Yarrowia lipolytica as a Platform for Punicic Acid Production.

Punicic acid (PuA) is a polyunsaturated fatty acid with significant medical, biological, and nutraceutical properties. The primary source of punicic acid is the pomegranate seed oil obtained from fruits of trees that are mainly cultivated in subtropical and tropical climates. To establish sustainable production of PuA, various recombinant microorganisms and plants have been explored as platforms with limited efficiencies. In this study, the oleaginous yeast Yarrowia lipolytica was employed as a host for PuA production. First, growth and lipid accumulation of Y. lipolytica were evaluated in medium supplemented with pomegranate seed oil, resulting in the accumulation of lipids up to 31.2%, consisting of 22% PuA esterified in the fraction of glycerolipids. In addition, lipid-engineered Y. lipolytica strains, transformed with the bifunctional fatty acid conjugase/desaturase from Punica granatum (PgFADX), showed the ability to accumulate PuA de novo. PuA was detected in both polar and neutral lipid fractions, especially in phosphatidylcholine and triacylglycerols. Promoter optimization for PgFADX expression resulted in improved accumulation of PuA from 0.9 to 1.8 mg/g of dry cell weight. The best-producing strain expressing PgFADX under the control of a strong erythritol-inducible promoter produced 36.6 mg/L PuA. These results demonstrate that the yeast Y. lipolytica is a promising host for PuA production.

Biorefining of rapeseed meal: A new and sustainable strategy for improving Cr(VI) biosorption on residual wastes from agricultural byproducts after phenolic extraction.

Phenolic recovery from agricultural byproducts has been highlighted due to their health-promoting bioactivities. However, uncontrolled discard of residues after extraction process would induce environmental pollution and bioresource waste. In this study, biorefining of phenolic-rich rapeseed meal (RSM) and its defatted sample (dRSM) was attempted by holistic utilization of phenolic extract and residue separately. Phenolic removal could significantly improve residues' Cr(VI) adsorption capacities by about 21%, which presented extended physical surface and more released functional groups. Moreover, simulating raw material by remixing 3% separated phenolic extracts or main component sinapic acid therein with corresponding residues further improved about 12% adsorption efficiencies. These indicated that the different present forms of phenolics had opposite effects on Cr(VI) removal. While natural conjugational form inhibited hosts' biosorption, free form had enhanced functions for either extract or residue. Four optimal adsorption parameters (pH, adsorbent dosage, contact time and initial Cr(VI) concentration), three kinetic (pseudo-first order, pseudo-second order and intra-particle diffusion) models and two isotherms (Langmuir and Freundlich) were used to reveal the adsorption process. The maximum Cr(VI) adsorption capacity on residues could reach about 100 mg/g, which was superior to that of most biosorbents derived from agricultural byproducts, even some biochar. Together with the residues' advantages with everlasting capacity after 3 adsorption-desorption cycles and excellent abilities for adsorbing multiple co-existed metal ions (Cr(VI), Cd(II), Cu(II), Pb(II), Ni(II) and Zn(II)), phenolic recovery was first proved to be a new and sustainable strategy for modifying biosorbents from agricultural byproducts with zero waste.

Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica.

Microbial production of lipids is one of the promising alternatives to fossil resources with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain (ß-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve ß-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding ß-ketothiolase in the producing strain, and the OCFA production was increased by 33% compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 â€‹g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.

Effects of Calcium and Manganese on Sporulation of Bacillus Species Involved in Food Poisoning and Spoilage.

Spores are resistant against many extreme conditions including the disinfection and sterilization methods used in the food industry. Selective prevention of sporulation of Bacillus species is an ongoing challenge for food scientists and fermentation technologists. This study was conducted to evaluate the effects of single and combined supplementation of calcium and manganese on sporulation of common pathogenic and food spoilage Bacillus species: B. cereus, B. licheniformis, B. subtilis and B. coagulans. Sporulation of Bacillus vegetative cells was induced on sporulation media supplemented with diverse concentrations of the minerals. Under the various mineral supplementation conditions, the degree of sporulation was quantified with colonies formed by the Bacillus spores. The results revealed that B. licheniformis and B. cereus displayed the weakest sporulation capabilities on media with minimal supplementation levels of calcium and manganese. The lowest sporulation of B. subtilis and B. coagulans was observed on media supplemented with the highest level of calcium and low levels of manganese. Depending on effect of supplementation on sporulation, the Bacillus species were divided into two distinct groups: B. licheniformis and B. cereus; and B. subtilis and B. coagulans. The information provides valuable insight to selectively reduce sporulation of Bacillus species undesirable in the food industry.

De novo Biosynthesis of Odd-Chain Fatty Acids in Yarrowia lipolytica Enabled by Modular Pathway Engineering.

Microbial oils are regarded as promising alternatives to fossil fuels as concerns over environmental issues and energy production systems continue to mount. Odd-chain fatty acids (FAs) are a type of valuable lipid with various applications: they can serve as biomarkers, intermediates in the production of flavor and fragrance compounds, fuels, and plasticizers. Microorganisms naturally produce FAs, but such FAs are primarily even-chain; only negligible amounts of odd-chain FAs are generated. As a result, studies using microorganisms to produce odd-chain FAs have had limited success. Here, our objective was to biosynthesize odd-chain FAs de novo in Yarrowia lipolytica using inexpensive carbon sources, namely glucose, without any propionate supplementation. To achieve this goal, we constructed a modular metabolic pathway containing seven genes. In the engineered strain expressing this pathway, the percentage of odd-chain FAs out of total FAs was higher than in the control strain (3.86 vs. 0.84%). When this pathway was transferred into an obese strain, which had been engineered to accumulate large amounts of lipids, odd-chain fatty acid production was 7.2 times greater than in the control (0.05 vs. 0.36 g/L). This study shows that metabolic engineering research is making progress toward obtaining efficient cell factories that produce odd-chain FAs.