Characterization of a pleiotropic regulator MtrA in Streptomyces avermitilis controlling avermectin production and morphological differentiation.

BACKGROUND: The macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated. RESULTS: In this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis. CONCLUSIONS: In this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.

Engineering and transcriptome study of Saccharomyces cerevisiae to produce ginsenoside compound K by glycerol.

Synthetic biology-based engineering of Saccharomyces cerevisiae to produce terpenoid natural products is an effective strategy for their industrial application. Previously, we observed that glycerol addition was beneficial for ginsenoside compound K (CK) production in a S. cerevisiae when it was fermented using the YPD medium. Here, we reconstructed the CK synthesis and glycerol catabolic pathway in a high-yield protopanaxadiol (PPD) S. cerevisiae strain. Remarkably, our engineered strain exhibited the ability to utilize glycerol as the sole carbon source, resulting in a significantly enhanced production of 433.1 ± 8.3 mg L-1 of CK, which was 2.4 times higher compared to that obtained in glucose medium. Transcriptomic analysis revealed that the transcript levels of several key genes involved in the mevalonate (MVA) pathway and the uridine diphosphate glucose (UDPG) synthesis pathway were up-regulated in response to glycerol. The addition of glycerol enhanced CK titers by augmenting the flux of the terpene synthesis pathway and facilitating the production of glycosyl donors. These results suggest that glycerol is a promising carbon source in S. cerevisiae, especially for the production of triterpenoid saponins.

Green conversion of delignified sorghum straw and polyethylene glycol into form-stable phase change materials with promising solar energy capture, transition, and storage capabilities.

Phase change materials (PCMs) have attracted considerable attention as a thermal energy management technology for thermal storage. However, achieving high energy-storing abilities, low leakage rates, and solar absorption abilities simultaneously in PCMs remains greatly challenging. This research proposed a green strategy for preparing sorghum straw-based PCMs. By facile delignification and solvothermal process, delignified sorghum straw (DSS) and carbon quantum dots (CQDs) derived from removal lignin are prepared. The obtained PEG@CQDs/DSS possessed considerable reusable stabilities, excellent photo-thermal conversion properties, and thermal energy management capacities due to the delicate micropores and intrinsic noncovalent interactions among components. Especially, the PEG@CQDs-7.5/DSS exhibited superior solar-thermal conversion capabilities (with conducive photo-thermal conversion efficiency ~90.84%), and kept stable after 100 cycles of heating and cooling, in which the melting enthalpy value is ~168.1 J/g (enthalpy efficiency of ~91.11%). In conclusion, the synthesized PCMs showed potential for application in energy-saving and building thermal management.

The association between smoking cessation and lifestyle/genetic variant rs6265 among the adult population in Taiwan.

Recent studies showed significant associations between socio-demographic, lifestyle factors, polymorphic variant rs6265, and smoking cessation behaviours. We examined rs6265 TT, TC and CC genotypes and their association with socio-demographic and other variables, including mental health status, drinking, exercise, and smoking behaviour among Taiwanese adults. Data on rs6265 were retrieved from the Taiwan Biobank, which contained genetic data collected between 2008 to 2019 from 20,584 participants (aged 30-70 years). Participants who smoked for more than 6 months prior to enrolment were categorized as smokers. If they had smoked and later quit for more than 6 months, they were classified as former smokers. Information regarding drinking, exercise, depression, and bipolar disorder were obtained through questionnaires and were categorized as either as affirmative (yes) or negative (no) responses. In contrast to previous studies, we found that the association between the polymorphism rs6265 and smoking behaviour was not significant (P-value = 0.8753). Males with lower education levels, young persons, and alcohol drinkers showed significant smoking behaviours (P-value < .0001). This population-based study indicates that rs6265 has no significant correlations with smoking cessation behaviour among adults in Taiwan.

Sustainable Biochar Nanosheets Derived from Sweet Sorghum Residues via Superbase Pretreatment.

