Advances in the biosynthesis of tetraacetyl phytosphingosine, a key substrate of ceramides.

Ceramides, formed by the dehydration of long-chain fatty acids with phytosphingosine and its derivatives, are widely used in skincare, cosmetics, and pharmaceuticals. Due to the exceedingly low concentration of phytosphingosine in plant seeds, relying on the extraction method is highly challenging. Currently, the primary method for obtaining phytosphingosine is the deacetylation of tetraacetyl phytosphingosine (TAPS) derived from fermentation. Wickerhamomyces ciferrii, an unconventional yeast from the pods of Dipteryx odorata, is the only known microorganism capable of naturally secreting TAPS, which is of great industrial value. In recent years, research and applications focused on modifying W. ciferrii for TAPS overproduction have increased rapidly. This review first describes the discovery history, applications, microbial synthesis pathway of TAPS. Research progress in using haploid breeding, mutagenesis breeding, and metabolic engineering to improve TAPS production is then summarized. In addition, the future prospects of TAPS production using the W. ciferrii platform are discussed in light of the current progress, challenges, and trends in this field. Finally, guidelines for future researches are also emphasized.

Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica.

Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.

Engineering Yarrowia lipolytica for sustainable Cis-13, 16-docosadienoic acid production.

Cis-13, 16-docosadienoic acid (DDA) is an omega-6 polyunsaturated fatty acid with great potential for application in medicine and health. Using microbial cell factories for DDA production is considered a viable alternative to extracting DDA from plant seeds. In this study, using Yarrowia lipolytica Po1f (Δku70) as a chassis, firstly, the adaptation of three elongases in Po1f (Δku70) were explored. Secondly, the DDA biosynthetic pathway was redesigned, resulting in a DDA content of 0.046 % of total fatty acids (TFAs). Thirdly, through the "push-pull" strategy, the DDA content increased to 0.078 % of TFAs. By enhancing the supply of acetyl-CoA, the DDA production in the engineered strain YL-7 reached 0.391 % of the TFAs (3.19 mg/L). Through optimizing the fermentation conditions, the DDA titer of YL-7 reached 29.34 mg/L. This research achieves the sustainable biological production of DDA in Y. lipolytica.

Harnessing oleaginous yeast to produce omega fatty acids.

Omega fatty acids are important for human health. They are traditionally extracted from animals or plants but can be alternatively produced using oleaginous yeast. Current efforts are producing yeast strains with similar fatty acid distributions and powerful lipogenesis capacity. The next step is to further make the process more competitive.

Exploiting synthetic biology platforms for enhanced biosynthesis of natural products in Yarrowia lipolytica.

With the rapid development of synthetic biology, researchers can design, modify, or even synthesize microorganisms de novo, and microorganisms endowed with unnatural functions can be considered "artificial life" and facilitate the development of functional products. Based on this concept, researchers can solve critical problems related to the insufficient supply of natural products, such as low yields, long production cycles, and cumbersome procedures. Due to its superior performance and unique physiological and biochemical characteristics, Yarrowia lipolytica is a favorable chassis cell used for green biomanufacturing by numerous researchers. This paper mainly reviews the development of synthetic biology techniques for Y. lipolytica and summarizes the recent research progress on the synthesis of natural products in Y. lipolytica. This review will promote the continued innovative development of Y. lipolytica by providing theoretical guidance for research on the biosynthesis of natural products.

Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid.

Punicic acid is a conjugated linolenic acid with various biological activities including antiobesity, antioxidant, anticancer, and anti-inflammatory effects. It is often used as a nutraceutical, dietary additive, and animal feed. Currently, punicic acid is primarily extracted from pomegranate seed oil, but it is restricted due to the extended growth cycle, climatic limitations, and low recovery level. There have also been reports on the chemical synthesis of punicic acid, but it resulted in a mixture of structurally similar isomers, requiring additional purification/separation steps. In this study, a comprehensive strategy for the production of punicic acid in Yarrowia lipolytica was implemented by pushing the supply of linoleic acid precursors in a high-oleic oil strain, expressing multiple copies of the fatty acid conjugase gene from Punica granatum, engineering the acyl-editing pathway to improve the phosphatidylcholine pool, and promoting the assembly of punicic acid in the form of triglycerides. The optimal strain with high oil production capacity and a significantly increased punicic acid ratio accumulated 3072.72 mg/L punicic acid, accounting for 6.19% of total fatty acids in fed-batch fermentation, providing a viable, sustainable, and green approach for punicic acid production to substitute plant extraction and chemical synthesis production.

Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.

Discovery of novel alkaline-tolerant xylanases from fecal microbiota of dairy cows.

