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:Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here, we operated two laboratory-scale sequence batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal (EBPR). Reactors formed two distinct biofilms, a floccular biofilm, consisting of small, loose, microbial aggregates, and a granular biofilm, forming larger, dense, spherical aggregates. Using metaproteomic methods we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. Both biofilms contained proteins that were indicative of core EBPR metabolisms and cellular function. To understand the proteomic differences between floccular and granular biofilm communities, we compared protein abundances that were statistically enriched in both biofilm states (alpha level = 0.05). Floccular biofilms were enriched with pathogenic secretion systems suggesting a previously unrecognized, highly competitive, mixed microbial community. Comparatively, granular biofilms revealed a high stress environment with evidence of nutrient starvation, phage predation pressure, extracellular polymeric substance (EPS) synthesis, and increased cell lysis. Granular biofilms enriched outermembrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement core EBPR metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter–enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.
Project description:Regulatory T cells (Tregs) are known to maintain survival and suppressive function in the presence of high levels of extracellular lactic acid. However, the effect of lactic acid on Treg induction is not known. We therfore evaluated the effect of lactic acid on Treg induction and observed an increased induction of Tregs in the presence of lactic acid. This increase occurred in a glycolysis-independent, acidity-dependent manner.
Project description:The goal of this study was to use global gene expression as a diagnostic tool to compare hepatic gene expression patterns in both male and female FHM in streams with the lowest and highest reproductive success, and potentially identify a suite of mRNA transcripts indicative of reproduction in a population The goal of this study was to compare differences in hepatic mRNA expression between gender at high and low egg-producing streams, not differences between individual streams. A k-means cluster analysis was performed using eggs/pair/day on the original 17 streams to delineate 3 clusters: high, medium and low. From that analysis, FHM from 6 of the original 17-streams used in Crago et al. (2010) were chosen for the microarray experiment (Fig. 1, Table 1). In this study the experimental condition is reproductive success; High versus Low reproductive success. The streams grouped into High Reproductive Success were Oak Creek-2007 (2313 eggs), Point Creek (1277 eggs), Meeme Creek (1164 eggs) and Baird Creek (967 eggs). The streams grouped into Low Reproductive Success were: Ashwaubenon Creek (0 eggs), Devils Creek (541 eggs) and Oak Creek-2006 (642 eggs). Multiple regression analysis using the 22 sediment and water quality characteristics measured in the 6 streams with the highest (n = 4 and lowest (n = 3) streams demonstrated that there were no differences amongst the streams in regards to measure sediment and water variables. .5 One array was run for each gender from each stream. So that Males from Point Creek were pooled and run on one array, males from Ashwaubenon Creek were run on a separate array, and so forth. There were 14 arrays used in this study, 7 for males, 7 for females from individual rivers. So that Males from Point Creek were pooled and run on one array, males from Ashwaubenon Creek were run on a separate array, and so forth. In the case of Oak Creek, which was sampled in both years, there was a large difference in egg production between two years. Therefore separate arrays were run for Oak Creek 2006 and Oak Creek 2007. All streams chosen had overall survival rates of at least 80% through the 21-day sampling period, except Devils River. The survival rate for Devils River was at 100% until four days prior to the end of the experiment when six fish died or escaped.