Two-dimensional (2D) sheet-like biochar as promising alternatives to graphene nanosheets has gained significant attention in materials science while being highly restricted by its complicated synthetic steps. In this study, the dimethyl sulfoxide/potassium hydroxide (DMSO/KOH) superbase system was first used to pretreat sweet sorghum residues (SS) and then carbonized to prepare sheet-like biochar. Ascribing to the strong nucleophilicity of DMSO/KOH, a synergistic effect was achieved by partially removing non-cellulosic components in SS and swelling the amorphous region of cellulose, leaving more layered cellulose behind (∼46.5 wt %), which was favorable for the formation of 2D biochar nanosheets with high graphitization degrees (∼93.1%). This strategy was also suitable for other biomass fibers (e.g., straw, wood powders, and nuclear shells) to obtain sheet-like biochar. The resulting sheet-like biochar could be compounded with cellulose nanofibers to achieve the structural design of composites and solve the molding problem of biochar, which was beneficial for dyeing wastewater treatment. Thus, this work provides insight into a simple strategy for developing 2D ultrathin sheet-like biochar from sustainable biomass wastes.

Spine, hip, and femoral neck bone mineral density in relation to vegetarian type and status among Taiwanese adults.

We determined the association of vegetarian type and status with bone mineral density (BMD) Z-scores at the spine, hip, and femoral neck. Compared to non-vegetarians, current vegetarians, especially vegans, lacto-vegetarians, and lacto-ovo-vegetarians had lower Z-scores at multiple sites. Sole reliance on a vegetarian diet might be detrimental to the bone. PURPOSE: The impact of vegetarian diets on BMD is contentious. We determined the association of vegetarian type and status with the spine, hip, and femoral neck BMD Z-scores. METHODS: We analyzed data from 20,110 Taiwan Biobank volunteers. BMD was measured using dual-energy X-ray absorptiometry (DXA). The vegetarian status (non-, former, and current vegetarians) and type (non-vegetarians, ovo-vegetarians, lacto-vegetarians, lacto-ovo-vegetarians, and vegans) were determined using questionnaires. RESULTS: The participants consisted of 12,910 women and 7200 men, with a mean age of 55.5 years. Based on vegetarian status (reference: non-vegetarians), current vegetarians had significantly lower BMD Z-scores at the spine (unstandardized regression coefficient, B = - 0.195, p = 0.006), left hip (B = - 0.125, p = 0.008), and right hip (B = - 0.100, p = 0.027), respectively. Based on vegetarian status and type (reference: non-vegetarians), current vegans and non-vegans had notably lower BMD Z-scores at specific skeletal sites. For non-vegans, the BMD Z-scores were significant at the spine (B = -0.184, p = 0.010), left hip (B = - 0.124, p = 0.010), and left femoral neck (B = - 0.125, p = 0.012). For current vegans, however, the BMD Z-scores were significant only at the right hip (B = - 0.232; p = 0.028). Nonetheless, after stratifying vegetarian diet into more subgroups, current vegans exhibited a significant reduction in BMD Z-scores at the spine and right hip, with B-coefficients of - 0.326 and - 0.238, respectively. Current lacto-vegetarians also had significantly lower Z-scores (p < 0.05) at the spine (B = - 0.459), left hip (B = - 0.313), and right hip (B = - 0.214). Moreover, current lacto-ovo-vegetarians had significantly lower Z-scores at the spine (B = - 0.175) and left hip (B = - 0.115). CONCLUSION: Current vegetarians, particularly vegans, lacto-vegetarians, and lacto-ovo-vegetarians, demonstrated significantly lower BMD Z-scores at various skeletal sites compared to non-vegetarians. Sole reliance on a vegetarian diet might be detrimental to the bone.

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM) is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, an SAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

The association between vegetarian diet and varicose veins might be more prominent in men than in women.

Background: Varicose veins (VVs), a common vascular disease is associated with a huge medical burden. The prevalence in women surpasses that in men. The role of vegetarian diets in the pathogenesis of the disease remains inconclusive. In this study, we examined the risk of VVs in vegetarian and non-vegetarian men and women. Methods: The study involved 9905 adults whose data were obtained from Taiwan Biobank between 2008 and 2020. Information on VVs, sex, and vegetarian diets was obtained from participants' self-responses to the Taiwan Biobank questionnaires. Results: The study subjects consisted of 4,142 men and 5,763 women. About 12% of men and 35% of women had VVs. Study participants were predominantly non-vegetarians (91.84% were men and 88.24% were women). Women had a higher risk of VVs than men. The odds ratio (OR); 95% confidence interval (CI) was 3.414; 2.995-3.891. There was a significant interaction between sex and vegetarian diets (p = 0.0034). Women were at higher risk of VVs than men both in the vegetarian (OR = 1.877, 95% CI = 1.270-2.774) and non-vegetarian (OR = 3.674, 95% CI = 3.197-4.223) groups. Based on vegetarian diets, only vegetarian men had a higher risk of VVs (OR = 1.453, 95% CI = 1.069 to 1.976). Based on the sex-stratified model, the risk of VVs was significantly higher in vegetarian men (OR = 1.457, 95% CI = 1.072-1.979), and in vegetarian and non-vegetarian women with corresponding ORs (95% CI) of 3.101 (2.528-3.803) and 3.599 (3.140-4.124), respectively. Conclusion: Women were more susceptible to varicose veins compared to men, regardless of diet. However, in terms of diet, only men who followed a vegetarian diet were at greater risk for developing VVs.