Xylo-oligosaccharides (XOS) are considered as a promising type of prebiotics that can be used in foods, feeds, and healthcare products. Xylanases play a key role in the production of XOS from xylan. In this study, we conducted a metagenomic analysis of the fecal microbiota from dairy cows fed with different types of fodders. Despite the diversity in their diets, the main phyla observed in all fecal microbiota were Firmicutes and Bacteroidetes. At the genus level, one group of dairy cows that were fed probiotic fermented herbal mixture-containing fodders displayed decreased abundance of Methanobrevibacter and increased growth of beneficial Akkermansia bacteria. Additionally, this group exhibited a high microbial richness and diversity. Through our analysis, we obtained a comprehensive dataset comprising over 280,000 carbohydrate-active enzyme genes. Among these, we identified a total of 163 potential xylanase genes and subsequently expressed 34 of them in Escherichia coli. Out of the 34 expressed genes, two alkaline xylanases with excellent temperature stability and pH tolerance were obtained. Notably, CDW-xyl-8 exhibited xylanase activity of 96.1 ± 7.5 U/mg protein, with an optimal working temperature of 55 â„ƒ and optimal pH of 8.0. CDW-xyl-16 displayed an activity of 427.3 ± 9.1 U/mg protein with an optimal pH of 8.5 and an optimal temperature at 40 â„ƒ. Bioinformatic analyses and structural modeling suggest that CDW-xyl-8 belongs to GH10 family xylanase, and CDW-xyl-16 is a GH11 family xylanase. Both enzymes have the ability to hydrolyze beechwood xylan and produce XOS. In conclusion, this metagenomic study provides valuable insights into the fecal microbiota composition of dairy cows fed different fodder types, revealing main microbial groups and demonstrating the abundance of xylanases. Furthermore, the characterization of two novel xylanases highlights their potential application in XOS production.

Combination of microbial and chemical synthesis for the sustainable production of ß-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

Personalized smart voice-based electronic prescription for remote at-home feedback management in cardiovascular disease rehabilitation: a multi-center randomized controlled trial.

Objective: To investigate the quality and efficacy of remote at-home rehabilitation for patients with cardiovascular disease (CVD) using personalized smart voice-based electronic prescription, and further explore the standardized health management mode of remote family cardiac rehabilitation. Trial design: A multicenter, randomized (1:1), non-blind, parallel controlled study. Methods: A total of 171 patients with CVD who were admitted to 18 medical institutions in China from April 2021 to October 2022 were randomly divided into a treatment group (86 cases) and a control group (85 cases) in a non-blinded experiment, based on the sequence of enrollment. The control group received routine at-home rehabilitation training, and the treatment group received remote feedback-based at-home cardiac rehabilitation management based on routine at-home rehabilitation training. The primary outcome was the difference in VO2peak (mL/min/kg) after 12 weeks. A linear mixed model was developed with follow-up as the dependent variable. Age and baseline data were utilized as covariates, whereas hospital and patient characteristics were adjusted as random-effect variables. As the linear mixed model can accommodate missing data under the assumption of random missing data, there was no substitute missing value for quantitative data. Results: A total of 171 participants, with 86 in the experimental group and 85 in the control group, were included in the main analysis. The analysis, which used linear mixing model, revealed significant differences in cardiopulmonary function indexes (VO2/kg peak, VO2peak, AT, METs, and maximum resistance) at different follow-up time (0, 4, and 12 weeks) in the experimental group (p < 0.05). In the control group, there was no significant difference in cardiopulmonary values at different follow-up time (0, 4, and 12 weeks; p > 0.05). VO2/kg peak (LS mean 1.49, 95%CI 0.09-2.89, p = 0.037) and other indicators of cardiopulmonary function (p < 0.05) were significantly different between the experimental group and the control group at week 12. The results were comparable in the complete case analysis. Conclusion: The remote home cardiac rehabilitation management mode using personalized smart voice-based electronic prescription provides several benefits to patients, including improvements in muscle strength, endurance, cardiopulmonary function, and aerobic metabolism. It also helps reduce risk factors for cardiovascular disease and enhances patients' self-management abilities and treatment compliance.Clinical trial registration: http://www.chictr.org.cn, identifier ChiCTR2100044063.

Reprogramming the fatty acid metabolism of Yarrowia lipolytica to produce the customized omega-6 polyunsaturated fatty acids.

Omega-6 polyunsaturated fatty acids (ω6-PUFAs), such as γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA), are indispensable nutrients for human health. Harnessing the lipogenesis pathway of Yarrowia lipolytica creates a potential platform for producing customized ω6-PUFAs. This study explored the optimal biosynthetic pathways for customized production of ω6-PUFAs in Y. lipolytica via either the Δ6 pathway from Mortierella alpina or the Δ8 pathway from Isochrysis galbana. Subsequently, the proportion of ω6-PUFAs in total fatty acids (TFAs) was effectively increased by bolstering the provision of precursors for fatty acid biosynthesis and carriers for fatty acid desaturation, as well as preventing fatty acid degradation. Finally, the proportions of GLA, DGLA and ARA synthesized by customized strains accounted for 22.58%, 46.65% and 11.30% of TFAs, and the corresponding titers reached 386.59, 832.00 and 191.76 mg/L in shake-flask fermentation, respectively. This work provides valuable insights into the production of functional ω6-PUFAs.

Systems Metabolic Engineering of Industrial Microorganisms.

