Enhancement of linalool production in Saccharomyces cerevisiae by utilizing isopentenol utilization pathway.

BACKGROUND: Linalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites. RESULTS: In this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20F96W-N127W (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20F96W-N127W genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg2+ addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation. CONCLUSIONS: The results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.

Variations in chemical fingerprints and major flavonoid contents from the leaves of thirty-one accessions of Hibiscus sabdariffa L.

The leaves of Hibiscus sabdariffa L. have been used as traditional folk medicines for treating high blood pressure and fever. There are many accessions of H. sabdariffa L. throughout the world. To assess the chemical variations of 31 different accessions of H. sabdariffa L., fingerprinting analysis and quantitation of major flavonoids were performed by high-performance liquid chromatography (HPLC). The HPLC method was validated for linearity, sensitivity, precision, repeatability and accuracy. A quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was applied for the characterization of major compounds. A total of 9 compounds were identified, including 6 flavonoids and 3 phenolic acids. In the fingerprint analysis, similarity analysis (SA) and principal component analysis (PCA) were used to differentiate the 31 accessions of H. sabdariffa L. Based on the results of PCA and SA, the samples No. 15 and 19 appeared much different from the main group. The total content of five flavonoids varied greatly among different accessions, ranging from 3.35 to 23.30 mg/g. Rutin was found to be the dominant compound and the content of rutin could contribute to chemical variations among different accessions. This study was helpful to understand the chemical variations between different accessions of H. sabdariffa L., which could be used for quality control. © 2015 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd.

GC×GC-TOFMS Analysis of Essential Oils Composition from Leaves, Twigs and Seeds of Cinnamomum camphora L. Presl and Their Insecticidal and Repellent Activities.

Interest in essential oils with pesticidal activity against insects and pests is growing. In this study, essential oils from different parts (leaves, twigs and seeds) of Cinnamomum camphora L. Presl were investigated for their chemical composition, and insecticidal and repellent activities against the cotton aphid. The essential oils, obtained by hydrodistillation, were analyzed by GC×GC-TOFMS. A total of 96 components were identified in the essential oils and the main constituents found in the leaves and twigs were camphor, eucalyptol, linalool and 3,7-dimethyl-1,3,7-octatriene. The major components found in the seeds were eucalyptol (20.90%), methyleugenol (19.98%), linalool (14.66%) and camphor (5.5%). In the contact toxicity assay, the three essential oils of leaves, twigs and seeds exhibited a strong insecticidal activity against cotton aphids with LC50 values of 245.79, 274.99 and 146.78 mg/L (after 48 h of treatment), respectively. In the repellent assay, the highest repellent rate (89.86%) was found in the seed essential oil at the concentration of 20 µL/mL after 24 h of treatment. Linalool was found to be a significant contributor to the insecticidal and repellent activities. The results indicate that the essential oils of C. camphora might have the potential to be developed into a natural insecticide or repellent for controlling cotton aphids.

Antioxidant activity of leaf extracts from different Hibiscus sabdariffa accessions and simultaneous determination five major antioxidant compounds by LC-Q-TOF-MS.

Hibiscus sabdariffa has gained attention for its antioxidant activity. There are many accessions of H. sabdariffa in the world. However, information on the quantification of antioxidant compounds in different accessions is rather limited. In this paper, a liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) method for simultaneous determination of five antioxidant compounds (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and isoquercitrin) in H. sabdariffa leaves was developed. The method was validated for linearity, sensitivity, precision, repeatability and accuracy. The validated method has been successfully applied for determination of the five analytes in eight accessions of H. sabdariffa. The eight accessions of H. sabdariffa were evaluated for their antioxidant activities by DPPH free radical scavenging assay. The investigated accessions of H. sabdariffa were rich in rutin and exhibited strong antioxidant activity. The two accessions showing the highest antioxidant activities were from Cuba (No. 2) and Taiwan (No. 5). The results indicated that H. sabdariffa leaves could be considered as a potential antioxidant source for the food industry. The developed LC-Q-TOF-MS method is helpful for quality control of H. sabdariffa.

High-Yield Recovery of Antioxidant Compounds from Bambusa chungii Culms Using Pressurized Hot Water Extraction.

A large amount of waste from Bambusa chungii culms is generated from the bamboo pulping industry, causing disposal problems. Nevertheless, bamboo culms are a suitable source of functional ingredients, such as antioxidant compounds. However, because of the high compactness and tightness in their material structure, obtaining phytochemicals from bamboo culms using conventional organic solvent extraction methods can be inefficient. In this research, we developed a pressurized hot water extraction (PHWE) method to recover 19 target phenolic compounds from Bambusa chungii culms. The extracted compounds were determined by ultra-high performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UPLC-QTOF-MS). The antioxidant potential of the extracts was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. We investigated the effects of temperature, extraction time, and the material-to-liquid ratio on PHWE, and these parameters were optimized with a Box-Behnken design experiment and response surface tool. The optimal extraction condition was found at 170 °C, with a 1:30 g/mL material-to-liquid ratio and a 14 min extraction time. Following these optimal parameters, the total yield of target phenolic compounds (TYPC) reached 3.85 mg/g of raw material, and the half-maximal inhibitory concentrations (IC50) for the DPPH and ABTS tests were 94.7 mg/L and 21.88 mg/L, respectively. The FRAP value was 1.23 µmol FSE/mg of dried extract. A strong correlation between TYPC and the antioxidant activity of the extract was confirmed. The TYPC and antioxidant capacity of the optimal PHWE extract of the Bambusa chungii culms were both considerably higher than those of extracts obtained from conventional solvent extractions. These results indicated that PHWE is an excellent green technique for recovering phenolic compounds from bamboo culms, and the PHWE extracts of Bambusa chungii culms may be a good source of natural antioxidants.

High-level production of linalool by engineered Saccharomyces cerevisiae harboring dual mevalonate pathways in mitochondria and cytoplasm.

Linalool, a valuable monoterpene alcohol, is widely used in cosmetics and flavoring ingredients. However, its scalable production by microbial fermentation is not yet achieved. In this work, considerable increase in linalool production was obtained in Saccharomyces cerevisiae by dual metabolic engineering of the mevalonic acid (MVA) pathway in both mitochondria and cytoplasm. A farnesyl pyrophosphate synthase mutant ERG20F96W/N127W and a linalool synthase from Cinnamomum osmophloeum (CoLIS) were introduced and meanwhile the endogenous ERG20 was down-regulated to prevent the competitive loss of precursor. In addition, overexpression of the proteins of CoLIS and ERG20F96W/N127W and another copy of the same enzymes CoLIS/ERG20F96W/N127W with mitochondrial localization signal (MLS) were carried out to further pull the flux to linalool. Finally, a maximum linalool titer of 23.45 mg/L was obtained in a batch fermentation with sucrose as carbon source. This combinatorial engineering strategy may provide hints for biosynthesis of other monoterpenes.

Co-valorization of paper mill sludge and corn steep liquor for enhanced n-butanol production with Clostridium tyrobutyricum Δcat1::adhE2.

In this study, hyper-butanol producing Clostridium tyrobutyricum Δcat1::adhE2 was used for butanol production from paper mill sludge (PMS) and corn steep liquor (CSL). Our results demonstrated that CSL can not only serve as a cheap nitrogen source, but also provide lactic acid that can be assimilated by C. tyrobutyricum for enhanced butanol production. Through a separate hydrolysis and fermentation, 16.5 g/L butanol with a yield of 0.26 g/g was obtained from PMS hydrolysates supplemented with 5% CSL. Further, a separate repeated hydrolysis was conducted to improve PMS hydrolysis rate and enhance sugar yield. Fermentation using hydrolysates from such process also generated high-level butanol with high yield. Our results suggested an innovative bioprocess for efficient biobutanol production from low-value waste streams.