Efficient Bioremediation of Petroleum-Contaminated Soil by Immobilized Bacterial Agent of Gordonia alkanivorans W33.

In this article, we report a method for preparing an immobilized bacterial agent of petroleum-degrading bacteria Gordonia alkanivorans W33 by combining high-density fermentation and bacterial immobilization technology and testing its bioremediation effect on petroleum-contaminated soil. After determining the optimal combination of MgCl2, CaCl2 concentration, and culture time in the fermentation conditions by conducting a response surface analysis, the cell concentration reached 7.48 × 109 CFU/mL by 5 L fed-batch fermentation. The W33-vermiculite-powder-immobilized bacterial agent mixed with sophorolipids and rhamnolipids in a weight ratio of 9:10 was used for the bioremediation of petroleum-contaminated soil. After 45 days of microbial degradation, 56.3% of the petroleum in the soil with 20,000 mg/kg petroleum content was degraded, and the average degradation rate reached 250.2 mg/kg/d.

Natural promoters and promoter engineering strategies for metabolic regulation in Saccharomyces cerevisiae.

Sharomyces cerevisiae is currently one of the most important foreign gene expression systems. S. cerevisiae is an excellent host for high-value metabolite cell factories due to its advantages of simplicity, safety, and nontoxicity. A promoter, as one of the basic elements of gene transcription, plays an important role in regulating gene expression and optimizing metabolic pathways. Promoters control the direction and intensity of transcription, and the application of promoters with different intensities and performances will largely determine the effect of gene expression and ultimately affect the experimental results. Due to its significant role, there have been many studies on promoters for decades. While some studies have explored and analyzed new promoters with different functions, more studies have focused on artificially modifying promoters to meet their own scientific needs. Thus, this article reviews current research on promoter engineering techniques and related natural promoters in S. cerevisiae. First, we introduce the basic structure of promoters and the classification of natural promoters. Then, the classification of various promoter strategies is reviewed. Finally, by grouping related articles together using various strategies, this review anticipates the future development direction of promoter engineering.

Associations Between Body Mass Index, WNT16 rs2908004 and Osteoporosis: Findings from Taiwan Biobank.

Purpose: Osteoporosis is a degenerative disease that affects women and men of all races. We studied the association between body mass index (BMI), rs2908004 polymorphism of the WNT16 gene, and osteoporosis using data from Taiwan Biobank (TWB). Patients and Methods: We analyzed data from 10,942 subjects aged 30 to 70. We defined osteoporosis based on a mean T-score of -2.5 and below in the hip. Body mass index was classified following the guidelines of the Health Promotion Administration. Imputation was carried out using the IMPUTE2 (v2.3.1) program. Multiple logistic regression was used for analysis. The odds ratios (ORs) and 95% confidence interval (CI) for osteoporosis were determined. Results: In the multivariate regression model, variant rs2908004 had a significant association with osteoporosis. That is, the rs2908004-GA+AA genotype was associated with lower osteoporosis risk than the GG genotype (OR, 0.651; 95% CI = 0.544 to 0.780). Compared to normal-weight, underweight was significantly associated with a higher risk of osteoporosis (OR, 6.517; 95% CI = 4.624 to 9.186) while overweight and obesity were protective (OR, 0.176; 95% CI = 0.140 to 0.221 and 0.057; 95% CI = 0.039 to 0.083, respectively). There was an interaction between rs2908004 and BMI (p = 0.0148). Subgroup analyses (using rs2908004-GG/normal-weight as the reference group) indicated ORs of 7.66 (95% CI = 5.153 to 11.394) in the rs2908004-GG/underweight group and 3.002 (95% CI = 1.509 to 5.974) in the rs2908004-GA+AA/underweight group (95% CI = 1.509 to 5.974). Odds ratios were substantially lower in rs2908004-GG/obese, rs2908004-GG/overweight, GA+AA/normal-weight, rs2908004-GA+AA/overweight, and rs2908004-GA+AA/obese groups, respectively. Conclusion: According to our study, underweight was associated with an increased risk of osteoporosis irrespective of WNT16 rs2908004 genotypes, while overweight and obesity were associated with a lower risk.

