Archer Daniels Midland Company

Kostyantyn Dmytruk

Andriy Sibirny

Charles Abbas

Marta Semkiv

Barbara Kshanovska

Olena Dmytruk

Andriy Y Voronovsky

Andriy A Sibirny

Described herein are new approaches for the selection ofstrains with increased ethanol production from hydrolyzed starch derived sugars. An industrial production strain ofAS400 was subjected to positive selection of mutants resistant to toxic concentrations of oxythiamine, trehalose, 3-bromopyruvate, glyoxylic acid, and glucosamine. The selected mutants are characterized by 5-8% increase in ethanol yield (g gof consumed glucose) as compared to the parental industrial ethanol-producing strain. A multiple-step selection approach that consisted of the sequential selection using glyoxylic acid, glucosamine and bromopyruvate as selective agents resulted in a 12% increase in ethanol yield during fermentation on industrial media. These results indicate that the selection methods provided herein are useful for producing a variety of strains that are promising candidates for industrial ethanol production.

Methods for the positive selection of ethanol overproducing mutants from

Described herein is a method to increase ethanol yield during alcoholic fermentation by expression a truncated versions of thePHO8 gene coding for vacuolar or cytosolic form of alkaline phosphatase, which expression lowers biomass accumulation while diverting greater carbon to ethanol production. Also described are as nucleic acid sequences and vectors for expression of the PHO8 gene and strains carrying the same. Strains containing intact PHO8 gene encoding vacuolar form of alkaline phosphatase, had slightly lower intracellular ATP levels with insignificant changes in biomass accumulation and up to 13% increase in ethanol production.

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Recombinant genetic constructs and strains ofhaving significantly increased ethanol productivity with a simultaneous decreased production of xylitol during high-temperature xylose fermentation are disclosed. The constructs include aXYL1 gene encoding xylose reductase mutated to decrease affinity of the enzyme toward NADPH. The modified version of XYL1 gene under control of a strong constitutive HpGAP promoter was overexpressed in a &#x394;xyl1 background. A recombinantstrain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase in the &#x394;xyl1 background was also constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were evaluated during high-temperature xylose fermentation (48&#xb0; C.). A significant increase in ethanol productivity (up to 7.4 times) was shown in the recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably: 0.9 mg&#x3c7; (L&#x3c7;h)versus 4.2 mg&#x3c7; (Lxh)by the wild type strain.

A new approach for increase of ethanol yield during alcoholic fermentation via decrease of biomass accumulation by using ATP degrading enzymes is described. The part of theSSB1 gene coding for cytosolic ATPase domain of ribosome associated chaperon cloned into expression cassette under control of the glycerol-3-phosphate dehydrogenase gene (GPD1) promoter was introduced into theBY4742 strain. The recombinant strains were tested for their ability to grow and produce ethanol during glucose anaerobic and aerobic cultivations. Strains overexpressing ATPase domain of SSB1 possessed decreased concentration of intracellular ATP. They accumulated elevated amounts of ethanol and were characterized by decreased biomass accumulation as compared to the wild-type strain under both anaerobic and aerobic conditions. Similarly, the apyrase gene apy fromencoding ATP/ADP hydrolyzing phosphatase and ATPase domain of SSB1 gene ofwere co-expressed under the control of galactose-inducible GAL1 promoter. The recombinantstrains revealed slight reduction of biomass accumulation, while specific ATPase activity, ethanol accumulation and yield during alcoholic galactose fermentation under semi anaerobic conditions were increased.

Growing community of inventors

Lviv, Ukraine

Kostyantyn Dmytruk