The effects of Metschnikowia pulcherrima and high glucose osmolality on S. cerevisiae pyruvate dehydrogenase pathway (PDH) by-pass were examined by varying the starting sugar concentration of synthetic grape juice and the inoculation ratio of S. cerevisiae to M. pulcherrima. The findings revealed that M. pulcherrima and osmolarity impacted S. cerevisiae’s PDH by-pass. The inoculation concentration of M. pulcherrima significantly affected pyruvate decarboxylase (PDC) activity and acs2 expression when the initial sugar concentration was 200 g L⁻¹ and 290 g L⁻¹. The osmolarity caused by the initial sugar (380 g L⁻¹) significantly influenced the enzymatic activity of S. cerevisiae, which decreased PDC and acetaldehyde dehydrogenase (ALD) activities while increasing Acetyl-CoA synthetase (ACS) activity. The reduction in acetic acid in the wine was caused by M. pulcherrima altering the initial sugar concentration faced by S. cerevisiae, which in turn affected enzymatic activity. The alteration of enzyme activity and accumulation of primary metabolites revealed why mixed fermentation could reduce the acetic acid content in wine by altering the enzymatic activity and affecting the expression of several key genes. The M. pulcherrima inoculation levels had no significant effect on the acetic acid and glycerol concentration in the same fermentation medium.

Algin is the most abundant substance in alga. Alginate lyase degrades algin and produces algin monosaccharides, disaccharides, and oligosaccharides, which are widely used in bioenergy, food, medicine, and other fields. In this study, one Exiguobacterium strain isolated from rotten kelp exhibited a robust ability to degrade the alga. The sequencing of this strain revealed the presence of three different types of algin alginate lyase. Nevertheless, the expression of three genes in Escherichia coli revealed a lower alginate lyase activity compared to that of the original strain. After codon optimization, the gene with the highest activity of the three was successfully expressed in Pichia pastoris to produce recombinant EbAlg664. The activity of the recombinant enzyme in 5 L high-density fermentation reached 1306 U/mg protein, 3.9 times that of the original Exiguobacterium strain. The results of the enzymatic analysis revealed that the optimal temperature and the pH range of recombinant EbAlg664 were narrower compared to the original strain. Additionally, the presence of Cu²⁺ and Co²⁺ enhanced the enzymatic activity, whereas Mg²⁺ and Fe³⁺ exhibited inhibitory effects on the recombinant alginate lyase. The study offers a theoretical and practical foundation for the industrial-scale production of engineered Pichia pastoris with high alginate lyase activity.