Jogging and weight training associated with increased high-density lipoprotein cholesterol levels in Taiwanese adults.

Background: Although previous studies have shown that aerobic and resistance exercise increase high-density lipoprotein cholesterol (HDL-C) levels, the optimal type of exercise has not been determined. Therefore, the purpose of this study was to investigate the association of jogging (a type of aerobic exercise) and weight training (a type of resistance exercise) with HDL-C levels in Taiwanese adults. Methods: The data used in this cross-sectional study were obtained from the Taiwan Biobank (TWB), which is a national health resource that contains the genetic information of Taiwanese volunteers aged 30-70 years. A total of 75,635 subjects (47,881 women and 27,754 men) were included in this study. The subjects were divided into four groups: jogging (n = 2,278), weight training (n = 522), mixed exercise (n = 519), and no exercise (n = 72,316). The TWB data were collected through questionnaires (e.g. basic characteristics, lifestyle factors, and disease history), biochemical tests, and anthropometric measurements. Results: Compared with no exercise, jogging, weight training, and mixed exercise were all associated with higher HDL-C levels (ß = 2.5470, 2.6249, and 3.2117, respectively). As seen, the ß value was highest for the mixed exercise group, followed by weight training and then jogging (p for trend <0.0001). Conclusions: In the current study, jogging and weight training were individually associated with higher levels of HDL-C. Engaging in both activities was associated with much higher levels of HDL-C. Our findings suggest that regular jogging and weight training might play an important role in increasing HDL-C levels.

RNA-Seq and Genome-Wide Association Studies Reveal Potential Genes for Rice Seed Shattering.

The loss of the shattering ability is one of the key events in rice domestication. The strength of the seed shattering ability is closely related to the harvest yield and the adaptability of modern mechanical harvesting methods. In this study, using a population of 587 natural rice cultivars, quantitative trait loci associated with seed shattering were detected by genome-wide association studies (GWASs). We consider the quantitative trait loci (QTLs) qBTS1 and qBTS3 to be the key loci for seed shattering in rice. Additionally, the abscission zone (AZ) and nonabscission zone (NAZ) of materials with a loss of shattering (DZ129) and easy shattering (W517) were subjected to RNA-Seq, and high-quality differential expression profiles were obtained. The AZ-specific differentially expressed genes (DEGs) of W517 were significantly enriched in plant hormone signal transduction, while the AZ-specific DEGs of DZ129 were enriched in phenylpropanoid biosynthesis. We identified candidate genes for the lignin-associated laccase precursor protein (LOC_Os01g63180) and the glycoside hydrolase family (LOC_Os03g14210) in the QTLs qBTS1 (chromosome 1) and qBTS3 (chromosome 3), respectively. In summary, our findings lay the foundation for the further cloning of qBTS1 and qBTS3, which would provide new insights into seed shattering in rice.

Novel nitrogen mustard-artemisinin hybrids with potent anti-leukemia action through DNA damage and activation of GPx.

The incidence of various types of cancers is increasing every year. Among these, leukemia is extremely common, and thus, developing novel drugs to combat leukemia is crucial. In this study, we designed and synthesized several hybrids and obtained a new lead molecule 5a, with a strong therapeutic effect on leukemia. The results indicated that most hybrids effectively inhibited the growth of leukemia cells, HCT-116, and A549 cancer cells with an IC50 of <10 µM. Among these hybrids, 5a and 4h showed significant anticancer activity against CCRF-CEM, with IC50 values of 0.895 µM and 0.555 µM, respectively. Particularly, 5a had lower toxicity to L02 than chlorambucil (CLB) and doxorubicin (Dox), and the high selectivity was also reflected in the normal human B lymphoblast cell line (IM9). Upon investigating the mechanism of action, we found that 5a downregulated Bcl-2 and caused DNA double-stranded breaks (DSBs) to induce several genotoxic stress responses. The results of the flow cytometry assay showed that 5a was a non-specific molecule in the cell cycle. Furthermore, 5a did not affect total ROS levels but significantly improved the activity of glutathione peroxidase (GPx). Preliminary studies showed that nitrogen mustard exerted an efficient effect, and 5a can combine the advantages of artemisinin and nitrogen mustard and exhibit effects superior to either. This study showed that 5a should be further investigated as a therapeutic compound against leukemia.

