Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids.

Odd-numbered fatty acids (FAs) have been widely used in nutrition, agriculture, and chemical industries. Recently, some studies showed that they could be produced from bacteria or yeast, but the products are almost exclusively odd-numbered long-chain FAs. Here we report the design and construction of two biosynthetic pathways in Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids (OMFAs) via ricinoleic acid and 10-hydroxystearic acid, respectively. The production of OMFAs was enabled by introducing a hydroxy fatty acid cleavage pathway, including an alcohol dehydrogenase from Micrococcus luteus, a Baeyer-Villiger monooxygenase from Pseudomonas putida, and a lipase from Pseudomonas fluorescens. These OMFA biosynthetic pathways were optimized by eliminating the rate-limiting step, generating heptanoic acid, 11-hydroxyundec-9-enoic acid, nonanoic acid, and 9-hydroxynonanoic acid at 7.83 mg/L, 9.68 mg/L, 9.43 mg/L and 13.48 mg/L, respectively. This work demonstrates the biological production of OMFAs in a sustainable manner in S. cerevisiae.

De Novo Biosynthesis of Free Vaccenic Acid with a Low Content of Oleic Acid in Saccharomyces cerevisiae.

Omega-7 (ω-7) fatty acids have potential application in the fields of nutraceutical, agricultural, and food industry. The natural ω-7 fatty acids are currently from plants or vegetable oils, which are unsustainable and limited by the availability of plant sources. Here, we developed an innovative biosynthetic route to produce vaccenic acid (C18:1 ω-7) while minimizing oleic acid (C18:1 ω-9) content in Saccharomyces cerevisiae. We have engineered S. cerevisiaeto produce C18:1 ω-7 by expressing a fatty acid elongase from Rattus norvegicus. To reduce the content of C18:1 ω-9, the endogenous desaturase Ole1 was replaced by the desaturase, which has specific activity on palmitoyl-coenzyme A (C16:0-CoA). Finally, the production of free C18:1 ω-7 was improved by optimizing the source of cytochrome b5 and overexpressing endoplasmic reticulum chaperones. After combining these strategies, the yield of C18:1 ω-7 was increased from 0 to 9.3 mg/g DCW and C18:1 ω-9 was decreased from 25.2 mg/g DCW to 1.6 mg/g DCW. This work shows a de novo synthetic pathway to produce the highest amount of free C18:1 ω-7 with a low content of C18:1 ω-9 in S. cerevisiae.

Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

De novo bio-production of odd-chain fatty acids in Saccharomyces cerevisiae through a synthetic pathway via 3-hydroxypropionic acid.

Odd-chain fatty acids (OCFAs) and their derivatives have attracted increasing attention due to their wide applications in the chemical, fuel, and pharmaceutical industry. However, most natural fatty acids are even-chained, and OCFAs are rare. In this work, a novel pathway was designed and established for de novo synthesis of OCFAs via 3-hydroxypropionic acid (3-HP) as the intermediate in Saccharomyces cerevisiae. First, the OCFAs biosynthesis pathway from 3-HP was confirmed, followed by an optimization of the precursor 3-HP. After combining these strategies, a de novo production of OCFAs at 74.8 mg/L was achieved, and the percentage of OCFAs in total lipids reached 20.3%, reaching the highest ratio of de novo-produced OCFAs. Of the OCFAs produced by the engineered strain, heptadecenoic acid (C17:1) and heptadecanoic acid (C17:0) accounted for 12.1% and 7.6% in total lipid content, respectively. This work provides a new and promising pathway for the de novo bio-production of OCFAs.

Engineering propionyl-CoA pools for de novo biosynthesis of odd-chain fatty acids in microbial cell factories.

Odd-chain fatty acids (OcFAs) and their derivatives have attracted great interest due to their wide applications in the food, pharmaceutical and petrochemical industries. Microorganisms can naturally de novo produce fatty acids (FAs), where mainly, even-chain with acetyl-CoA instead of odd-chain with propionyl-CoA is used as the primer. Usually, the absence of the precursor propionyl-CoA is considered the main reason that limits the efficient production of OcFAs. It is thus crucial to explore/evaluate/identify promising propionyl-CoA biosynthetic pathways to achieve large-scale biosynthesis of OcFAs. This review discusses the latest advances in microbial metabolism engineering toward producing propionyl-CoA and considers future research directions and challenges toward optimized production of OcFAs.

Metabolic engineering of threonine catabolism enables Saccharomyces cerevisiae to produce propionate under aerobic conditions.

BACKGROUND: Propionate is widely used as a preservative in the food and animal feed industries. Propionate is currently produced by petrochemical processes, and fermentative production of propionate remains challenging. METHODS AND RESULTS: In this study, a synthetic propionate pathway was constructed in the budding yeast Saccharomyces cerevisiae, for propionate production under aerobic conditions. Through expression of tdcB and aldH from Escherichia coli and kivD from Lactococcus lactis, L-threonine was converted to propionate via 2-ketobutyrate and propionaldehyde. The resulting yeast aerobically produced 0.21 g L-1 propionate from glucose in a shake flask. Subsequent overexpression of pathway genes and elimination of competing pathways increased propionate production to 0.37 g L-1 . To further increase propionate production, carbon flux was pulled into the propionate pathway by weakened expression of pyruvate kinase (PYK1), together with overexpression of phosphoenolpyruvate carboxylase (ppc). The final propionate production reached 1.05 g L-1 during fed-batch fermentation in a fermenter. CONCLUSIONS AND IMPLICATIONS: In this work, a yeast cell factory was constructed using synthetic biology and metabolic engineering strategies to enable propionate production under aerobic conditions. Our study demonstrates engineered S. cerevisiae as a promising alternative for the production of propionate and its derivatives.

