutant strains which supported growth of unsaturated-fatty-acid-auxotrophic cells ( Saccharomyces cerevisiae KD115) layered on the mutant colonies were screened. The mutant strain excreted palmitoleic acid as triglyceride form at a significantly high level, corresponding to about double level of the parental strain.

Purification and characterization of a carboxymethylcellulose-degrading enzyme secreted by a yeast strain newly isolated from soil

Abstract Eight yeast strains (MUT-series) possessing high abilities to utilize carboxymethylcellulose (CMC) were obtained from 127 strains previously isolated from soil. A strain MUT-6 secreted a carboxymethylcellulose-degrading enzyme (CMCase) and accumulated oligosaccharides in a CMC-medium. The CMCase was purified from the culture fluid of the yeast as a single protein (MW: 34,000 daltons) in SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the enzyme was as follows: H-Lys-Leu-Pro-Tyr-Leu-Gly-Gly-Val-Asn-Leu-Ala-Gly-Thr-Asp-Phe-Gly-Ile-Asp-Ile-Tyr- Gly-. Optimal conditions for enzyme reaction were 65–70°C for temperature and 4.3 for pH. Heat stability was remarkably high; half-life time at 98°C (pH 6.0) was 30 min. Products from hydrolysis of CMC by the CMCase were di-, tri-, and tetra-oligosaccharides without mono-mer, and could be metabolized by a transformant of Saccharomyces cerevisiae with a plasmid carrying the BGL 1 gene encoding cellobiase of Saccharomycopsis fibuligera. Cellubiose, as well as CMC, was found to be an effective inducer for CMCase production.

Methylalkylketone Fermentation from Palm-Kernel Oil by Penicillium decumbens IFO 7091

Abstract To make the production of methylalkylketones (MAKs) from palm-kernel oil by practical fermentation using Penicillium decumbens IFO 7091, the accumulation of MAKs was studied. The optimal culture conditions were temperature, 28°C, and initial pH, 8.2, in the basal medium. The MAKs accumulated were methylpentylketone, methylheptylketone, methylnonylketone, and methylundecylketone; in these MAKs, methylnonylketone is major. Under the culture conditions described above, the microorganism accumulated 2.05 g of MAKs with consumption of 5.14 g of fatty acids in 8 d of shaking-culture in 100 ml of medium with 10 g of palm-kernel oil. Approximately 47% of the constituent total fatty acids of palm-kernel oil are layric acid, it was metabolized rapidly and converted to methylnonylketone in a high yield, over 46.4% (formed methylnonylketone/consumed lauric acid), while long chain fatty acids, such as palmitic, stearic, oleic, and linoleic acids, were hardly metabolized with little MAK accumulation. In a 5- l jar fermentor, in which 270 g of palm-kernel oil were used as substrate, 58.7 g of MAKs were obtained from 144.5 g of consumed fatty acids after 7 d of culture.

Expression of the carboxymethylcellulase gene, CMC1, from Cryptococcus flavus in Saccharomyces cerevisiae

Abstract A cDNA copy of the carboxymethylcellulase (CMCase 1) gene, CMC1 , of the yeast Cryptococcus flavus was expressed in Saccharomyces cerevisiae under the control of the glyceraldehyde-3-phosphate dehydrogenase gene ( GAP ) promoter derived from S. cerevisiae . In glucose medium, extracellular production of a large amount of CMCase 1 was achieved by S. cerevisiae cells transformed with YEp-plasmid carrying CMC1 . Two major proteins (36 kDa and 34 kDa) secreted in the medium both had CMCase activity and immuno-responsibility to anti-CMCase 1-antibody. From an analysis of amino acid sequences, it was found that the 36 kDa protein is a pro-CMCase 1 having an N-terminal residue, Ala, the 19th amino acid of the peptide deduced from the nucleotide sequence of CMC1 on the cDNA, and that the 34 kDa protein is a mature CMCase 1. Alteration of the cell morphology of S. cerevisiae was induced by transformation with YEp-plasmid carrying CMC1 .

Formation of monohydroxy-n-nonane-2-ones from tricaprin by Fusarium avenaceum f. sp. fabae IFO 7158

Abstract Fusarium avenaceum f. sp. fabae IFO 7158 metabolized tricaprin with accumulation of n-nonane-2-one and three unknown minor compounds in the medium. The three compounds were identified to be 8-hydroxy-n-nonane-2-one, 7-hydroxy-n-nonane-2-one, and 6-hydroxy-n-nonane-2-one. The accumulation of the monohydroxy-n-nonane-2-ones took place with a decrease in n-nonane-2-one (C9:0-2-one) once formed from capric acid (C10:0). Thus, it is speculated that hydroxylations of n-nonane-2-one at ω2, ω3, and ω4 carbons gave the 8-hydroxy-, 7-hydroxy-, and 6-hydroxy-n-nonane-2-ones, respectively. Even though the microorganism could utilize well for growth all of the synthetic triglycerides, such as tricaproin, tricaprylin, tricaprin, trilaurin, trimyristin, and tripalmitin, the monohydroxy-n-alkane-2-one was formed exclusively from tricaprin. The maximal accumulation of monohydroxy-n-nonane-2-ones, a mixture of 8-hydroxy-, 7-hydroxy- and 6-hydroxy-n-nonane-2-ones in an approximate ratio of 10:7:0.5, was 139 mg from 1.0 g tricaprin. Finally, a novel metabolic pathway for the medium chain fatty acid in the fungus is discussed.

Subterminal hydroxylation of ketoalkanes by fungi

Abstract Fusarium avenaceum f. sp. fabae IFO-7158 metabolized ketoalkanes and accumulated two or three monohydroxy forms from each ketoalkane. The positions for hydroxylation tended to shift to the ketonic group with an increase in the carbon number of the ketoalkane alkyl chain. In the cases of hexyl, heptyl and octyl groups, mainly the ω2 and ω3 positions were hydroxylated. For nonyl and undecyl groups, the substrates were converted into ω2-, ω3-, and ω4-derivatives and ω3-, ω4-, and ω-derivatives, respectively. Subterminal hydroxylation of n-undecan-2-one was also carried out by Penicillium, Aspergillus, Trichoderma, and Cladosporium. Cleavage reactions of a CC bond accompanied by subterminal hydroxylation of n-undecan-2-one and n-tridecan-2-one.