Screening and Remodeling of Enone Oxidoreductase for High Production of 2(or 5)-Ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-Furanone in Saccharomyces Cerevisiae.

Owing to its unique fragrance, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is widely used as a food flavoring agent and has high demand. Enone oxidoreductase is a vital enzyme involved in HEMF production. In this study, an enone oxidoreductase from Naumovozyma dairenensis CBS 421 (NDEO) was used for HEMF production for the first time. The mutant NDEOT183W,K290W was obtained through semirational protein engineering, which increased the HEMF yield by 75.2%. Finally, the engineered strain BM4 produced the highest HEMF yield, 194.42 mg L-1 in 132 h. Our study revealed that HEMF production can be improved in Saccharomyces cerevisiae and that this is an efficient method to improve the activity of enone oxidoreductase, which is important for the industrial synthesis of furanone.

Heterologous biosynthesis of taraxerol by engineered Saccharomyces cerevisiae.

Taraxerol is an oleanane-type pentacyclic triterpenoid compound distributed in many plant species that has good effects on the treatment of inflammation and tumors. However, the taraxerol content in medicinal plants is low, and chemical extraction requires considerable energy and time, so taraxerol production is a problem. It is a promising strategy to produce taraxerol by applying recombinant microorganisms. In this study, a Saccharomyces cerevisiae strain WKde2 was constructed to produce taraxerol with a titer of 1.85 mg·l-1, and the taraxerol titer was further increased to 12.51 mg·l-1 through multiple metabolic engineering strategies. The endoplasmic reticulum (ER) size regulatory factor INO2, which was reported to increase squalene and cytochrome P450-mediated 2,3-oxidosqualene production, was overexpressed in this study, and the resultant strain WTK11 showed a taraxerol titer of 17.35 mg·l-1. Eventually, the highest reported titer of 59.55 mg·l-1 taraxerol was achieved in a 5 l bioreactor. These results will serve as a general strategy for the production of other triterpenoids in yeast.

Metabolic Engineering of Saccharomyces cerevisiae for Heterologous Carnosic Acid Production.

Carnosic acid (CA), a phenolic tricyclic diterpene, has many biological effects, including anti-inflammatory, anticancer, antiobesity, and antidiabetic activities. In this study, an efficient biosynthetic pathway was constructed to produce CA in Saccharomyces cerevisiae. First, the CA precursor miltiradiene was synthesized, after which the CA production strain was constructed by integrating the genes encoding cytochrome P450 enzymes (P450s) and cytochrome P450 reductase (CPR) SmCPR. The CA titer was further increased by the coexpression of CYP76AH1 and SmCPR ∼t28SpCytb5 fusion proteins and the overexpression of different catalases to detoxify the hydrogen peroxide (H2O2). Finally, engineering of the endoplasmic reticulum and cofactor supply increased the CA titer to 24.65 mg/L in shake flasks and 75.18 mg/L in 5 L fed-batch fermentation. This study demonstrates that the ability of engineered yeast cells to synthesize CA can be improved through metabolic engineering and synthetic biology strategies, providing a theoretical basis for microbial synthesis of other diterpenoids.

Biosynthesis of valerenic acid by engineered Saccharomyces cerevisiae.

OBJECTIVE: To produce valerenic acid (VA) in Saccharomyces cerevisiae by engineering a heterologous synthetic pathway. RESULT: Valerena-4,7(11)-diene synthase (VDS) derived from Valeriana officinalis (valerian) was expressed in S. cerevisiae to generate valerena-4,7(11)-diene as the precursor of VA. By overexpressing the key genes of the mevalonate pathway ERG8, ERG12 and ERG19, and integrating 4 copies of MBP (maltose-binding protein)-VDS-ERG20 gene expression caskets into the genome, the production of valerena-4,7(11)-diene was improved to 75 mg/L. On this basis, the cytochrome P450 monooxygenase LsGAO2 derived from Lactuca sativa was expressed to oxidize valerena-4,7(11)-diene to produce VA, and the most effective VA production strain was used for fermentation. The yield of VA reached 2.8 mg/L in the flask and 6.8 mg/L in a 5-L bioreactor fed glucose. CONCLUSIONS: An S. cerevisiae strain was constructed and optimized to produce VA, but the valerena-4,7(11)-diene oxidation by LsGAO2 is still the rate-limiting step for VA synthesis that needs to be further optimized in future studies.

