Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

De Novo Synthesis of Chrysin in Saccharomyces cerevisiae.

Chrysin, a flavonoid, has been found to have been widely used in the health food field. But at present, chrysin production is hindered by the low availability of precursors and the lack of catalytic enzymes with high activity. Therefore, ZmPAL was initially screened to synthesize trans-cinnamic acid with high catalytic activity and specificity. To enhance the supply of precursors, the shikimic acid and chorismic acid pathway genes were overexpressed. Besides, the expression of the intracellular and mitochondrial carbon metabolism genes CIT, MAC1/3, CTP1, YHM2, RtME, and MDH was enhanced to increase the intracellular acetyl-CoA content. Chrysin was synthesized through a novel gene combination of ScCPR-EbFNSI-1 and PcFNSI. Finally, de novo synthesis of chrysin was achieved, reaching 41.9 mg/L, which is the highest reported concentration to date. In summary, we identified efficient enzymes for chrysin production and increased it by regulating acetyl-CoA metabolism in mitochondria and the cytoplasm, laying a foundation for future large-scale production.

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3'-hydroxylase/cytochrome P450 reductase (F3'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid ß-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Spiritual care needs and their attributes among Chinese inpatients with advanced breast cancer based on the Kano model: a descriptive cross-sectional study.

BACKGROUND: Numerous previous research have established the need for spiritual care among patients with cancer globally. Nevertheless, there was limited research, primarily qualitative, on the spiritual care needs of Chinese inpatients with advanced breast cancer. Furthermore, the need for spiritual care was rarely explored using the Kano model. To better understand the spiritual care needs and attributes characteristics of inpatients with advanced breast cancer, this study examined the Kano model. METHODS: A descriptive cross-sectional design study was conducted in the oncology departments of three tertiary grade-A hospitals in China from October 2022 to May 2023. To guarantee high-quality reporting of the study, the Strengthening the Reporting of Observational Studies in Epidemiology Checklist was used. Data on the demographic characteristics questionnaire, the Nurse Spiritual Therapeutics Scale (NSTS), and the Kano model-based Nurse Spiritual Therapeutics Attributes Scale (K-NSTAs) were collected through convenience sampling. The Kano model, descriptive statistics, two independent samples t-tests, and one-way analysis of variance were used to analyze the data. RESULTS: The overall score for spiritual care needs was 31.16 ± 7.85. The two dimensions with the highest average scores, "create a good atmosphere" (3.16 ± 0.95), and the lowest average scores, "help religious practice" (1.72 ± 0.73). The 12 items were distributed as follows: three attractive attributes were located in Reserving Area IV; five one-dimensional attributes were distributed as follows: three one-dimensional attributes were located in Predominance Area I, and two were found in Improving Area II; two must-be attributes were located in Improving Area II; and two indifference attributes were located in Secondary Improving Area III. CONCLUSION: The Chinese inpatients with advanced breast cancer had a middle level of spiritual care needs, which need to be further improved. Spiritual care needs attributes were defined, sorted, categorized, and optimized accurately and perfectly by the Kano model. And "create a good atmosphere" and "share self-perception" were primarily one-dimensional and must-be attributes. In contrast, the items in the dimensions of "share self-perception" and "help thinking" were principally attractive attributes. Nursing administrators are advised to optimize attractive attributes and transform indifference attributes by consolidating must-be and one-dimensional attributes, which will enable them to take targeted spiritual care measures based on each patient's characteristics and unique personality traits.

YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica.

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

Efficient synthesis of squalene by cytoplasmic-peroxisomal engineering and regulating lipid metabolism in Yarrowia lipolytica.

Squalene, a high-value acyclic triterpenoid compound, is broadly used in the food and medical industries. Although the large acetyl-CoA pool and hydrophobic space of Yarrowia lipolytica are suitable for the accumulation of squalene, the current production level in Y. lipolytica is still not sufficient for industrial production. In this study, two rounds of multicopy integration of genes encoding key enzymes were performed to enhance squalene anabolic flux in the cytoplasm. Furthermore, the mevalonate pathway was imported into peroxisomes through the compartmentalization strategy, and the production of squalene was significantly increased. By augmenting the acetyl-CoA supply in peroxisomes and the cytoplasm, the squalene was boosted to 2549.1 mg/L. Finally, the squalene production reached 51.2 g/L by fed-batch fermentation in a 5-L bioreactor. This is the highest squalene production reported to date for microbial production, and this study lays the foundation for the synthesis of steroids and squalene derivatives.

Remodelling metabolism for high-level resveratrol production in Yarrowia lipolytica.

Resveratrol is a polyphenol with numerous applications in food, pharma, and cosmetics. Lack of precursors and low titer are the main problems hindering industrial scale resveratrol production. Based on previous prescreening, expressing the combination of FjTAL, Pc4CL1 and VvSTS achieved the best resveratrol titer. This was further improved to 235.1 mg/L through engineering the shikimic acid pathway, applying a modular enzyme assembly of Pc4CL1 and VvSTS, enhancing p-coumaric acid supply and diverting glycolytic flux toward erythrose-4-phosphate. The titer was increased to 819.1 mg/L following two rounds of multicopy integration of resveratrol biosynthesis and malonyl-CoA supply, respectively. The titer reached 22.5 g/L with a yield on glucose of 65.5 mg/g using an optimum fed-batch strategy in a 5 L bioreactor with morphology control. This research is the highest report on the de novo production of resveratrol in Yarrowia lipolytica and the findings lay a solid foundation for other producing polyphenols.

Two-stage oxygen supply strategy for enhancing fed-batch production of pyrroloquinoline quinone in Hyphomicrobium denitrificans FJNU-6.

Oxygen is a vital parameter for pyrroloquinoline quinone (PQQ) biosynthesis. In this study, the effects of oxygen supply on the biosynthesis of PQQ were first investigated systematically with Hyphomicrobium denitrificans FJNU-6. Following a kinetic analysis of the specific cell growth rate (µx) and specific PQQ formation rate (µp) in 5 L benchtop fermentation systems at various oxygen supply levels ranging from 0 to 60%, a novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity. Moreover, the transcription of genes involved in methanol oxidation and PQQ biosynthesis was analyzed throughout the process to outline the effect of oxygen supply on cell metabolism. Furthermore, with constant feeding of methanol at 0-1 g/L after the initial methanol was consumed completely, the PQQ concentration and productivity reached 1070 mg/L and 7.64 mg/L/h, respectively, after 140 h in a 5-L fermenter. The two-stage oxygen supply strategy developed in this study provides an effective and economical strategy for the industrial production of PQQ.Key Points• A novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity.•The transcription of genes involved in methanol oxidation and PQQ biosynthesis was regulated by changes in oxygen supply.• This study offers an effective and economical strategy for industrial or large-scale production of PQQ.

Folium Sennae and emodin reverse airway smooth muscle contraction.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

[Ethacrynic acid inhibits airway smooth muscle contraction in mice].

The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca2+ concentration ([Ca2+]i) in ASM cells. The results showed that EA significantly inhibited the high K+ (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) µmol/L and (56.22 ± 7.62) µmol/L, respectively. EA decreased the K+ and ACh-induced elevation of [Ca2+]i from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca2+ influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca2+ influx and decreases of [Ca2+]i, leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.

NS8593 inhibits Ca2+ permeant channels reversing mouse airway smooth muscle contraction.

AIMS: This study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism. MAIN METHODS: ASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system. KEY FINDINGS: High K+ (80 mM K+) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs). SIGNIFICANCE: Our results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator.