Research Tools : Fermentation


Yeast-Based Intein Platform for Drug Production

UW–Madison researchers have engineered non-self-cleaving Mxe GyrA inteins shown to significantly improve the production of fusion proteins from Saccharomyces cerevisiae. The novel inteins were developed through directed evolution, and they enhance fusion protein display (up to 3x) and secretion levels (up to 30x) compared to the wild type intein. The new yeast-based platform provides a robust alternative to bacterial intein expression systems.

Small Molecule Catalysts of Oxidative Protein Folding

UW–Madison researchers have synthesized novel catalysts for use in the production of high value recombinant proteins including antibodies and other biologics. The small molecules contain binding features that mimic PDI and are nearly optimal for catalyzing disulfide bond formation. Namely, pendant “R” groups provide affinity for the hydrophobic substrate (unfolded protein) but not the product (folded protein), enabling the catalysts to operate like a true enzyme.

The novel catalysts and method can be used in vivo or in vitro.

More Efficient Ethanol Production from Mixed Sugars Using Spathaspora Yeast

UW–Madison researchers have developed a method for producing ethanol using Spathaspora passalidarum yeast to ferment xylose or cellobiose, even when mixed with glucose.

The ethanol is converted from biomass or other lignocellulosic material from agricultural residues, fast-growing hardwoods and processing byproducts. Sugars, lignin and other components are first extracted from this feedstock using standard methods to form mixtures rich in different sugars. The mixture is contacted with a Spathaspora yeast cell under oxygen-limiting conditions suitable to allow the yeast to ferment a portion of the xylose and/or cellobiose into ethanol.

Xylose-Fermenting Recombinant Yeast Strains

UW-Madison researchers have developed a xylose-fermenting recombinant yeast strain that expresses xylose reductase, xylitol dehydrogenase and xylulokinase but has reduced expression of the phosphatase PHO13. This strain of S. cerevisiase can increase conversion of xylose-containing biomass into ethanol and is not inhibited by xylulokinase overexpression.

Xylose-Fermenting, Recombinant Yeast Strains for Use in Ethanol Production

UW-Madison researchers have now devised a way to increase ethanol production from plant biomass by engineering the glucose-fermenting yeast Saccharomyces cerevisiae to ferment xylose. To create the recombinant strains, the inventors transformed S. cerevisiae with three genes from the xylose-fermenting yeast Pichia stipitis. The genes, called XYL1, XYL2 and XYL3, encode the enzymes xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XR), respectively. To overcome problems associated with XR over-expression, transformants were selected for rapid growth on xylose, which is correlated with moderate XR expression and high ethanol production. To direct carbon use to the production of ethanol rather than cell mass, the strains were further engineered for down-regulation of respiration.

Thermostable Barley Alpha-Glucosidase for Improved Ethanol Production

UW-Madison researchers have developed a mutant barley alpha-glucosidase with increased thermal stability. They developed thermostable forms of the enzyme using site directed mutagenesis. Sites for mutagenesis were selected through comparisons with the sequences of other, more thermostable, alpha-glucosidase proteins.

Method for Preventing Superoxide Damage to Cells and Oxygen-Labile Proteins

UW-Madison researchers have developed a method of protecting cells and oxygen-labile enzymes from oxidative damage. YggX is a protein identified from Salmonella enterica serovar Typhimurium. Elevated levels of the YggX protein increase the resistance of Salmonella enterica to superoxide stress and reduce damage to its DNA. Also, when high levels of this protein are present in the cell, enzymes that are normally susceptible to oxidative damage remain active. Engineering cells to overexpress the YggX protein or its homolog renders the cells more resistant to oxidative damage. The YggX protein or its homolog could also be used to protect an oxygen-labile protein from superoxide damage by co-expressing YggX with the oxygen-labile protein.

Sham-Sensitive Terminal Oxidase Gene From Xylose-Fermenting Yeast

UW-Madison researchers have developed an additional xylose-fermenting, mutant yeast strain capable of increased ethanol production, which may be used to convert xylose in xylose-containing media into ethanol. This mutant also disrupts respiration in Pichia stipitis, but through an alternate pathway called the SHAM-sensitive respiratory pathway. The researchers created the mutant by removing or replacing at least part of the functional SHAM-sensitive terminal oxidase gene natively present in the parent strain with nonfunctional DNA.

Disruption of the Cytochrome C Gene in Xylose-Fermenting Yeast

UW-Madison researchers have developed a xylose-fermenting, mutant yeast strain capable of increased ethanol production, as well as a method for converting xylose in xylose-containing media into ethanol.  The invention uses a mutant strain of Pichia stipitis that exhibits reduced expression of functional c type cytochrome. This disruption of the cytochrome c gene routes a large fraction of the yeast's reducing power into fermentative activity, increasing the ethanol production rate from xylose two-fold.

Expression System and Fermentation Processes For Overexpression Of Holo-Acyl Carrier Protein

UW-Madison researchers have created eight bacterial expression plasmids from either E. coli or spinach, which express both apo-ACP and E. coli holo-ACP synthase, the enzyme responsible for the post-translational modification of ACP. Therefore, the plasmids allow completion of the required modification. The researchers' expression system enables the production of ACPs in high yields by using chemically defined minimal medium and specialized fermentation procedures.