Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes. </p

モクシツ バイオマス カラ ノ コウコウリツ エタノール セイサン オ メザシタ タンパクシツ コウガク ニ ヨル キシロース タイシャ コウソ ノ コウオン アンテイカ ニ カンスル ケンキュウ

京都大学0048新制・課程博士博士(エネルギー科学)甲第13178号エネ博第152号新制||エネ||36(附属図書館)UT51-2007-H451京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻(主査)教授 牧野 圭祐, 教授 吉川 暹, 助教授 小瀧 努学位規則第4条第1項該当Doctor of Energy ScienceKyoto UniversityDA

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing proteinengineered NADH-preferring xylose reductase from Pichia stipitis

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD+-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(kcat/Km with NADH)/(kcat/Km with NADPH)] compared with the wild-type (WT), which was due to decrease of kcat with NADPH in the R276H mutant and increase of Km with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20% increase of ethanol production and 52% decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP+ and NADH/NAD+ ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae