Direct Production of Difructose Anhydride IV from Sucrose by Co-fermentation of Recombinant Yeasts

A functional sweetener, difructose anhydride IV (DFA IV), is enzymatically produced from sucrose via levan by levansucrase (LSRase) followed by levan fructotransferase (LFTase). Here, we have demonstrated a consolidated production system for the direct conversion of DFA IV from sucrose using the co-culture of two recombinant yeast strains secreting LSRase from Bacillus subtilis and LFTase from Arthrobacter ureafaciens, respectively. To ensure secretory production of the enzymes, target protein-specific translational fusion partners (TFP) were employed, and the selected strains produced 3.8 U/mL of LSRase and 16.0 U/mL LFTase activity into the fermentation broth. To optimise the direct production, sucrose concentration and cell ratios were investigated. In the optimised conditions, 64.3 g/L crude DFA IV was directly produced from 244.7 g/L sucrose using co-fermentation of recombinant yeasts. These results promise an efficient production titre, yield, and DFA IV productivity in an industrially applicable method.

Effect of MIG1 Gene Deletion on Glucose Repression in Baker’s Yeast

Mig1p, a zinc finger class of DNA-binding protein, plays a critical role in glucose repression for maltose utilization in Baker’s yeast. Maltose is the hydrolyzate of starch, which is the main component of dough. In this study, the recombinant yeast strains with MIG1 gene deletion were constructed, and the maltose metabolism of the parental and mutant strains in batch cultivations were investigated. Our results show that the degree of glucose repression of mutants △MIG1α and △MIG1a are reduced by 49.88% and 41.59% respectively compared to their parental strains, suggesting that MIG1 deletion can partially relieve glucose repression of maltose metabolism.

Enhanced Susceptibility to Antifungal Oligopeptides in Yeast Strains Overexpressing ABC Multidrug Efflux Pumps

ABSTRACT The susceptibility to several oligopeptide and amino acid antifungals of a Saccharomyces cerevisiae strain carrying multiple deletions in yeast multidrug resistance genes was compared to transformants containing the CDR1, CDR2, or MDR1 genes that encode the major Candida albicans drug efflux pumps. Recombinant yeast strains overexpressing Cdr1p and Cdr2p showed enhanced susceptibilities to all tested oligopeptide antifungals. The enhanced susceptibilities of multidrug-resistant yeast strains to oligopeptide antifungals corresponded to higher rates of oligopeptide uptake. Yeast cells overexpressing Cdr1p or Cdr2p effluxed protons at higher rates than the reference cells lacking these ABC transporters. An increased plasma membrane electrochemical gradient caused by the functional overexpression of Cdr1p or Cdr2p appeared to increase cellular susceptibility to oligopeptide antifungals by stimulating their uptake via oligopeptide permeases.

Production of l-Ascorbic Acid by Metabolically Engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii

ABSTRACT Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, l-galactono-1,4-lactone, or l-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and d-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme l-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous d-arabinono-1,4-lactone oxidase as well as l-galactose dehydrogenase are capable of producing about 100 mg of l-ascorbic acid liter−1, converting 40% (wt/vol) of the starting compound l-galactose.