Insights into the Correlation between Microbial Community Succession and Pericarp Degradation during Pepper (Piper nigrum L.) Peeling Process via Retting.

White pepper, used both as a seasoning in people's daily diets and as a medicinal herb, is typically produced by removing the pericarp of green pepper through the retting process. However, the mechanism of the retting process for peeling remains unclear. Therefore, this study aimed to investigate the changes in physicochemical factors, microbial community succession effects, and metabolites of the pepper pericarp during the pepper peeling process. The findings indicated that pre-treatment involving physical friction before the retting process effectively reduced the production time of white pepper. During the retting process, the pectinase activity increased, leading to a decrease in the pectin content in the pepper pericarp. There was a significant correlation observed between the changes in pH, pectin content, and peeling rate and the Shannon diversity index of bacteria and fungi. Prevotella, Lactococcus, and Candida were the dominant microbial genera during the retting. The functional predictions suggested that the monosaccharides degraded from the pepper pericarp could have been utilized by microbes through sugar metabolism pathways. Metabolomic analysis showed that the metabolic pathways of carbohydrates and amino acids were the main pathways altered during the pepper peeling process. The verification experiment demonstrated that the degradation of pectin into galacturonic acid by polygalacturonase was identified as the key enzyme in shortening the pepper peeling time. The structure of the pepper pericarp collapsed after losing the support of pectin, as revealed by scanning electron microscopy. These results suggest that the decomposition of the pepper pericarp was driven by key microbiota. The succession of microbial communities was influenced by the metabolites of the pepper pericarp during retting. These findings provide new insights into the retting process and serve as an important reference for the industrial production of white pepper.

MaiJiTong granule attenuates atherosclerosis by reducing ferroptosis via activating STAT6-mediated inhibition of DMT1 and SOCS1/p53 pathways in LDLR-/- mice.

BACKGROUND AND PURPOSE: Atherosclerosis is the primary pathological basis of cardiovascular disease. Ferroptosis is a regulated form of cell death, a process of lipid peroxidation driven by iron, which can initiate and promote atherosclerosis. STAT6 is a signal transducer that shows a potential role in regulating ferroptosis, but, the exact role in ferroptosis during atherogenesis remains unclear. The Traditional Chinese Medicine Maijitong granule (MJT) is used for treating cardiovascular disease and shows a potential inhibitory effect on ferroptosis. However, the antiatherogenic effect and the underlying mechanism remain unclear. In this study, we determined the role of STAT6 in ferroptosis during atherogenesis, investigated the antiatherogenic effect of MJT, and determined whether its antiatherogenic effect was dependent on the inhibition of ferroptosis. METHODS: 8-week-old male LDLR-/- mice were fed a high-fat diet (HFD) at 1st and 10th week, respectively, to assess the preventive and therapeutic effects of MJT on atherosclerosis and ferroptosis. Simultaneously, the anti-ferroptotic effects and mechanism of MJT were determined by evaluating the expression of genes responsible for lipid peroxidation and iron metabolism. Subsequently, we reanalyzed microarray data in the GSE28117 obtained from cells after STAT6 knockdown or overexpression and analyzed the correlation between STAT6 and ferroptosis. Finally, the STAT6-/- mice were fed HFD and injected with AAV-PCSK9 to validate the role of STAT6 in ferroptosis during atherogenesis and revealed the antiatherogenic and anti-ferroptotic effect of MJT. RESULTS: MJT attenuated atherosclerosis by reducing plaque lesion area and enhancing plaque stability in both preventive and therapeutic groups. MJT reduced inflammation via suppressing inflammatory cytokines and inhibited foam cell formation by lowering the LDL level and promoting ABCA1/G1-mediated lipid efflux. MJT ameliorated the ferroptosis by reducing lipid peroxidation and iron dysregulation during atherogenesis. Mechanistically, STAT6 negatively regulated ferroptosis by transcriptionally suppressing SOCS1/p53 and DMT1 pathways. MJT suppressed the DMT1 and SOCS1/p53 via stimulating STAT6 phosphorylation. In addition, STAT6 knockout exacerbated atherosclerosis and ferroptosis, which abolished the antiatherogenic and anti-ferroptotic effects of MJT. CONCLUSION: STAT6 acts as a negative regulator of ferroptosis and atherosclerosis via transcriptionally suppressing DMT1 and SOCS1 expression and MJT attenuates atherosclerosis and ferroptosis by activating the STAT6-mediated inhibition of DMT1 and SOCS1/p53 pathways, which indicated that STAT6 acts a novel promising therapeutic target to ameliorate atherosclerosis by inhibiting ferroptosis and MJT can serve as a new therapy for atherosclerosis treatment.

