Conduction of a chemical structure-guided metabolic phenotype analysis method targeting phenylpropane pathway via LC-MS: Ginkgo biloba and soybean as examples.

The phenylpropane pathway (PPP) is one of the most extensively investigated metabolic routes. This pathway biosynthesizes many important active ingredients such as phenylpropanoids and flavonoids that affect the flavor, taste and nutrients of food. How to elucidate the metabolic phenotype of PPP is fundamental in food research and development. In this study, we designed a structural periodical table filled with 103 metabolites produced from PPP. All of them especially the 62 structural isomers were qualified and quantified with high resolution and sensitivity via multiple reaction mode in liquid chromatography tandem triple quadrupole mass spectrometry. Ginkgo biloba and soybean were used as samples for the practical application of this method: The delicate spatial-temporal metabolic balance of PPP from ginkgo biloba has been first elucidated; It is first confirmed that the salt and draught stresses could redirect the biosynthesis trend of PPP to produce more isoflavones in soybean leaves.

Producing Designer Oils in Industrial Microalgae by Rational Modulation of Co-evolving Type-2 Diacylglycerol Acyltransferases.

Microalgal oils, depending on their degree of unsaturation, can be utilized as either nutritional supplements or fuels; thus, a feedstock with genetically designed and tunable degree of unsaturation is desirable to maximize process efficiency and product versatility. Systematic profiling of ex vivo (in yeast), in vitro, and in vivo activities of type-2 diacylglycerol acyltransferases in Nannochloropsis oceanica (NoDGAT2s or NoDGTTs), via reverse genetics, revealed that NoDGAT2A prefers saturated fatty acids (SFAs), NoDGAT2D prefers monounsaturated fatty acids (MUFAs), and NoDGAT2C exhibits the strongest activity toward polyunsaturated fatty acids (PUFAs). As NoDGAT2A, 2C, and 2D originated from the green alga, red alga, and eukaryotic host ancestral participants of secondary endosymbiosis, respectively, a mechanistic model of oleaginousness was unveiled, in which the indigenous and adopted NoDGAT2s formulated functional complementarity and specific transcript abundance ratio that underlie a rigid SFA:MUFA:PUFA hierarchy in triacylglycerol (TAG). By rationally modulating the ratio of NoDGAT2A:2C:2D transcripts, a bank of N. oceanica strains optimized for nutritional supplement or fuel production with a wide range of degree of unsaturation were created, in which proportion of SFAs, MUFAs, and PUFAs in TAG varied by 1.3-, 3.7-, and 11.2-fold, respectively. This established a novel strategy to simultaneously improve productivity and quality of oils from industrial microalgae.

Efficacy of head space solid-phase microextraction coupled to gas chromatography-mass spectrometry method for determination of the trace extracellular hydrocarbons of cyanobacteria.

Hydrocarbons are widespread in cyanobacteria, and the biochemical synthetic pathways were recently identified. Intracellular fatty alka(e)nes of cyanobacteria have been detected by liquid-liquid extraction (LLE) coupled to gas chromatography-mass spectrometry (GC/MS). However, whether fatty alka(e)nes can be released to cyanobacterial culture media remains to be clarified. This work develops a sensitive method for analyzing the trace level of extracellular hydrocarbons in cyanobacterial culture media by head space solid-phase microextraction (HS-SPME) coupled to GC/MS. Headspace (HS) extraction mode using polydimethylsiloxane fiber to extract for 30min at 50°C was employed as the optimal extraction conditions. Five cyanobacterial fatty alka(e)nes analogs including pentadecene (C15:1), pentadecane (C15:0), heptadecene (C17:1), heptadecane (C17:0), nonadecane (C19:0) were analyzed, and the data obtained from HS-SPME-GC/MS method were quantified using internal standard peak area comparisons. Limits of detection (LOD), limits of quantitation (LOQ), linear dynamic range, precisions (RSD) and recovery for the analysis of extracellular fatty alka(e)nes of cyanobacteria by HS-SPME-GC/MS were evaluated. The LODs limits of detection (S/N = 3) varied from 10 to 21 ng L-1. The correlation coefficients (r) of the calibration curves ranged from 0.9873 to 0.9977 with a linearity from 0.1 to 50 µg L-1. The RSD values were ranging from 7.8 to 14.0% and from 4.0 to 8.8% at 1.0 µg L-1 and 10.0 µg L-1 standard solutions, respectively. Comparative analysis of extracellular fatty alka(e)nes in the culture media of model cyanobacteria Synechocystis sp. PCC 6803 demonstrated that sensitivity of HS-SPME-GC/MS method was significantly higher than LLE method. Finally, we found that heptadecane can be released into the culture media of Synechocystis sp. PCC 6803 at the later growth period.

Functional characterization of MpaG', the O-methyltransferase involved in the biosynthesis of mycophenolic acid.

Mycophenolic acid (MPA, 1) is a clinically important immunosuppressant. In this report, a gene cluster mpa' responsible for the biosynthesis of 1 was identified from Penicillium brevicompactum NRRL 864. The S-adenosyl-L-methionine-dependent (SAM-dependent) O-methyltransferase encoded by the mpaG' gene was functionally and kinetically characterized in vitro. MpaG' catalyzes the methylation of demethylmycophenolic acid (DMMPA, 6) to form 1. It also showed significant substrate flexibility by methylating two structural derivatives of 6 prepared by organic synthesis.

New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

Purification and characteristics of xyloglucanase and five other cellulolytic enzymes from Trichoderma reesei QM9414.

By combining anion-exchange chromatography with gel filtration, an effective method for purification of wild-type xyloglucanase and five other cellulolytic enzymes from strain QM9414 of Trichoderma reesei was established. Characterization by enzyme activity assay, SDS-PAGE, and mass spectrometry identified the purified proteins as cellobiohydrolases I and II, endoglucanases I and II, a xyloglucanase, and ß-xylosidase, of which the xyloglucanase was purified for the first time from the mutant strain QM9414. This method holds great promise to study the mechanism of cellulolytic enzymes, to investigate the synergistic action between cellulase and other cellulolytic enzymes, and to better exploit enzyme preparations for degradation of lignocellulose.

Characterization of the bafilomycin biosynthetic gene cluster from Streptomyces lohii.

New hope for old bones: The plecomacrolide bafilomycin has been explored for decades as an anti-osteoporotic. However, its structural complexity has limited the synthesis of analogues. The cloning of the bafilomycin biosynthetic gene cluster from the environmental isolate Streptomyces lohii opens the door to the production of new analogues through bioengineering.

Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry.

Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work.

Strain construction for ethanol production from dilute-acid lignocellulosic hydrolysate.

In order to construct a strain that converts sugar mixture and resist/metabolize inhibitors in lignocellulosic dilute-acid hydrolysate, the biotechnology of inactive intergeneric fusion between Saccharomyces cerevisiae and Pachysolen tannophilis was performed. Fusant 1 was successfully obtained as a hybrid strain, which was screened out by xylose and mixed sugar (xylose and glucose) fermentation. This strain showed good abilities of ethanol production, ethanol tolerance, and resistance to the toxic inhibitors presenting in the hydrolysate. The maximum volumetric yield of ethanol and yield of xylitol in mixed sugar was 9.52 g/l and 0.44 g/g, respectively. The results indicated that the constructed strain Fusant 1 was a good producer for ethanol and xylitol from lignocellulosic dilute-acid hydrolysate.