Biomedical subjects

Patricia J Slininger

Publications and source records attributed to Patricia J Slininger.

4 recordsLinked to original sources

PubMed2026

Structural genome variation drives adaptation of the xylose-fermenting yeast Scheffersomyces stipitis to lignocellulosic hydrolysates.

Second-generation (2G) bioethanol from lignocellulosic feedstocks is a sustainable alternative to fossil fuels. However, its production is constrained by the poor performance of industrial microbes in hydrolysates that are generated during biomass pretreatment. Scheffersomyces stipitis is a native xylose fermenting yeast and a promising platform for 2G bioethanol production, and adaptive evolution under hydrolysate stress has yielded strains with enhanced performance. However, the chromosomal basis of this adaptation is unknown. Here, we demonstrate that chromosome scale structural variation, rather than point mutations, underlies the improved phenotype of the evolved strains. By integrating long- and short-read genome sequencing, we identify two major chromosomal rearrangements in the top performing isolate: a reciprocal translocation between chromosomes 1 and 2 that disrupts the NUDIX hydrolase gene YSA1, and the formation of a mitotically stable 175 kb minichromosome derived from chromosome 5. Functional analyses show that disruption of YSA1 enhances xylose utilisation and ethanol yield, while the minichromosome contributes to improved performance in hydrolysate conditions. These findings provide direct evidence that balanced rearrangements and minichromosome formation can be selected during prolonged stress and can generate adaptive phenotypes. Taken together, our study establishes genome reorganisation as a key driver of adaptation in S. stipitis.

Xylose↗

PubMed2006

Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR.

Gene expression analysis provides significant insight to understand regulatory mechanisms of biology, yet acquisition and reproduction of quality data, as well as data confirmation and verification remain challenging due to a lack of proper quality controls across different assay platforms. We present a set of six universal external RNA quality controls for microbial mRNA expression analysis that can be applied to both DNA oligo microarray and real-time qRT-PCR including using SYBR Green and TaqMan probe-based chemistry. This set of controls was applied for Saccharomyces cerevisiae and Pseudomonas fluorescens Pf-5 microarray assays and qRT-PCR for yeast gene expression analysis. Highly fitted linear relationships between detected signal intensity and mRNA input were described. Valid mRNA detection range, from 10 to 7000 pg and from 100 fg to 1000 pg were defined for microarray and qRT-PCR assay, respectively. Quantitative estimation of mRNA abundance was tested using randomly selected yeast ORF including function unknown genes using the same source of samples by the two assay platforms. Estimates of mRNA abundance by the two methods were similar and highly correlated in an overlapping detection range from 10 to 1000 pg. The universal external RNA controls provide a means to compare microbial gene expression data derived from different experiments and different platforms for verification and confirmation. Such quality controls ensure reliability and reproducibility of gene expression data, and provide unbiased normalization reference for validation, quantification, and estimate of variation of gene expression experiments. Application of these controls also improves efficiency and facilitates high throughput applications of gene expression analysis using the qRT-PCR assay.

Animals↗

PubMed2006

Nitrogen source and mineral optimization enhance D: -xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124.

Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61+/-9 g/l) by providing C:N in the vicinity of approximately 57-126:1 and optimizing the combination of urea and amino acids to supply 40-80 % nitrogen from urea and 60-20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others.

Amino Acids↗

PubMed2005

Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains.

Furfural and hydroxymethylfurfural (HMF) are representative inhibitors among many inhibitive compounds derived from biomass degradation and saccharification for bioethanol fermentation. Most yeasts, including industrial strains, are susceptible to these inhibitory compounds, especially when multiple inhibitors are present. Additional detoxification steps add cost and complexity to the process and generate additional waste products. To promote efficient bioethanol production, we studied the mechanisms of stress tolerance, particularly to fermentation inhibitors such as furfural and HMF. We recently reported a metabolite of 2,5-bis-hydroxymethylfuran as a conversion product of HMF and characterized a dose-dependent response of ethanologenic yeasts to inhibitors. In this study, we present newly adapted strains that demonstrated higher levels of tolerance to furfural and HMF. Saccharomyces cerevisiae 307-12H60 and 307-12H120 and Pichia stipitis 307 10H60 showed enhanced biotransformation ability to reduce HMF to 2,5-bis-hydroxymethylfuran at 30 and 60 mM, and S. cerevisiae 307-12-F40 converted furfural into furfuryl alcohol at significantly higher rates compared to the parental strains. Strains of S. cerevisiae converted 100% of HMF at 60 mM and S. cerevisiae 307-12-F40 converted 100% of furfural into furfuryl alcohol at 30 mM. The results of this study suggest a possible in situ detoxification of the inhibitors by using more inhibitor-tolerant yeast strains for bioethanol fermentation. The development of such tolerant strains provided a basis and useful materials for further studies on the mechanisms of stress tolerance.

Biotransformation↗