Engineering Transaldolase in <i>Pichia stipitis</i> to Improve Bioethanol Production

In our effort to improve the efficiency and yield of xylose-to-ethanol bioconversion in <i>Pichia stipitis</i>, the transaldolase (TAL) in the pentose phosphate pathway was identified as a rate-limiting enzyme for improvement. A mutant containing the Q263R change was first obtained by directed evolution with 5-fold increase of activity, which was then incorporated into <i>P. stipites</i> <i>via</i> the pYDS vector to produce a genetically stable strain for fermentation on xylose. In comparison with the parental strain, TAL-Q263R­(+) increases ethanol prodcution by 36% and 100% as measured by volumetric production rate and specific production rate, respectively. Thus improving the transaldolase activity in <i>P. stipitis</i> can significantly increase the rate and yield of xylose conversion to ethanol

Effective Sugar Nucleotide Regeneration for the Large-Scale Enzymatic Synthesis of Globo H and SSEA4

We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in <i>E. coli</i> and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics