Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides.

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.

Characterization of a New Flavone and Tyrosinase Inhibition Constituents from the Twigs of Morus alba L.

The twigs of Morus alba L. were found to show strong tyrosinase inhibition activity, and the responsible active components in the extract were further investigated in this study. A flavone, named morusone (1), and sixteen known compounds 2-17 were isolated from M. alba twigs and their structures were identified by interpretation of the corresponding ESI-MS and NMR spectral data. In the tyrosinase inhibitory test, the compounds steppogenin (IC50 0.98 ± 0.01 µM), 2,4,2',4'-tetrahydroxychalcone (IC50 0.07 ± 0.02 µM), morachalcone A (IC50 0.08 ± 0.02 µM), oxyresveratrol (IC50 0.10 ± 0.01 µM), and moracin M (8.00 ± 0.22 µM) exhibited significant tyrosinase inhibition activities, much stronger than that of the positive control kojic acid. These results suggest that M. alba twig extract should served as a good source of natural tyrosinase inhibitors for use in foods as antibrowning agents or in cosmetics as skin-whitening agents.

Structure characterization of phospholipids and lipid A of Pseudomonas putida KT2442.

Pseudomonas putida KT2442 is an important bacterium for producing various types of polyhydroxyalkanoate polymers. Phospholipids and lipid A in membranes of P. putida play important roles in stress responses, but detailed structural information of these lipids is not known. In this study, phospholipids and lipid A were isolated from P. putida KT2442, and their structures were analyzed using thin layer chromatography, high performance liquid chromatography, and electrospray ionization/mass spectrometry. Major phospholipids in P. putida KT2442 were phosphatidylethanolamine (79.9%), phosphatidylglycero1 (12.7%), and cardiolipin (7.4%), with C16:1 and/or C18:1 acyl chains. Four lipid A species were found in P. putida KT2442: two are hexa-acylated, and the other two are penta-acylated. Compared with lipid A of P. aeruginosa, P. putida lipid A has less hydroxylation on the secondary acyl chains and less modification. Therefore, P. putida lipid A could be used as a base structure to investigate lipid A modification of P. aeruginosa for understanding its pathogenesis.

Exploring the characteristics, digestion behaviors, and nutraceutical potential of the underutilized Chimonanthus praecox (L.) link kernel oil: A combined in vitro and in vivo study.

Chimonanthus praecox (L.) Link kernel oil (LMO) has the potential to expand the variety of nutraceutical plant oils available and provide support for the application of functional food. This study aimed to assess the edible potential of LMO by examining its physicochemical characteristics, digestion behaviors, and nutraceutical properties. The results revealed that LMO has a high oil content of 40.84% and is particularly rich in linoleic acid (53.37-56.30%), oleic acid (22.04-25.08%) and triacylglycerol (TAG) of linoleic acid -palmitoleic acid- oleic acid (10.57-12.70%). The quality characteristics and phytochemical composition of LMO were found to be influenced by variety and extraction methods used. In simulated in vitro digestion tests, LMO showed a better lipid release rate and degree. Animal studies further demonstrated that LMO led to better TAG and cholesterol excretion compared to soybean oil and camellia oleifera oil. Overall, this study highlights the potential of LMO as a high-quality edible oil.

Typing of blood-group antigens on neutral oligosaccharides by negative-ion electrospray ionization tandem mass spectrometry.

Blood-group antigens, such as those containing fucose and bearing the ABO(H)- and Lewis-type determinants expressed on the carbohydrate chains of glycoproteins and glycolipids, and also on unconjugated free oligosaccharides in human milk and other secretions, are associated with various biological functions. We have previously shown the utility of negative-ion electrospay ionization tandem mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) for typing of Lewis (Le) determinants, for example, Le(a), Le(x), Le(b), and Le(y) on neutral and sialylated oligosaccharide chains. In the present report, we extended the strategy to characterization of blood-group A-, B-, and H-determinants on type 1 and type 2 and also on type 4 globoside chains to provide a high sensitivity method for typing of all the major blood-group antigens, including the A, B, H, Le(a), Le(x), Le(b), and Le(y) determinants, present in oligosaccharides. Using the principles established, we identified two minor unknown oligosaccharide components present in the products of enzymatic synthesis by bacterial fermentation. We also demonstrated that the unique fragmentations derived from the D- and (0,2)A-type cleavages observed in ESI-CID-MS/MS, which are important for assigning blood-group and chain types, only occur under the negative-ion conditions for reducing sugars but not for reduced alditols or under positive-ion conditions.

Identification of three genes encoding for the late acyltransferases of lipid A in Cronobacter sakazakii.

Lipid A, the hydrophobic anchor of lipopolysaccharide, is an essential component in the outer membrane of most Gram-negative bacteria. Food-borne pathogen Cronobacter sakazakii synthesizes two lipid A species, differing by the length of the secondary acyl chain. In this work, we identified three genes ESA02293, ESA02951 and ESA01386 encoding for the late acyltransferases of lipid A biosynthesis pathway in C. sakazakii. Based on the sequence alignment, proteins YP_001438378.1 encoded by ESA02293, YP_001439016.1 encoded by ESA02951, and YP_001437482.1 encoded by ESA01386 are homologous to E. coli LpxL, LpxP and LpxM, respectively. Functions of the three acyltransferases were confirmed by overexpressing the genes in E. coli, isolating lipid As and analyzing their structures using an ESI/MS. C. sakazakii LpxL and LpxM transfer a C14:0 secondary acyl chain to the 2'- and 3'-position of lipid A, respectively. C. sakazakii LpxP can transfer either a C16:1 or a C14:0 secondary acyl chains to the 2'-position of lipid A.

Discovery of two transglutaminases derived from same zymogen for the Streptomyces hygroscopicus and analysis of their formation processes.

BACKGROUND: Transglutaminase (TGase) is secreted as a zymogen (Pro-TGase) and is then processed by removal of its N-terminal region through exogenous proteolytic activity. In this study it was discovered that the Pro-TGase from Streptomyces hygroscopicus was also activated by its TGase (processed through exogenous proteolytic activity), resulting in a different active form. RESULTS: The two TGases exhibited different ionic strengths, hydrophobicities, Km values and stabilities. Circular dichroism spectral analysis showed that the two enzymes had non-identical secondary structures, while liquid chromatography/mass spectrometry (LC-MS) analysis indicated that they differed in molecular mass by 111 Da. The formation of the TGase activated from Pro-TGase by TGase was delayed compared with that of TGase processed through exogenous proteolytic activity. Furthermore, it was found that the TGase activated from Pro-TGase by TGase did not activate Pro-TGase. CONCLUSION: Two TGases derived from the same zymogen from S. hygroscopicus were discovered. These two active forms of TGase may be due to different activation processes: one of them is catalysed by its own active TGase, while the other is activated by an exogenous protease.

Triacylglycerol regioisomers containing palmitic acid analyzed by ultra-performance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry: Comparison of standard curve calibration and calculation equation.

Triacylglycerol (TAG) regioisomers containing palmitic acid (16:0) was identified using ultra-performance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry (UPSFC-Q-TOF-MS) and quantified using calibration curve method and calculation equation method. There were negative linear correlation between [RA-A]+/[RA-A]++[RA-B]+ and content of sn-A-B-A (%) for AAB/ABA type TAGs, [Rsn-1 FA-sn-3 FA]+/[RB-C]++[RA-C]++[RA-B]+ and content of fatty acid (FA) at sn-2 position (%) for BAC/ABC/ACB type TAGs. The difference between calculation equation and standard curve method was acceptable. The TAG regioisomers in human milk, mammalian milk, lard and fish oil were identified and quantified using the developed methods. This study provided a reliable and facile method for analysis of the TAG regioisomers, which was capable of the selection of oil materials for infant formula production.

Comprehensive lipidomic analysis of milk polar lipids using ultraperformance supercritical fluid chromatography-mass spectrometry.

Polar lipids in milk are receiving increasing interest due to their bioactivities. However, milk polar lipids present a wide range of physical-chemical properties at different concentrations, making their analysis challenging. In this study, we presented a comprehensive lipidomic method using ultraperformance supercritical fluid chromatography (UPSFC)-quadrupole-time of flight-mass spectrometry (Q-TOF-MS), which enabled the separation of 18 lipid classes (including nonpolar lipids, cholesterol, ceramide, glycerophospholipids, sphingomyelin, and gangliosides) within 10 min. The method was used to analyze the polar lipids in seven samples, including human milk, other mammalian milk and milk fat globule membrane ingredients, identifying 14 lipid classes containing 219 lipid molecular species. A mass spectrometry data processing strategy applicable for high-throughput studies was also developed and validated.

Transcriptome Analysis of Bacillus licheniformis for Improving Bacitracin Production.

This study aims to find the targets that may influence the production of bacitracin based on RNA sequencing in Bacillus licheniformis. Transcriptional profiling revealed that (i) the expression of the bacT gene, encoding a type II thioesterase (TEIIbac), was positively correlated with bacitracin production and (ii) the oxygen uptake exhibited significant influence on precursor synthesis. The verified experiments showed that the overexpression of TEIIbac with an endogenous promoter increased the bacitracin A titer by 37.50%. Furthermore, the increase of oxygen availability through Vitreoscilla hemoglobin (VHb) expression increased the bacitracin A titer by 126.67% under oxygen-restricted conditions. From the transcriptome perspective, the results of this paper demonstrate that TEIIbac and oxygen supply are crucial to bacitracin production. This study also provides insights into the construction of chassis cells for the industrial production of secondary metabolites with a preference for aerobic conditions.

Combining Precursor-Directed Engineering with Modular Designing: An Effective Strategy for De Novo Biosynthesis of l-DOPA in Bacillus licheniformis.

3-Hydroxy-l-tyrosine (l-DOPA) is a promising drug for treating Parkinson's disease. Tyrosine hydroxylase catalyzes the microbial synthesis of l-DOPA, which is hindered by the efficiency of catalysis, the supply of cofactor tetrahydrobiopterin, and the regulation of the pathway. In this study, the modular engineering strategy in Bacillus licheniformis was identified to effectively enhance l-DOPA production. First, the catalytic efficiency of biocatalyst tyrosine hydroxylase from Streptosporangium roseum DSM 43021 (SrTH) was improved by 20.3% by strengthening its affinity toward tetrahydrobiopterin. Second, the tetrahydrobiopterin supply pool was increased by bottleneck gene expression, oxygen transport facilitation, budC (encoding meso-2,3-butanediol dehydrogenase) deletion, and tetrahydrobiopterin regeneration using a native YfkO nitroreductase. The strain 45ABvC successfully produced tetrahydrobiopterin, which was detected as pterin (112.48 mg/L), the oxidation product of tetrahydrobiopterin. Finally, the yield of precursor l-tyrosine reached 148 mg/gDCW, with an increase of 71%, with the deletion of a novel spliced transcript 41sRNA associated with the regulation of the shikimate pathway. The engineered strain 45ABvCS::PD produced 167.14 mg/L (2.41 times of wild-type strain) and 1290 mg/L l-DOPA in a shake flask and a 15 L bioreactor, respectively, using a fermentation strategy on a mixture of carbon sources. This study holds great potential for constructing a microbial source of l-DOPA and its high-value downstream pharmaceuticals.

Adenylation domains of nonribosomal peptide synthetase: A potential biocatalyst for synthesis of dipeptides and their derivatives.

Dipeptides and their derivatives are important functional compounds that can be applied to fields such as medicine and food. As biological macromolecules, the adenylation domains of nonribosomal peptide synthetase (NRPS) can recognize and activate various building blocks, such as amino acids, for the biosynthesis of nonribosomal peptides. In this way, the amide bond formation can be achieved through a nucleophilic reaction where the adenylation domain serves as a biocatalyst and is further used to conduct dipeptide synthesis. In this study, the adenylation domains (BAA2, BBA2, and BCA4) of bacitracin synthetase were predicted and expressed. The substrate evaluation results showed that adenylation domains displayed broad substrate selectivity for amino acids in vitro. Furthermore, the use of dipeptide synthesis in adenylation domains suggested that the polarity of amino acids could have an influence on nucleophilic reactions. Finally, L-alanyl-L-glutamine and aspartame were successfully synthesized through catalysis by the adenylation domains BAA2 and BCA4, respectively. This study expands on approaches to the synthesis of functional dipeptides and their derivatives based on the chemoenzymatic process.

Construction of an Escherichia coli mutant producing monophosphoryl lipid A.

Lipid A is a major component in the outer membrane of most gram-negative bacteria. Monophosphoryl lipid A contains no phosphate group at 1-position and can be used as an adjuvant. We constructed an Escherichia coli mutant CW001 by integrating a gene lpxE into the chromosome of E. coli W3110. The gene lpxE encodes an enzyme LpxE which removes the 1-phosphate group of lipid A. CW001 predominantly produces 1-dephosphorylated lipid A in vivo, as adjudged by thin layer chromatography and electro-spray ionization mass spectrometry. This study not only is important for the development of lipid A adjuvants but also provides a novel method for integration of heterologous genes into the chromosome of E. coli.

Identification and Quantification of Triacylglycerols Using Ultraperformance Supercritical Fluid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry: Comparison of Human Milk, Infant Formula, Other Mammalian Milk, and Plant Oil.

Triacylglycerol (TAG) components in human milk during different lactation periods, infant formulas with different fat sources, other mammalian milk (cow, goat, donkey, and yak milk), and plant oil (sunflower, rapeseed, corn, soybean, palm, palm kernel, and coconut oil) were analyzed and compared using ultraperformance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry (UPSFC-Q-TOF-MS). We identified 191 TAGs (86, 102, 101, and 54 TAGs in human milk, infant formula, mammalian milk, and plant oil, respectively). TAGs esterified with palmitic acid (16:0) were major TAG structures in human milk (59.08% of total TAGs) and contained 30 TAG types. The sn-O/P/O regioisomer constituted more than 80% of the O/P/O content of human milk, whereas the sn-O/O/P levels were higher in other samples. The carbon number (CN) 52 content was higher than the CN 54 content in human milk, with the opposite observed in infant formula. TAGs with CN < 40 content were abundant in cow, goat, and yak milk; donkey milk was rich in CN 52 content. TAGs composed of medium-chain fatty acids (MCFAs) and long-chain fatty acids (LCFAs) were rich in human milk, while TAGs with three MCFAs were rich in infant formula. The TAG characteristics of infant formula were directly related to its fat resource. TAGs with fewer double bonds were abundant in the plant oil formula; however, highly unsaturated TAGs were prominent in the cow and goat milk formulas, similar to plant oil and mammalian milk. Significant differences in the TAG distribution were observed among the different species.

Characterization and determination of free phytosterols and phytosterol conjugates: The potential phytochemicals to classify different rice bran oil and rice bran.

Phytosterols are important beneficial compounds found in rice bran (RB) and rice bran oil (RBO). Although relationships have been confirmed between the forms of phytosterols and their bioactivities, the analysis of different forms of phytosterols in RB and RBO has been lacking. In this study, high temperature gas chromatography-mass spectrometry (HTGC-MS) was combined with the single standard to determine multi-components (SSDMC) method to determine free sterols (FSs) and steryl glycosides (SGs) in RB and RBO. High-performance liquid chromatography (HPLC) was used to determine steryl ferulates (SFs). There was clear variation in the composition of FS, SF and SG, indicating that different forms of phytosterols can discriminate between different RB and RBO. The developed method may be also useful for the detection of other compounds of interest in oils, oil seeds or cereals.

Exploring the relationship between potato components and Maillard reaction derivative harmful products using multivariate statistical analysis.

The correlation between potato components and Maillard reaction derivative harmful products (MRDHPs) formation during heat-processing was assessed in nine commercial potato varieties in China. Principal component analysis (PCA) combined with canonical correlation analysis (CCA) approach was performed to explore their relationships. The variables contributing most to the PCA results were extracted for CCA, and the results indicated that several amino acids, including lysine, tryptophan, alanine, phenylalanine, aspartate, and glutamate, have significant impacts on acrylamide and ß-carboline heterocyclic amine formation. Moreover, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, α-solanine, and α-chaconine were also important factors associated with acrylamide and ß-carboline heterocyclic amine formation. Optimally using raw potato varieties based on the impacts of these factors can help control MRDHP formation during thermal processing. For the first time, such approach was applied, which may be a useful tool for discovering the correlation of food components and MRDHPs.

