Improving and Streamlining Gene Editing in Yarrowia lipolytica via Integration of Engineered Cas9 Protein.

The oleaginous yeast Yarrowia lipolytica is a prominent subject of biorefinery research due to its exceptional performance in oil production, exogenous protein secretion, and utilization of various inexpensive carbon sources. Many CRISPR/Cas9 genome-editing systems have been developed for Y. lipolytica to meet the high demand for metabolic engineering studies. However, these systems often necessitate an additional outgrowth step to achieve high gene editing efficiency. In this study, we introduced the eSpCas9 protein, derived from the Streptococcus pyogenes Cas9(SpCas9) protein, into the Y. lipolytica genome to enhance gene editing efficiency and fidelity, and subsequently explored the optimal expression level of eSpCas9 gene by utilizing different promoters and selecting various growth periods for yeast transformation. The results demonstrated that the integrated eSpCas9 gene editing system significantly enhanced gene editing efficiency, increasing from 16.61% to 86.09% on TRP1 and from 33.61% to 95.19% on LIP2, all without the need for a time-consuming outgrowth step. Furthermore, growth curves and dilution assays indicated that the consistent expression of eSpCas9 protein slightly suppressed the growth of Y. lipolytica, revealing that strong inducible promoters may be a potential avenue for future research. This work simplifies the gene editing process in Y. lipolytica, thus advancing its potential as a natural product synthesis chassis and providing valuable insights for other comparable microorganisms.

Lonicera caerulea Pomace Alleviates DSS-Induced Colitis via Intestinal Barrier Improvement and Gut Microbiota Modulation.

The objective of this investigation was to appraise the mitigative effects of Lonicera caerulea pomace (LCP)-supplemented diets on Dextran Sulfate Sodium (DSS)-induced colitis, and to discuss the potential mechanisms. LCP, a by-product of fruit juice processing, harbors a higher content of polyphenols and dietary fiber compared to the L. caerulea pulp. In a murine model of colitis, the LCP diet attenuated the symptoms of colitis, as evidenced by the reduction in the disease activity index (DAI), extension of colon length, and amelioration of histopathological damage. The anti-inflammatory attributes of LCP were substantiated by a decrease in myeloperoxidase (MPO) activity and suppression of inflammatory cytokine expressions within the colon. Meanwhile, LCP mediated the repair of the intestinal barrier, characterized by the upregulation of gene expressions of tight junction (TJ) proteins and Muc2. Furthermore, LCP altered the composition of the gut microbiota, manifested in increased alpha diversity, enhancement of the abundance of beneficial bacteria (Akkermansia, Coprococcus and Bifidobacterium), and diminishment in the abundance of pathogenic bacteria (Escherichia, Enterococcus, Mucispirillum and Clostridium). Dietary LCP also increased the concentrations of SCFAs within the intestinal luminal contents of colitis mice. Given the affirmative impact of LCP on colitis, LCP may possess great potential in promoting intestinal health.

Lonicera Caerulea Juice Alleviates Alcoholic Liver Disease by Regulating Intestinal Flora and the FXR-FGF15 Signaling Pathway.

Alcoholic liver disease (ALD) is a growing public health issue with high financial, social, and medical costs. Lonicera caerulea, which is rich in polyphenolic compounds, has been shown to exert anti-oxidative and anti-inflammatory effects. This study aimed to explore the effects and mechanisms of concentrated Lonicera caerulea juice (LCJ) on ALD in mice. ALD was established in mice via gradient alcohol feeding for 30 days. The mice in the experimental group were given LCJ by gavage. The reduction of aspartate transaminase (AST) and alanine transaminase (ALT) in the serum of mice indicated that LCJ has a liver-protective effect. LCJ improved the expression of AMPK, PPARα, and CPT1b in ALD mice to reduce the liver lipid content. Additionally, LCJ increased the expression of farnesoid X receptor (FXR), fibroblast growth factor 15 (FGF15), and fibroblast growth factor receptor 4 (FGFR4), which lowers the expression of cytochrome P450 7A1 (CYP7A1) and lessens bile acid deposition in the liver. In mice, LCJ improved the intestinal barrier by upregulating the expression of mucins and tight junction proteins in the small intestine. Moreover, it accelerated the restoration of microbial homeostasis in both the large and small intestines and increased short-chain fatty acids in the cecum. In conclusion, LCJ alleviates ALD by reducing liver and serum lipid accumulation and modulating the FXR-FGF15 signaling pathway mediated by gut microbes.

