Loss of a Functional Mitochondrial Pyruvate Carrier in Komagataella phaffii Does Not Improve Lactic Acid Production from Glycerol in Aerobic Cultivation.

Cytosolic pyruvate is an essential metabolite in lactic acid production during microbial fermentation. However, under aerobiosis, pyruvate is transported to the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) and oxidized in cell respiration. Previous reports using Saccharomyces cerevisiae or Aspergillus oryzae have shown that the production of pyruvate-derived chemicals is improved by deleting the MPC1 gene. A previous lactate-producing K. phaffii strain engineered by our group was used as a host for the deletion of the MPC1 gene. In addition, the expression of a bacterial hemoglobin gene under the alcohol dehydrogenase 2 promoter from Scheffersomyces stipitis, known to work as a hypoxia sensor, was used to evaluate whether aeration would supply enough oxygen to meet the metabolic needs during lactic acid production. However, unlike S. cerevisiae and A. oryzae, the deletion of Mpc1 had no significant impact on lactic acid production but negatively affected cell growth in K. phaffii strains. Furthermore, the relative quantification of the VHb gene revealed that the expression of hemoglobin was detected even in aerobic cultivation, which indicates that the demand for oxygen in the bioreactor could result in functional hypoxia. Overall, the results add to our previously published ones and show that blocking cell respiration using hypoxia is more suitable than deleting Mpc for producing lactic acid in K. phaffii.

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae.

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.