Project description:D-lactic acid is a three-carbon organic acid with a chiral structure and can improve the thermostability of polylactic acid. Microorganisms such as the methylotrophic yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of D-lactic acid, have been engineered to produce it in high titers. However, tolerance to D-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to D-lactic acid and leads to increased D-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high D-lactic acid concentrations. Furthermore, integrating a D-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that can produce D-lactic acid at a titer of 5.12 0.35 g/L in 48 hours , a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to D-lactic acid including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of D-lactic acid by manipulating yeast flocculation.
Project description:Lactic acid bacteria (LAB) belong to an economically important group of Gram-positive microorganisms, whose main characteristic is the production of lactic acid by carbohydrates fermentation. Lactobacillus paraplantarum CRL 1905 is a LAB isolated from quinoa sourdoughs with biotechnological potential as a starter or probiotic. Inorganic phosphate (Pi) is an essential nutrient for most bacteria cell functions and it is involved in many regulatory processes. The aim of the project was to evaluate the influence of environmental Pi concentration in different physiological and molecular aspects of the CRL 1905 strain. Phenotypic and proteomic data provide new insights to understand the adaptations in several metabolic pathways that CRL 1905 experiments in response to differential Pi conditions.
Project description:Lactococcus lactis is the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme in aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures: The acetate pathway contributes to biomass yield in respiration, without affecting medium pH. The acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways could lead to fine tuning respiration growth, with improved yield and stability.