Xylose fermentation by Saccharomyces cerevisiae using endogenous xylose-assimilating genes.

OBJECTIVES: To genetically engineer Saccharomyces cerevisiae for improved ethanol productivity from glucose/xylose mixtures. RESULTS: An endogenous gene cassette composed of aldose reductase (GRE3), sorbitol dehydrogenase (SOR1) and xylulose kinase (XKS1) with a PGK1 promoter and a terminator was introduced into two S. cerevisiae strains, a laboratory strain (CEN.PK2-1C) and an industrial strain (Kyokai No. 7). The engineered Kyokai No. 7 strain (K7-XYL) exhibited a higher sugar consumption rate (1.03 g l(-1) h(-1)) and ethanol yield (63.8 %) from a glucose and xylose mixture compared to the engineered CEN.PK2-1C strain. Furthermore, K7-XYL produced a larger amount of ethanol (39.6 g l(-1)) compared to K7-SsXYL (32 g l(-1)) with integrated xylose reductase and xylitol dehydrogenase from a xylose-assimilating yeast Scheffersomyces stipitis instead of GRE3 and SOR1. CONCLUSION: The created S. cerevisiae strain showed sufficient xylose-fermenting ability to be used for efficient ethanol production from glucose/xylose.

Kinetic modeling and sensitivity analysis for higher ethanol production in self-cloning xylose-using Saccharomyces cerevisiae.

We constructed a xylose-utilizing Saccharomyces cerevisiae strain using endogenous xylose-assimilating genes (strain K7-XYL). Such self-cloning yeast is expected to make a great contribution to cost reduction of ethanol production processes. However, it is difficult to modify self-cloning yeast for optimal performance because the available gene source is limited. To improve the ethanol productivity of our self-cloning yeast, a kinetic model of ethanol production was constructed and sensitivity analysis was performed. Alcohol dehydrogenase (ADH1) was identified as a metabolic bottleneck reaction in the ethanol production pathway. An ADH1 overexpression strain (K7-XYL-ADH1) was constructed and evaluated in YP (yeast extract 10 g/L, peptone 20 g/L) medium containing 50 g/L xylose as the sole carbon source. Strain K7-XYL-ADH1 showed higher ethanol productivity (13.8 g/L) than strain K7-XYL (12.5 g/L). Then, K7-XYL-ADH1 was evaluated in YP medium containing 80 g/L glucose and 50 g/L xylose; however, the ethanol productivity did not change relative to that of K7-XYL (K7-XYL 46.3 g/L, K7-XYL-ADH1 45.9 g/L). We presumed that due to the presence of glucose, the internal redox balance of the cells had changed. On culturing in an aerated 5-L jar fermentor to change the internal redox balance of cells, strain K7-XYL-ADH1 showed higher ethanol productivity than K7-XYL (K7-XYL 45.0 g/L, K7-XYL-ADH1 49.4 g/L). Our results confirmed that ADH1 was a metabolic bottleneck in the ethanol production pathway. By eliminating the bottleneck, self-cloning yeast showed almost the same ethanol productivity as genetically modified yeast.

Residue 345 of dibenzothiophene (DBT) sulfone monooxygenase is involved in C-S bond cleavage specificity of alkylated DBT sulfones.

Rhodococcus erythropolis IGTS8 that possesses dibenzothiophene sulfone monooxygenase mutated at residue 345 (Q345A), can degrade octyl sulfide on which the wild strain cannot grow. Residue 345 and the neighbouring residues were changed by site-directed mutagenesis. Only DszA changed at residue 345 gave an altered C-S bond cleavage pattern of 3-methyl DBT sulfone. This residue is therefore involved in C-S bond cleavage specifically for alkylated DBT sulfone.

Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65.

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (Cx-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C8-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C6-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated Cx-DBTs is the transfer process from the oil phase into the cell.