Effects of different n-6/n-3 polyunsaturated fatty acids ratios on lipid metabolism in patients with hyperlipidemia: a randomized controlled clinical trial.

Background and aims: Intake of n-3 polyunsaturated fatty acids (PUFA) is helpful for cardiometabolic health. It improves lipid metabolism, and increasing n-3 PUFA is often considered beneficial. However, the role of n-6/n-3 in the regulation of lipid metabolism has been much debated. Therefore, this study was performed on the effect of different proportions of n-6/n-3 diet on lipid metabolism, and quality of life in patients with hyperlipidemia, aiming to explore appropriate proportions of n-6/n-3 to provide the theoretical basis for the development and application of nutritional blended oil in the future. Methods: These 75 participants were randomized and assigned into three groups, which received dietary oil with high n-6/n-3 PUFA ratios (HP group: n-6/n-3 = 7.5/1), dietary oil with middle n-6/n-3 PUFA ratios (MP group: n-6/n-3 = 2.5/1) or low n-6/n-3 PUFA ratios (LP group: n-6/n-3 = 1/2.5). All patients received dietary guidance and health education were monitored for hyperlipidemia. Anthropometric, lipid and blood glucose parameters and quality of life were assessed at baseline and 60 days after intervention. Result: After 60 days, high-density lipoprotein cholesterol (HDL-c) level was increased (p = 0.029) and Total cholesterol (TC) level was decreased (p = 0.003) in the MP group. In the LP group, TC level was decreased (p = 0.001), TG level was decreased (p = 0.001), but HDL-c level was not significantly increased. At the end of intervention, quality of life' score was improved in both MP and LP groups (p = 0.037). Conclusion: Decreasing the intake of edible oil n-6/n-3 ratio can improve blood lipids and quality of life. This is significant for the prevention of cardiovascular disease (CVD). It is also essential to note that an excessive reduction of the n-6/n-3 ratio does not further improve the blood lipid metabolism. In addition, the application of perilla oil in nutritional blended oil has particular significance. Clinical trial registration: https://www.chictr.org.cn/indexEN.html, identifier ChiCTR-2300068198.

Engineering the ß-Oxidation Pathway in Yarrowia lipolytica for the Production of trans-10, cis-12-Conjugated Linoleic Acid.

trans-10, cis-12-Conjugated linoleic acid (t10, c12-CLA) is an octadecadienoic acid with various biological benefits. Previously, linoleic acid isomerase from Propionibacterium acnes (PAI) was overexpressed in Yarrowia lipolytica (Y. lipolytica) to produce t10, c12-CLA. However, the t10, c12-CLA yield was restricted by the peroxisomal ß-oxidation pathway. In this study, to minimize the degradation of t10, c12-CLA, four genetically modified strains of Y. lipolytica (Δpox2-oPAI, Δpox3-oPAI, Δpox2Δpox3-oPAI, and Δpex10-oPAI) were constructed and compared in terms of production stability and yield of t10, c12-CLA using safflower seed oil as substrates. The Δpex10-oPAI strain exhibited the best results, as revealed by the reduction of the t10, c12-CLA degradation rate from 58.5 to 18.6 mg/L/h. Additionally, the YLUpex10mP recombinant strain bearing six copies of oPAI combined with PEX10 deletion enhanced t10, c12-CLA production to 7.4 g/L and exhibited a CLA degradation rate of 19.7 mg/L/h, a 78% decrease from that of the control strain. Finally, in a bioreactor containing low-cost volatile fatty acids as partial carbon sources, the t10, c12-CLA content in the YLUpex10mP strain increased to 9.7 g/L, 1.3 times higher than in flasks. To our knowledge, this is the highest t10, c12-CLA yield through microbial synthesis reported to date.

Enhanced Soluble Expression of Linoleic Acid Isomerase by Coordinated Regulation of Promoter and Fusion Tag in Escherichia coli.