Characterization of lipid A Cronobacter sakazakii.

We have characterized the structure of lipid A in food-borne pathogen Cronobacter sakazakii BAA-894. Lipid A molecules were purified by mild acid hydrolysis, DEAE-cellulose anion exchange chromatography and preparative thin-layer chromatography. Its structure was analyzed by electrospray ionization/mass spectrometry. Two lipid A species have been found in C. sakazakii BAA-894. The majority has a structure of disaccharide of glucosamine, phosphorylated at 1- and 4'-positions, and acylated with primary 3-hydroxymyristoyl chains at 2-, 3-, 2'- and 3'-positions and secondary myristoyl residues at 2'- and 3'- positions. The other contains a secondary lauroyl chain at 2'-position in place of the myristoyl residue.

Identification of α-Tocopherol and Its Oxidation Products by Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry.

This study aimed to determine α-tocopherol (α-T) and its thermal oxidation products simultaneously. A novel method based on an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed. This approach was achieved by means of a BEH C18 analytical column under gradient elution conditions with eluents of acetonitrile/isopropanol (1:9, v/v) and acetonitrile/water (4:6, v/v). Compounds were elucidated through exact molecular mass and fragmentation ions obtained from the Q-TOF-MS detector. Two oxidation products, α-tocopheryl quinone and 5-formyl-γ-tocopherol, were identified, and one new compound was determined. This approach offered a simple, precise, and reliable method to determine oxidation products of α-T, which may give a way to understand the mechanism of the thermal oxidative process of α-T.

Caffeic acid assists microwave heating to inhibit the formation of mutagenic and carcinogenic PhIP.

The inhibitory effect of caffeic acid on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) was investigated in chemical model systems under microwave heating (MW). A mechanistic study was subsequently carried out to identify the inhibitory mechanism. The results showed that both for conductive heating (CV) and MW, the inhibition of PhIP increased with the concentration of caffeic acid but decreased with the prolongation of heating time. The inhibition on PhIP under MW was always higher than under CV, which were dominated by the difference in dielectric loss (ε″). UPLC-MS analysis showed that caffeic acid releases a CO2 molecule to produce 4-vinylcatechol which can form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. The structure of adduct was characterized as 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. The findings indicate that trapping of phenylacetaldehyde by 4-vinylcatechol is a key mechanism of caffeic acid in inhibiting PhIP formation.

LC-MS/MS for simultaneous detection and quantification of Amadori compounds in tomato products and dry foods and factors affecting the formation and antioxidant activities.

In order to establish an efficient detection method to evaluate the formation of Amadori compounds (ACs) in food products and study the potential health effects, an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method using caffeine as internal standard was developed to determine eight ACs. The detection limits ranged from 0.0179 to 0.0887 mg/L for the ACs. The accuracy of the method was tested through measuring recovery of the spiked samples that varied from 81.90 ± 2.98% to 108.74 ± 2.34%. This method was further applied to detect ACs in 10 food products. Results showed that dry fruits and vegetables were rich in ACs, the total content of ACs varied from 1.36 ± 0.26 to 3415.91 ± 147.96 mg/100 g. The total amount of ACs in tomato juice heated under vacuum condition showed significant increment (P < 0.05) in 25 min at 80 °C comparing with that under atmospheric pressure due to the rapid loss of water. Besides, the amino acid content shows positive correlation with the corresponding AC formation in Maillard reaction during food drying. After heated at fixed water activity (Aw) for 4 hr by sous-vide process, the ACs content in tomato powder increased significantly and the antioxidant activity improved as well. PRACTICAL APPLICATION: Results of this study provided a valuable tool to evaluate the formation of ACs in complex dry food products, facilitated the quality control of food products. The knowledge obtained will offer useful information to food processors. The synthesized ACs would facilitate further study into the antioxidant activities and potential health effects of specified AC.