Project description:The yeast strain SJP-SNU was investigated as a probiotic and was characterized with respect to growth temperature, bile salt resistance, hydrogen sulfide reducing activity, intestinal survival ability and chicken embryo pathogenicity. In addition, we determined the complete genomic and mitochondrial sequences of SJP-SNU and conducted comparative genomics analyses. SJP-SNU grew rapidly at 37 °C and formed colonies on MacConkey agar containing bile salt. SJP-SNU reduced hydrogen sulfide produced by Salmonella serotype Enteritidis and, after being fed to 4-week-old chickens, could be isolated from cecal feces. SJP-SNU did not cause mortality in 10-day-old chicken embryos. From 13 initial contigs, 11 were finally assembled and represented 10 chromosomal sequences and 1 mitochondrial DNA sequence. Comparative genomic analyses revealed that SJP-SNU was a strain of Pichia kudriavzevii. Although SJP-SNU possesses pathogenicity-related genes, they showed very low amino acid sequence identities to those of Candida albicans. Furthermore, SJP-SNU possessed useful genes, such as phytases and cellulase. Thus, SJP-SNU is a useful yeast possessing the basic traits of a probiotic, and further studies to demonstrate its efficacy as a probiotic in the future may be warranted.
Project description:RNA-seq libraries were constructed for three samples, including (I) ΔURA3 strain grown in SD medium containing 2% w/v glucose(pH=5.0);(II)ΔURA3 strain grown in SD medium containing 2%w/v glucose and 2% w/v succinic acid(pH=5.0);(III)ΔURA3 strain grown in SD medium containing 2% w/v succinic acid(pH=5.0). For preparation of RNA samples, ΔURA3 cells grown overnight were inoculated into 50 ml liquid Synthetic Dextrose (SD) medium with the initial inoculation amount of OD600= 0.1, and cultured for 24 hours to the logarithmic growth phase (OD600= 2-3). The cells were collected by centrifugation at 6000g for 5 minutes. After washing twice with phosphate buffer saline (PBS), they were inoculated into new 50 mL SD medium containing carbon sources of different combinations of glucose and succinic acid .The pH of the culture sample is adjusted to 5.0 with NaOH. After flask culturing at 30°C and 250 rpm for an additional three hours, the yeast cells were collected by centrifugation for total RNA isolation and Illumina RNA-seq library construction. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The 150-nt paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). Each sample contains two biological replicates.
Project description:Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids. Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ± 5 mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ± 17.7 mg/L in shake flasks, and 1232 ± 64 mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid.
Project description:In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate-degrading capacity. The mutant strain TY1322 shows a biomass-specific phytate degradation of 1.26 mmol g-1 h-1 after 8 h of cultivation in a high-phosphate medium, which is about 8 times higher compared with the wild-type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46°C) and in the presence of ox bile (2% w/v), indicating this strain's ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55°C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non-genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification.
Project description:Renewable and low-cost lignocellulosic wastes have attractive applications in bioethanol production. The yeast Saccharomyces cerevisiae is the most widely used ethanol-producing microbe; however, its fermentation temperature (30-35°C) is not optimum (40-50°C) for enzymatic hydrolysis in the simultaneous saccharification and fermentation (SSF) process. In this study, we successfully performed an SSF process at 42°C from a high solid loading of 20% (w/v) acid-impregnated steam explosion (AISE)-treated rice straw with low inhibitor concentrations (furfural 0.19 g l-1 and acetic acid 0.95 g l-1 ) using an isolate Pichia kudriavzevii SI, where the ethanol titre obtained (33.4 gp l-1 ) was nearly 39% greater than that produced by conventional S. cerevisiae BCRC20270 at 30°C (24.1 gp l-1 ). In addition, P. kudriavzevii SI exhibited a high conversion efficiency of > 91% from enzyme-saccharified hydrolysates of AISE-treated plywood chips and sugarcane bagasse, although high concentrations of furaldehydes, such as furfural 1.07-1.21 g l-1 , 5-hydroxymethyl furfural 0.20-0.72 g l-1 and acetic acid 4.80-7.65 g l-1 , were present. This is the first report of ethanol fermentation by P. kudriavzevii using various acid-treated lignocellulosic feedstocks without detoxification or added nutrients. The multistress-tolerant strain SI has greater potential than the conventional S. cerevisiae for use in the cellulosic ethanol industry.
Project description:A 23-year-old female, who had undergone a sleeve gastrectomy two weeks earlier, presented with abdominal complaints. A CT scan showed portal vein thrombosis, bowel ischemia, and intra-abdominal sepsis. Anastomosis and antibiotic therapy were not successful, and the patient went into multi-organ failure and died. Multiple cultures revealed a yeast fungus confirmed as Pichia kudriavzevii using rRNA gene sequencing. We report the first case of peritonitis in association with P kudriavzevii. In addition to the abdominal complications and surgical interventions, the yeast was found to have significantly contributed to the patient's death. SIMILAR CASES PUBLISHED: None. CONFLICT OF INTEREST: None.
Project description:Most proteogenomic approaches for mapping single amino acid polymorphisms (SAPs) require construction of a sample-specific database containing protein variants predicted from the next-generation sequencing (NGS) data. We present a new strategy for direct SAP detection without relying on NGS data. Among the 348 putative SAP peptides identified in an industrial yeast strain, 85.6% of SAP sites were validated by genomic sequencing.