PAI is a linoleic acid isomerase from Propionibacterium acnes and is the key enzyme in the synthesis of trans10, cis12-conjugated linoleic acid. However, the majority of the expressed PAI in Escherichia coli occurs in its nonfunctional form in inclusion bodies, limiting the biosynthesis of conjugated linoleic acid. In an attempt to improve the solubility of recombinant PAI in Escherichia coli, three promoters representing different transcriptional strengths (T7, CspA, and Trc), paired with three fusion tags, (His6, MBP, and Fh8), respectively, were investigated in this study. Among the nine recombinant strains, Escherichia coli BL21 (DE3) (pET24a-Mpai), containing the T7 promoter and MBP fusion tag, led to a considerable increase in PAI solubility to 86.2%. MBP-PAI was purified 41-fold using affinity column chromatography. The optimum catalytical conditions of MBP-PAI were 37 °C and pH 7.5 with the addition of 1 mmol/L Tween-20. Most of the tested metal ions inhibited MBP-PAI activity. The apparent kinetic parameters (Km and Vmax) were measured with linoleic acid concentrations ranging from 71 µM to 1428 µM. The substrate linoleic acid did not exert any inhibitory effect on MBP-PAI. The Km of MBP-PAI was 253.9 µmol/L, and the Vmax was 2253 nmol/min/mg. This study provided a new method for improving the solubility of the recombinant linoleic acid isomerase in Escherichia coli.

Beneficial Effects of Three Dietary Cyclodextrins on Preventing Fat Accumulation and Remodeling Gut Microbiota in Mice Fed a High-Fat Diet.

Globally, obesity and its metabolic complications, which are intimately linked to diet, are major public health problems. Cyclodextrins (CDs) are cyclic oligosaccharides consisting of (α-1,4)-linked D-glucopyranose units that can reduce fat bioavailability and affect metabolism by improving intestinal flora as prebiotics. We compared the effects of three CDs on preventing fat accumulation and remodeling gut microbiota in a high-fat diet-fed C57BL/6J mouse model. α-CD maximized energy expenditure by 12.53%, caused the RER value to drop from 0.814 to 0.788, and increased the proportion of fatty acid oxidation for energy supply. ß-CD supplementation resulted in a marked 24.53% reduction in weight gain and a decrease in epididymal-fat-relative weight from 3.76% to 2.09%. It also minimized ectopic fat deposition and improved blood lipid parameters. γ-CD maximized the concentration of SCFAs in the cecum from 6.29 to 15.31 µmol/g. All three CDs reduced the abundance ratio of Firmicutes and Bacteroidetes to a low-fat diet level, increased the abundance of Lactobacillus and Akkermansia, and reduced the abundance of Allobaculum and Ruminococcus. These findings imply that a combination of multiple CDs may exert superior effects as a potential strategy for obesity prevention.

Characterization of Pinus densiflora var. zhangwuensis S.J.Zhang, C.X.Li & X.Y.Yuan complete chloroplast genome.

Pinus densiflora var. zhangwuensis S.J.Zhang, C.X.Li & X.Y.Yuan, a tree species with promising afforestation potential in northern China. Here, we assembled and annotated the complete chloroplast (cp) genome of P. densiflora var. zhangwuensis using the Oxford Nanopore sequencing technology. The cp genome was 119,725 bp in length, exhibiting a typical quadripartite structure with a large single-copy (LSC: 65,552 bp) and a small single-copy (SSC: 53,183 bp) separated by a pair of inverted repeats regions (IRA and IRB: each of 495 bp) region. The overall GC content is 37.3%. The genome was predicted to encode 112 distinct genes, including 72 protein-coding, 36 tRNA, and four rRNA genes. Maximum-likelihood (ML) phylogenetic for cp genome sequences of 18 Pinaceae species revealed that P. densiflora var. zhangwuensis was closely related to Pinus sylvestris.

Production of trans-10,cis-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of Yarrowia lipolytica.

OBJECTIVE: To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica. RESULTS: Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l-1, 100 g wet cells l-1, and 25 g LA l-1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l-1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l-1. CONCLUSION: Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

Production of conjugated linoleic acid by heterologous expression of linoleic acid isomerase in oleaginous fungus Mortierella alpina.