Fabrication of efficient alginate composite beads embedded with N-doped carbon dots and their application for enhanced rare earth elements adsorption from aqueous solutions.

The increasing demand of rare earth elements in agriculture and industry raises the potential environmental pollution with their inevitable toxicity and biological accumulation. It is necessary to develop an efficient and low-cost technique to effectively remove rare earth elements. N-doped carbon dots (CDs) were green-synthesized using one-pot domestic microwave oven and novel alginate composite (ALG@CDs) beads were fabricated by embedding the N-doped CDs into pure alginate hydrogel beads. The synthetic materials were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and zeta potential measurement. The ALG@CDs beads could effectively adsorb rare earth elements from aqueous solutions. Taking the adsorption of rare earth element Gd (III) as an example, initial concentration, adsorbent dosage, pH, temperature and contact time were optimized. The adsorption isotherms and kinetics fitted to Langmuir isotherm model (R2 > 0.9991) and pseudo-second-order kinetic model (R2 = 0.9918), respectively. The adsorption capacity of ALG@CDs-3 (201.21 mg/g) for Gd (III) was much higher than that of bare ALG beads (124.38 mg/g) according to the maximum theoretical adsorption capacity of Langmuir model. Competitive adsorption results indicated that ALG@CDs beads had stronger affinity to Gd (III) than to Al (III), Co (II), Ni (II), Zn (II), and Mg (II). The adsorbent could be easily collected and reused for five adsorption-regeneration cycles. The results indicated that the fabricated ALG@CDs beads are the promising materials for adsorption or enrichment of rare earth elements from waste water.

Isolation and purification of alkaloids from lotus leaves by ionic-liquid-modified high-speed countercurrent chromatography.

An effective high-speed countercurrent chromatography method was successfully established by using ionic liquids as the modifier of the two-phase solvent system. Adding a small amount of ionic liquids significantly shortens the separation time and improves the separation efficiency. The conditions of ionic-liquid-modified high-speed countercurrent chromatography including solvent systems, types and content of added ionic liquids, and ionic liquids posttreatment were investigated. The established method was successfully applied to separate alkaloids from lotus leaves using a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water/[C4 mim][BF4 ] (1:5:1:5:0.15, v/v/v/v/v). Four alkaloids pronuciferine (1.7 mg), N-nornuciferine (4.3 mg), nuciferine (3.1 mg), and roemerine (2.1 mg) were obtained with the purities of 90.53, 92.25, 99.86, and 98.63%, respectively, from 100 mg crude extract of lotus leaves. The results indicated that the ionic-liquid-modified high-speed countercurrent chromatography method was suitable for alkaloid separation from lotus leaves and would be a promising method for the separation of alkaloids from other natural products.

Separation of phenolic acids from sugarcane rind by online solid-phase extraction with high-speed counter-current chromatography.

Sugarcane rind contains some functional phenolic acids. The separation of these compounds from sugarcane rind is able to realize the integrated utilization of the crop and reduce environment pollution. In this paper, a novel protocol based on interfacing online solid-phase extraction with high-speed counter-current chromatography (HSCCC) was established, aiming at improving and simplifying the process of phenolic acids separation from sugarcane rind. The conditions of online solid-phase extraction with HSCCC involving solvent system, flow rate of mobile phase as well as saturated extent of absorption of solid-phase extraction were optimized to improve extraction efficiency and reduce separation time. The separation of phenolic acids was performed with a two-phase solvent system composed of butanol/acetic acid/water at a volume ratio of 4:1:5, and the developed online solid-phase extraction with HSCCC method was validated and successfully applied for sugarcane rind, and three phenolic acids including 6.73 mg of gallic acid, 10.85 mg of p-coumaric acid, and 2.78 mg of ferulic acid with purities of 60.2, 95.4, and 84%, respectively, were obtained from 150 mg sugarcane rind crude extracts. In addition, the three different elution methods of phenolic acids purification including HSCCC, elution-extrusion counter-current chromatography and back-extrusion counter-current chromatography were compared.

A general separation method of phenolic acids using pH-zone-refining counter-current chromatography and its application to oat bran.

pH-zone-refining counter-current chromatography technique for the separation of natural and synthetic mixtures has been widely used, especially for organic acids and alkaloids. Phenolic acids are very important compounds due to the potential treatment for a wide variety of diseases. However, there is not a general method for their separation. In this work, the conditions of pH-zone-refining counter-current chromatography, involving solvent systems, concentration of retainer and eluter, flow rate of mobile phase as well as sample pretreatment, were optimized to improve extraction efficiency and reduce separation time. Finally a general separation method for seven common phenolic acids has been established using pH-zone-refining counter-current chromatography. The separation of these phenolic acids was performed with a two-phase solvent system composed of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4.75: 0.25: 5, where 3mM trifluoroacetic acid was added to the organic stationary phase as a retainer and 3mM NH4OH was added to the aqueous mobile phase as an eluter. As a result, seven phenolic acids, including syringic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were successfully separated with the purities of 95.9%, 67.3%, 96.9%, 82.4%, 97.0%, 91.0%, and 97.2%, respectively. The established general method has been applied to the crude sample of oat bran pretreated with AB-8 resin. A total of 49.5mg of syringic acid, 109.2mg of p-coumaric acid and 184.5mg of ferulic acid were successfully purified in one run from 1.22g crude extract with the purities of 95.2%, 93.0%, and 91.8%, respectively.

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH-zone-refining countercurrent chromatography.

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH-zone-refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two-phase solvent system composed of methyl-tert-butyl-ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH4 OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH-zone-refining CCC, a slightly polar two-phase solvent system composed of methyl-tert-butyl-ether/acetonitrile/n-butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH4 OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH-zone-refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.