The green and sustainable production of chemicals, materials, fuels, food, and pharmaceuticals has become a key solution to the global energy and environmental crisis [...].

Combining orthogonal plant and non-plant fatty acid biosynthesis pathways for efficient production of microbial oil enriched in nervonic acid in Yarrowia lipolytica.

Nervonic acid has proven efficacy in brain development and the prevention of neurodegenerative diseases. Here, an alternative and sustainable strategy for nervonic acid-enriched plant oil production was established. Different ß-ketoacyl-CoA synthases and heterologous Δ15 desaturase were co-expressed, combined with the deletion of the ß-oxidation pathway to construct orthogonal plant and non-plant nervonic acid biosynthesis pathways in Yarrowia lipolytica. A "block-pull-restrain" strategy was further applied to improve the supply of stearic acid as the precursor of the non-plant pathway. Then, lysophosphatidic acid acyltransferase from Malania oleifera (MoLpaat) was identified, which showed specificity for nervonic acid. Endogenous LPAAT was exchanged by MoLPAAT resulted in 17.10 % nervonic acid accumulation. Finally, lipid metabolism was engineered and cofactor supply was increased to boost the lipid accumulation in a stable null-hyphal strain. The final strain produced 57.84 g/L oils with 23.44 % nervonic acid in fed-batch fermentation, which has the potential to substitute nervonic acid-enriched plant oil.

Efficient production of cordycepin by engineered Yarrowia lipolytica from agro-industrial residues.

Cordycepin, a nucleoside compound with a variety of biological activities, has been extensively applied in the nutraceutical and pharmaceutical industries. The advancement of microbial cell factories using agro-industrial residues provides a sustainable pathway for cordycepin biosynthesis. Herein, the cordycepin production was enhanced by the modification of glycolysis and pentose phosphate pathway in engineered Yarrowia lipolytica. Then, cordycepin production based on economical and renewable substrates (sugarcane molasses, waste spent yeast, and diammonium hydrogen phosphate) was analyzed. Furthermore, the effects of C/N molar ratio and initial pH on cordycepin production were evaluated. Results indicated that the maximum cordycepin productivity of 656.27 mg/L/d (72 h) and cordycepin titer was 2286.04 mg/L (120 h) by engineered Y. lipolytica in the optimized medium, respectively. The cordycepin productivity in the optimized medium was increased by 28.81% compared with the original medium. This research establishes a promising way for efficient cordycepin production from agro-industrial residues.

High-Level Production of Patchoulol in Yarrowia lipolytica via Systematic Engineering Strategies.

Patchoulol is an important sesquiterpene alcohol with a strong and lasting odor, which has led to prominent applications in perfumes and cosmetics. In this study, systematic metabolic engineering strategies were adopted to create an efficient yeast cell factory for patchoulol overproduction. First, a baseline strain was constructed by selecting a highly active patchoulol synthase. Subsequently, the mevalonate precursor pool was expanded to boost patchoulol synthesis. Moreover, a method for downregulating squalene synthesis based on Cu2+-repressible promoter was optimized, which significantly improved the patchoulol titer by 100.9% to 124 mg/L. In addition, a protein fusion strategy resulted in a final titer of 235 mg/L in shake flasks. Finally, 2.864 g/L patchoulol could be produced in a 5 L bioreactor, representing a remarkable 1684-fold increase compared to the baseline strain. To our knowledge, this is the highest patchoulol titer reported so far.

New Anti-Prelog Stereospecific Whole-Cell Biocatalyst for Asymmetric Reduction of Prochiral Ketones.

The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog's rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca2+ or K+ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications.

Combination of a Push-Pull-Block Strategy with a Heterologous Xylose Assimilation Pathway toward Lipid Overproduction from Lignocellulose in Yarrowia lipolytica.

The production of biodiesel using microbial lipids derived from renewable lignocellulosic biomass is considered a promising strategy to reduce environmental pressure and promote the green energy transition. The hydrolysates of lignocellulosic biomass are rich in glucose and xylose, which makes it crucial to efficiently utilize both sugars. Here, we combined metabolic engineering and adaptive laboratory evolution (ALE) to construct an engineered Yarrowia lipolytica strain that can efficiently produce lipids from glucose and xylose. First, the "Push-Pull-Block" strategy was adopted to increase lipid content to 73.42% of the dry cell weight (DCW). Then, a heterologous xylose-utilization pathway was integrated into the engineered strain, which was subjected to ALE. The final evolved strain could accumulate 53.64% DCW of lipids from xylose, and the lipid titer reached 16.25 g/L. This work sheds light on the potential of microbial lipid overproduction from lignocellulose using engineered Y. lipolytica.

Multienzymatic synthesis of nylon monomers from vegetable oils.

Nylons are important polyamide (PA) materials that can be polymerized from different monomers. Bio-based nylon monomers are traditionally obtained through chemical conversion from vegetable oils, but they can be more sustainably obtained through multienzymatic catalysis. For large-scale application of this process, enzyme engineering and process innovation must be combined.