OBJECTIVE: To increase the commercial value of oleaginous fungus Mortierella alpina by incorporation of trans-10,cis-12 conjugated linoleic acid (CLA) into the polyunsaturated fatty acids (PUFAs) of M. alpina via Propionibacterium acnes isomerase (PAI) conversion. RESULTS: The PAI gene and the codon optimization version were heterologously expressed in M. alpina via Agrobacterium tumefaciens-mediated transformation (ATMT). Coding usage modification significantly improved the translation of PAI transcripts and trans-10,cis-12 CLA was produced up to 1.2 mg l(-1), which corresponds to approx. 0.05% of the total fatty acid (TFA). Since PAI prefers free linoleic acid as a substrate rather than any other forms, 5 µM long-chain acyl CoA synthetase inhibitor was added and the trans-10,cis-12 CLA content increased approx. 24-fold to 29 mg l(-1), reaching up to 1.2% (w/w) of the TFA in M. alpina. CONCLUSION: Heterologous expression of PAI in M. alpina by ATMT methods is a practicable way in biosynthesis of CLA and this system may be a feasible platform for industrial production of CLA.

Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid.

BACKGROUND: Conjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains. RESULTS: We have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l. CONCLUSIONS: We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.

[Physiological characteristics of Pinus densiflora var. zhangwuensis and Pinus sylvestris var. mongolica seedlings on sandy lands under salt-alkali stresses].

For the popularization of Pinus densiflora var. zhangwuensis, a new afforestation tree species on the desertified and salinized-alkalized lands in Northern China, and to evaluate the salinity-alkalinity tolerance of the tree species and to better understand the tolerance mechanisms, a pot experiment with 4-year old P. densiflora var. zhangwuensis and P. sylvestris var. mongolica was conducted to study their seedlings growth and physiological and biochemical indices under the effects of three types salt (NaCl, Na2CO3, and NaHCO3 ) stresses and of alkali (NaOH) stress. Under the salt-alkali stresses, the injury level of P. densiflora var. zhangwuensis was lower, and the root tolerance index was higher. The leaf catalase (CAT) activity increased significantly by 22. 6 times at the most, as compared with the control; the leaf malondialdehyde (MDA) content had no significant increase; the leaf chlorophyll (Chl) content had a smaller decrement; and the leaf water content (LWC) increased slightly. P. sylvestris var. mongolica responded differently to the salt-alkali stresses. Its leaf CAT activity had less change, MDA content increased significantly, Chl content had significant decrease, and LWC decreased slightly. It was suggested that P. densi-flora var. zhangwuensis had a greater salinity-alkalinity tolerance than P. sylvestris var. mongolica. The higher iron concentration in P. densiflora var. zhangwuensis needles enhanced the CAT activity and Chl content, whereas the higher concentrations of zinc and copper were associated with the stronger salinity-alkalinity tolerance.

De novo synthesis of trans-10, cis-12 conjugated linoleic acid in oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Conjugated linoleic acid (CLA) has many well-documented beneficial physiological effects. Due to the insufficient natural supply of CLA and low specificity of chemically produced CLA, an effective and isomer-specific production process is required for medicinal and nutritional purposes. RESULTS: The linoleic acid isomerase gene from Propionibacterium acnes was expressed in Yarrowia lipolytica Polh. Codon usage optimization of the PAI and multi-copy integration significantly improved the expression level of PAI in Y. lipolytica. The percentage of trans-10, cis-12 CLA was six times higher in yeast carrying the codon-optimized gene than in yeast carrying the native gene. In combination with multi-copy integration, the production yield was raised to approximately 30-fold. The amount of trans-10, cis-12 CLA reached 5.9% of total fatty acid yield in transformed Y. lipolytica. CONCLUSIONS: This is the first report of production of trans-10, cis-12 CLA by the oleaginous yeast Y. lipolytica, using glucose as the sole carbon source through expression of linoleic acid isomerase from Propionibacterium acnes.

Genome characterization of the oleaginous fungus Mortierella alpina.

Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.