Effect of noninvasive embryo viability testing versus conventional IVF on the live birth rate in IVF/ICSI patients: a study protocol for a double-blind, multicenter, randomized controlled trial.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) was demonstrated to be superior to conventional IVF in reducing the incidence of miscarriage and abnormal offspring after the first embryo transfer (ET). PGT-A requires several embryo trophectoderm cells, but its negative impacts on embryo development and long-term influence on the health conditions of conceived children have always been a concern. As an alternative, noninvasive PGT-A (niPGT-A) approaches using spent blastocyst culture medium (SBCM) achieved comparable accuracy with PGT-A in several pilot studies. The main objective of this study is to determine whether noninvasive embryo viability testing (niEVT) results in better clinical outcomes than conventional IVF after the first embryo transfer. Furthermore, we further investigated whether niEVT results in higher the live birth rate between women with advanced maternal age (AMA, > 35 years old) and young women or among patients for whom different fertilization protocols are adopted. METHODS: This study will be a double-blind, multicenter, randomized controlled trial (RCT) studying patients of different ages (20-43 years) undergoing different fertilization protocols (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]). We will enroll 1140 patients at eight reproductive medical centers over 24 months. Eligible patients should have at least two good-quality blastocysts (better than grade 4 CB). The primary outcome will be the live birth rate of the first embryo transfer (ET). Secondary outcomes will include the clinical pregnancy rate, ongoing pregnancy rate, miscarriage rate, cumulative live birth rate, ectopic pregnancy rate, and time to pregnancy. DISCUSSION: In this study, patients who undergo noninvasive embryo viability testing (niEVT) will be compared to women treated by conventional IVF. We will determine the effects on the pregnancy rate, miscarriage rate, and live birth rate and adverse events. We will also investigate whether there is any difference in clinical outcomes among patients with different ages and fertilization protocols (IVF/ICSI). This trial will provide clinical evidence of the effect of noninvasive embryo viability testing on the clinical outcomes of the first embryo transfer. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR) Identifier: ChiCTR2100051408. 9 September 2021.

Human leukocyte antigen-G in gynaecological tumours.

Gynaecological tumours that threaten the health of women, especially when advanced and recurrent, have remained mostly intractable to existing treatments. Therefore, new therapeutic targets are urgently needed. Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex class I molecule typically expressed in foetuses for protection against destruction by the maternal immune system. HLA-G is also expressed under pathological conditions, such as in solid tumours, and may participate in tumour development and serve as a novel immune checkpoint in cancer. Furthermore, it is expressed in most gynaecological tumours. Therefore, inhibiting HLA-G and its receptors to block the immune escape pathway could represent a new strategy in cancer immunotherapy. To the best of our knowledge, this review is the first to summarize recent research findings on HLA-G in gynaecological oncology. We highlight the fact that HLA-G is expressed in gynaecological tumour tissues, wherein it inactivates immune effectors involved in tumour progression. Further studies on HLA-G in gynaecological oncology are needed to incorporate HLA-G into the design and evaluation of immunotherapy for malignant gynaecological diseases.

Enhancement of ß-Caryophyllene Biosynthesis in Saccharomyces cerevisiae via Synergistic Evolution of ß-Caryophyllene Synthase and Engineering the Chassis.

ß-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. ß-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of ß-caryophyllene synthase (CPS) is one of the main restrictive factors for ß-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the ß-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in Vmax and Kcat. The Kcat/Km of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in ß-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to ß-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6T1025N to improve the transmembrane transport of ß-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of ß-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a ß-caryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of ß-caryophyllene production by yeast.

Preimplantation genetic testing for aneuploidy helps to achieve a live birth with fewer transfer cycles for the blastocyst FET patients with unexplained recurrent implantation failure.

PURPOSE: This retrospective cohort study aimed to investigate the value of preimplantation genetic testing for aneuploidy (PGT-A) as a screening test for patients suffering from unexplained recurrent implantation failure (RIF). METHODS: After screening patients in one reproductive medicine center, twenty-nine, forty-nine and thirty-eight women (< 40 years old) who had suffered unexplained RIF with PGT-A, or RIF without PGT-A, or no RIF with PGT-A were included. The clinical pregnancy rate and live birth rate per transfer, the conservative and optimal cumulative clinical pregnancy rates (CCPR) and live birth rates (CLBR) after three blastocyst FETs were analyzed. RESULTS: The live birth rate per transfer was significantly higher in the RIF + PGT-A group than that in the RIF + NO PGT-A group (47.6% vs. 24.6%, p = 0.014). After 3 cycles of FET, RIF + PGT-A group had significantly higher conservative CLBR and optimal CLBR compared to the RIF + NO PGT-A group (69.0% vs. 32.7%, p = 0.002 and 73.7% vs. 57.5%, p = 0.016), but had similar conservative and optimal CLBRs compared to the NO RIF + PGT-A group. The number of FET cycles required when half women achieved a live birth was 1 in the PGT-A group and 3 in RIF + NO PGT-A group. The miscarriage rates were not different between the RIF + PGT-A and RIF + NO PGT-A, RIF + PGT-A and NO RIF + PGT-A groups. CONCLUSION: PGT-A did be superior in reducing the number of transfer cycles required to achieve a similar live birth rate. Further studies to identify the RIF patients who would benefit most from PGT-A are necessary.

Follicular helper T lymphocytes in the endometria of patients with reproductive failure: Association with pregnancy outcomes and inflammatory status of the endometria.

PROBLEM: The phenotypes and functions of B and CD4+ T-helper cell subsets during chronic inflammation of the endometria remain largely unexplored. This study aimed to investigate the characteristics and functions of follicular helper T (Tfh) cells to understand the pathological mechanisms of chronic endometritis (CE). METHOD OF STUDY: Eighty patients who underwent hysteroscopic and histopathological examinations for CE were divided into three groups-those with positive results for hysteroscopy and CD138 staining (DP), negative results for hysteroscopy but positive CD138 staining (SP), and negative results for hysteroscopy and CD138 staining (DN). The phenotypes of B cells and CD4+ T-cell subsets were analyzed using flow cytometry. RESULTS: CD38+ and CD138+ cells were mainly expressed in the non-leukocyte population of the endometria, and the endometrial CD19+ CD138+ B cells were fewer than the CD3+ CD138+ T cells. The percentage of Tfh cells increased with chronic inflammation in the endometria. Additionally, the elevated percentage of Tfh cells correlated with the number of miscarriages. CONCLUSIONS: CD4+ T cells, particularly Tfh cells, may be critical in chronic endometrial inflammation and affect its microenvironment, thereby regulating endometrial receptivity, compared to B cells.

The Serotonergic System and Amyotrophic Lateral Sclerosis: A Review of Current Evidence.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the premature death of motor neurons. Serotonin (5-HT) is a crucial neurotransmitter, and its dysfunction, whether as a contributor or by-product, has been implicated in ALS pathogenesis. Here, we summarize current evidence linking serotonergic alterations to ALS, including results from post-mortem and neuroimaging studies, biofluid testing, and studies of ALS animal models. We also discuss the possible role of 5-HT in modulating some important mechanisms of ALS (i.e. glutamate excitotoxity and neuroinflammation) and in regulating ALS phenotypes (i.e. breathing dysfunction and metabolic defects). Finally, we discuss the promise and limitations of the serotonergic system as a target for the development of ALS biomarkers and therapeutic approaches. However, due to a relative paucity of data and standardized methodologies in previous studies, proper interpretation of existing results remains a challenge. Future research is needed to unravel the mechanisms linking serotonergic pathways and ALS and to provide valid, reproducible, and translatable findings.

A bibliometric analysis of the global trends and hotspots for the ketogenic diet based on CiteSpace.

The ketogenic diet (KD) is a potential nutritional therapy that is frequently utilized in various conditions. More and more studies are being done on KD in recent years. However, as far as we know, few studies have made an effort to offer a thorough synthesis and assessment of this topic. This paper aims to do a rigorous and thorough evaluation of the knowledge structure, development trend, and research hotspot of scientific outputs connected to KD. The bibliographic records connected to KD from January 1, 2001 to April 22, 2022 were collected using the core collection database of Web of Science. The complex data input, that consisted of the amount of publications, journals, authors, institutions, countries, keywords and cited references, was generated and analyzed visually using CiteSpace. A total of 2676 literatures on the KD were published between 2001 and 2022. The most KD-related publications were found in Epilepsia and Epilepsia Research. The authors with the most KD-related papers are Kossoff EH and Rho J. The United States is the country with the most publications, and Johns Hopkins University, Johns Hopkins University Hospital, and Johns Hopkins Medical Institutions are the institutions with the most articles. The high frequency keywords are "KD," "ketone body," "children," "efficacy," "weight loss," "low carbohydrate diet," "metabolism," "epilepsy," "beta hydroxybutyrate," and "modified atkins diet." The 2018 study by Kossoff EH on epilepsia and the 2017 study by Puchalska P on ketone body metabolism earned 127 and 114 citations, respectively. The results of this bibliometric analysis provide information on the state and trends in KD and may be used by researchers to pinpoint hot issues and discover new areas of study.

Outcomes associated with different surgical approaches to radical hysterectomy: A systematic review and network meta-analysis.

OBJECTIVE: To evaluate the efficacy and safety of five different approaches to cervical cancer surgery. METHODS: We conducted a systematic search for comparative studies on different radical hysterectomy types for cervical cancer in PubMed, Embase, the Cochrane Library, and Web of Science databases. All included observational studies used survival analyses to compare clinical outcomes of patients undergoing different radical hysterectomy types. All studies were assessed by the Newcastle-Ottawa Scale with scores of at least seven points. We extracted the relevant data and conducted a network meta-analysis to compare clinical outcomes among five surgical approaches. RESULTS: Thirty studies (n = 11 353) were included. Robotic surgery had the lowest blood loss volume and hospitalization duration; open surgery had the shortest operative time. Vaginal assisted laparoscopic surgery was associated with the highest number of resected lymph nodes and lowest rate of perioperative complications. Survival outcomes and tumor recurrence outcomes were similar among the approaches. CONCLUSION: The current approaches to cervical cancer surgery have comparable efficacies.

Transcriptomic analysis of mid-secretory endometrium reveals essential immune factors associated with pregnancy after single euploid blastocyst transfer.

PURPOSE: Implantation is a limiting factor for treatment success in assisted reproduction. Both embryonic and endometrial factors contribute to implantation. Embryonic factors have often been ignored in previous studies about the role of endometrium in implantation. In this study, we sought to identify the endometrial genes associated with negative pregnancy outcomes following the transfer of a single euploid blastocyst. METHODS: Computational analyses of the transcriptomes of mid-secretory endometria from nine pregnant and seven non-pregnant patients in a cycle preceding the transfer of a single euploid blastocyst in a vitrified-warmed cycle were performed. RESULTS: Principal component analysis of two reported endometrial receptivity gene sets showed close clustering of the pregnant and non-pregnant samples. Differential gene expression analysis and co-expression module analysis identified 131 genes associated with the pregnancy status. The endometrial signatures identified highlight the importance of immune and metabolic regulation in pregnancy outcome. Network analysis identified 20 hub genes that could predict pregnancy outcomes with 88.9% sensitivity and 85.7% specificity. Single-cell gene expression analysis highlighted the regulation of endometrial natural killer (NK) cells, T cells, and macrophages during embryo implantation. Immune cell abundance analysis supported the dysregulation of cytotoxic immune cells in the endometria of non-pregnant women. CONCLUSIONS: We reported the first endometrial gene signature associated with pregnancy after elimination of embryo aneuploidy and highlighted the importance of the endometrial immune microenvironment and metabolic status in pregnancy outcomes.

Single-cell Sequencing Reveals Clearance of Blastula Chromosomal Mosaicism in In Vitro Fertilization Babies.

Although chromosomal mosaic embryos detected by trophectoderm (TE) biopsy offer healthy embryos available for transfer, high-resolution postnatal karyotyping and chromosome testing of the transferred embryos are insufficient. Here, we applied single-cell multi-omics sequencing for seven infants with blastula chromosomal mosaicism detected by TE biopsy. The chromosome ploidy was examined by single-cell genome analysis, with the cellular identity being identified by single-cell transcriptome analysis. A total of 1616 peripheral leukocytes from seven infants with embryonic chromosomal mosaicism and three control ones with euploid TE biopsy were analyzed. A small number of blood cells showed copy number alterations (CNAs) on seemingly random locations at a frequency of 0%-2.5% per infant. However, none of the cells showed CNAs that were the same as those of the corresponding TE biopsies. The blastula chromosomal mosaicism may be fully self-corrected, probably through the selective loss of the aneuploid cells during development, and the transferred embryos can be born as euploid infants without mosaic CNAs corresponding to the TE biopsies. The results provide a new reference for the evaluations of transferring chromosomal mosaic embryos in certain situations.

Corrigendum: Identification of key endometrial microRNAs and their target genes associated with pathogenesis of recurrent implantation failure by integrated bioinformatics analysis.

[This corrects the article DOI: 10.3389/fgene.2022.919301.].

Cat8 Response to Nutritional Changes and Interaction With Ehrlich Pathway Related Factors.

Cat8 is an important transcription factor regulating the utilization of non-fermentative carbon sources in Saccharomyces cerevisiae. However, our previous studies found that Cat8 may play a critical role in nitrogen metabolism, but the regulatory mechanism has not been elucidated. In this study, the nuclear localization and analysis of regulatory activity showed that the Cat8 function relies on Snf1 kinase. In the fermentation with glucose or glycerol as carbon sources under phenylalanine (Phe) induction, by comparing the changes of cellular gene expression and Cat8 target gene binding profiles after Cat8 overexpression, enhanced transcription was shown among key genes involved in the Ehrlich pathway (e.g., ARO9, ARO10, and ADH2) and its upstream and downstream related factors (e.g., GAP1, AGP1, GAT1, PDR12, and ESPB6), indicating that Cat8 participated in the regulation of nitrogen metabolism. Moreover, highly active Cat8 interacts with transcriptional activator Aro80 and GATA activator Gat1 coordinately to regulate the transcription of ARO10. Altogether, our results showed that Cat8 may act as a global transcription factor in response to nutritional changes, regulating both carbon and nitrogen utilization. This provides a new insight for us to explore the regulation of cell nutrient metabolism networks in yeast.

Identification of Key Endometrial MicroRNAs and Their Target Genes Associated With Pathogenesis of Recurrent Implantation Failure by Integrated Bioinformatics Analysis.

Purpose: Recurrent implantation failure (RIF) is an enormous challenge for in vitro fertilization (IVF) clinicians. An understanding of the molecular mechanisms of RIF helps to predict prognosis and develop new therapeutic strategies. The study is designed to identify diagnostic biomarkers for RIF as well as the potential mechanisms underlying RIF by utilizing public databases together with experimental validation. Methods: Two microarray datasets of RIF patients and the healthy control endometrium were downloaded from the Gene Expression Omnibus (GEO) database. First, differentially expressed microRNAs (miRNAs) (DEMs) were identified and their target genes were predicted. Then, we identified differentially expressed genes (DEGs) and selected hub genes through protein-protein interaction (PPI) analyses. Functional enrichment analyses of DEGs and DEMs were conducted. Furthermore, the key DEMs which targeted these hub genes were selected to obtain the key miRNA-target gene network. The key genes in the miRNA-target gene network were validated by a single-cell RNA-sequencing dataset of endometrium from GEO. Finally, we selected two miRNA-target gene pairs for further experimental validation using dual-luciferase assay and quantitative polymerase chain reaction (qPCR). Results: We identified 49 DEMs between RIF patients and the fertile group and found 136,678 target genes. Then, 325 DEGs were totally used to construct the PPI network, and 33 hub genes were selected. Also, 25 DEMs targeted 16 key DEGs were obtained to establish a key miRNA-target gene network, and 16 key DEGs were validated by a single-cell RNA-sequencing dataset. Finally, the target relationship of hsa-miR-199a-5p-PDPN and hsa-miR-4306-PAX2 was verified by dual-luciferase assay, and there were significant differences in the expression of those genes between the RIF and fertile group by PCR (p < 0.05). Conclusion: We constructed miRNA-target gene regulatory networks associated with RIF which provide new insights regarding the underlying pathogenesis of RIF; hsa-miR-199a-5p-PDPN and hsa-miR-4306-PAX2 could be further explored as potential biomarkers for RIF, and their detection in the endometrium could be applied in clinics to estimate the probability of successful embryo transfer.