Construction and optimization of Saccharomyces cerevisiae for synthesizing forskolin.

Forskolin, one of the primary active metabolites of labdane-type diterpenoids, exhibits significant medicinal value, such as anticancer, antiasthmatic, and antihypertensive activities. In this study, we constructed a Saccharomyces cerevisiae cell factory that efficiently produced forskolin. First, a chassis strain that can accumulate 145.8 mg/L 13R-manoyl oxide (13R-MO), the critical precursor of forskolin, was constructed. Then, forskolin was produced by integrating CfCYP76AH15, CfCYP76AH11, CfCYP76AH16, ATR1, and CfACT1-8 into the 13R-MO chassis with a titer of 76.25 µg/L. We confirmed that cytochrome P450 enzymes (P450s) are the rate-limiting step by detecting intermediate metabolite accumulation. Forskolin production reached 759.42 µg/L by optimizing the adaptations between CfCYP76AHs, t66CfCPR, and t30AaCYB5. Moreover, multiple metabolic engineering strategies, including regulation of the target genes' copy numbers, amplification of the endoplasmic reticulum (ER) area, and cofactor metabolism enhancement, were implemented to enhance the metabolic flow to forskolin from 13R-MO, resulting in a final forskolin yield of 21.47 mg/L in shake flasks and 79.33 mg/L in a 5 L bioreactor. These promising results provide guidance for the synthesis of other natural terpenoids in S. cerevisiae, especially for those containing multiple P450s in their synthetic pathways. KEY POINTS: • The forskolin biosynthesis pathway was optimized from the perspective of system metabolism for the first time in S. cerevisiae. • The adaptation and optimization of CYP76AHs, t66CfCPR, and t30AaCYB5 promote forskolin accumulation, which can provide a reference for diterpenoids containing complex pathways, especially multiple P450s pathways. • The forskolin titer of 79.33 mg/L is the highest production currently reported and was achieved by fed-batch fermentation in a 5 L bioreactor.

Risk of gallstones based on ABCG8 rs11887534 single nucleotide polymorphism among Taiwanese men and women.

BACKGROUND: Gallstones are abnormal masses caused by impaired metabolism of cholesterol, bilirubin, or bile salts in the gallbladder or biliary tract. ATP-binding cassette subfamily G member 8 (ABCG8) is a protein that regulates cholesterol efflux from the liver. Genome-wide association studies (GWAS) and meta-analyses of GWAS revealed the ABCG8 rs11887534 variant as the most common genetic determinant of gallstones in humans. These findings have not been extensively replicated in Taiwanese. Therefore, we appraised the relationship between gallstones and rs11887534 in a relatively large Taiwanese sample. METHODS: We retrieved data collected through questionnaires, physical and biochemical tests from the Taiwan Biobank Bank (TWB). The study participants comprised 7388 men and 13,880 women who voluntarily enrolled in the Taiwan Biobank project between 2008 and 2019. Gallstones were self-reported. RESULTS: The overall sample size was 21,268 comprising 938 gallstone patients and 20,330 non-gallstone individuals. Among the participants, 20,640 had the GG and 628 had the GC + CC genotype. At p-value < 0.05, the baseline genotypes and gallstone status between men and women were not significantly different. The risk of gallstones was higher in participants having the GC + CC compared to the GG genotype: odds ratio (OR); 95% confidence interval (CI) = 1.698; 1.240-2.325), but was lower in men compared to women (OR = 0.763; 95% CI = 0.638-0.913). Compared to men with the rs11887534 GG genotype, women with the GG and GC + CC genotypes had a higher risk of gallstone (OR; 95% CI = 1.304; 1.087-1.565 for GG and 2.291; 1.514-3.467 for GC + CC). The positive association between GC + CC and gallstones was retained after we restricted the analysis to the female participants (OR; 95% CI = 1.789 = 1.208-2.648). Hormone use was associated with an elevated risk of gallstones (OR; 95% CI = 1.359; 1.107-1.668). Relative to GG and no hormone use, we found a significantly high risk among hormone users with the GC + CC genotype (OR; 95% CI = 3.596; 1.495-8.650). CONCLUSIONS: The rs11887534 GC + CC genotype was independently associated with a higher risk of gallstones. This risk was much higher among women, especially those who used hormones for various gynecological purposes.