Enhancement of Intracellular Accumulation of Copper by Biogenesis of Lipid Droplets in Saccharomyces cerevisiae Revealed by Transcriptomic Analysis.

Copper is an essential micronutrient for life, whose homeostasis is rigorously regulated to meet the demands of normal biological processes and to minimize the potential toxicity. Copper enriched by yeast is regarded as a safe and bioavailable form of copper supplements. Here, a Saccharomyces cerevisiae mutant strain H247 with expanded storage capability of copper was obtained through atmospheric and room-temperature plasma treatment. Transcriptomic analyses found that transcriptional upregulation of DGA1 might be the major contributor to the enhancement of intracellular copper accumulation in strain H247. The positive correlation between biogenesis of lipid droplets and intracellular accumulation of copper was confirmed by overexpression of the diacylglycerol acyltransferase encoding genes DGA1 and LRO1 or knockout of DGA1. Lipid droplets are not only the storage pool of copper but might prompt the copper trafficking to mitochondria, vacuoles, and Golgi apparatus. These results provide new insights into the sophisticated copper homeostatic mechanisms and the biological functions of lipid droplets.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and ß-alanine metabolism.

Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431-fold and 9-fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and ß-alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3-4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl-CoA supply and ß-alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3-4-CPS generated from expressing ß-caryophyllene synthase in SQ3-4 produced 11.86 ± 0.09 mg l-1 ß-caryophyllene, while strain SQ3-5 resulted from down-regulation of ERG1 in SQ3-4 produced 408.88 ± 0.09 mg l-1 squalene in shake flask cultivations. Strain SQ3-5 produced 4.94 g l-1 squalene in fed-batch fermentation in cane molasses medium, indicating the promising potential for cost-effective production of squalene.

Whole Genome Sequencing and RNA-seq-Driven Discovery of New Targets That Affect Carotenoid Synthesis in Phaffia rhodozyma.

Carotenoids are unsaturated compounds with terpene groups. Among them, astaxanthin has strong antioxidant properties. It is widely used in aquaculture, food, medicine, and cosmetics with a broad market prospect. Phaffia rhodozyma is an important microorganism that synthesizes astaxanthin, but its wild strains have low pigment content, long growth cycle, and low fermentation temperature. Therefore, it is important to research the genetic improvement of the physiological and biochemical properties of P. rhodozyma. In this study, the atmospheric and room temperature plasma mutagenesis technology was adopted, through the functional evolution of the carotenoid production performance; then, through the comparative analysis of the genomics and transcriptomics of the wild strain and evolved strain, the key factor GST1 gene that affects carotenoid synthesis was discovered.

Enhancement of Copper Uptake of Yeast Through Systematic Optimization of Medium and the Cultivation Process of Saccharomyces cerevisiae.

Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100 mg/L copper ions with 3.5 to 5.0 g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.

Engineering of cis-Element in Saccharomyces cerevisiae for Efficient Accumulation of Value-Added Compound Squalene via Downregulation of the Downstream Metabolic Flux.

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (PADH1) and its enhanced variant PUADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter PERG1(M5) or PERG11(M3), respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.