Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol.

Eugenol, the main component of essential oil from the Syzygium aromaticum clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway. To overcome this, we developed a novel biosynthetic pathway for converting eugenol into vanillin, by introducing cinnamoyl-CoA reductase (CCR), which catalyzes conversion of coniferyl aldehyde to feruloyl-CoA. This approach bypasses the need for two catalysts, namely coniferyl aldehyde dehydrogenase and feruloyl-CoA synthetase, thereby eliminating inhibition while simplifying the pathway. To further improve efficiency, we enhanced CCR catalytic efficiency via directed evolution and leveraged an artificialvanillin biosensor for high-throughput screening. Switching the cofactor preference of CCR from NADP+ to NAD+ significantly improved pathway efficiency. This newly designed pathway provides an alternative strategy for efficiently biosynthesizing feruloyl-CoA-derived natural products using eugenol.

Engineering and application of LacI mutants with stringent expressions.

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).

Designing glucose utilization "highway" for recombinant biosynthesis.

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from "glucose repression" are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Efficient 3-D Finite Element Modeling of Periodic XBAR Resonators.

An efficient technique is presented for 3-D finite element modeling of large-scale periodic excited bulk acoustic resonator (XBAR) resonators in the time-harmonic domain. In this technique, a domain decomposition scheme is used to decompose the computational domain into many small subdomains whose FE subsystems can be factorized with a direct sparse solver at a low cost. Transmission conditions (TCs) are enforced to interconnect adjacent subdomains, and a global interface system is formulated and solved iteratively. To accelerate the convergence, a second-order TC (SOTC) is designed to make the subdomain interfaces transparent for propagating and evanescent waves. An effective forward-backward preconditioner is constructed that when combined with the SOTC significantly reduce the number of iterations at no additional cost. Numerical results are given to demonstrate the accuracy, efficiency, and capability of the proposed algorithm.

Six Spain Thymus essential oils composition analysis and their in vitro and in silico study against Streptococcus mutans.

BACKGROUND: Streptococcus mutans is a well-known oral pathogen that plays a critical role in the development of dental caries. Many studies have been directed to discover the chemical compounds present in natural products to inhibit the growth and biofilm formation activity of S. mutans. Thymus essential oils exhibit good inhibition on the growth and pathogenesis of S. mutans. However, details about the active compounds in Thymus essential oil and the inhibition mechanism still remain unclear. The aim of this study was to investigate the antimicrobial activity of 6 Thymus species (Three samples of Thymus vulgaris, two samples of Thymus zygis, and one sample of Thymus satureioides essential oils) on S. mutans, to identify the potential active components, and to reveal the underlying mechanism. METHODS: The composition of Thymus essential oils was analyzed by gas chromatography-mass spectrometry. And its antibacterial effect was evaluated based on the bacterial growth, acid production, biofilm formation and genetic expression of virulence factors by S. mutans. Potential active components of the Thymus essential oil were identified using molecular docking and correlation analysis. RESULTS: GC-MS analysis showed that the major components in the 6 Spain Thymus essential oils were linalool, α-terpineol, p-cymene, thymol and carvacrol. MIC and MBC analysis showed that 3 Thymus essential oils showed very sensitive antimicrobial activity, and were chosen for further analysis. The 3 Thymus essential oil exhibited a significant inhibitory effect on acid production, adherence and biofilm formation of S. mutans and the expression of virulence genes, such as brpA, gbpB, gtfB, gtfC, gtfD, vicR, spaP and relA. Correlation analysis showed that phenolic components, such as carvacrol and thymol, were positively related to DIZ value, which suggests that they are the potential antimicrobial components. Molecular docking between the Thymus essential oil components and virulence proteins also found that carvacrol and thymol exhibited strong binding affinity with functional domains of virulence genes. CONCLUSIONS: Thymus essential oil showed significant inhibition against the growth and pathogenesis of S. mutans depending on their composition and concentration. And phenolic compounds, such as carvacrol and thymol, are the major active components. Thymus essential oil could be used in oral healthcare products as a potential anti-caries ingredient.

Engineering an SspB-mediated degron for novel controllable protein degradation.

Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory. Limited high-quality tools are available for controllable protein degradation. By coupling structure-guided modeling and directed evolution, we establish state-of-the-art high-throughput screening strategies for engineering both degradation efficiency and SspB-ssrA binding specificity of this degron. The reliability of our approach is confirmed by functional validation of both SspB and ssrA mutants using fluorescence assays and metabolic engineering of itaconic acid or ferulic acid biosynthesis. Isothermal titration calorimetry analysis and molecular modeling revealed that an appropriate instead of excessively strong interaction between SspB and ssrA benefited degradation efficiency. Mutated SspB-ssrA pairs with 7-22-fold higher binding KD than the wild-type pair led to higher degradation efficiency, revealing the advantage of directed evolution over rational design in degradation efficiency optimization. Furthermore, an artificial SspB-ssrA pair exhibiting low crosstalk of interactions with the wild-type SspB-ssrA pair was also developed. Efforts in this study have demonstrated the plasticity of SspB-ssrA binding pocket for designing high-quality controllable protein degradation tools. The obtained mutated degrons enriched the tool box of metabolic engineering designs.

Whole-Cell Biosensors Aid Exploration of Vanillin Transmembrane Transport.

Transcriptional regulatory protein (TRP)-based whole-cell biosensors are widely used nowadays. Here, they were demonstrated to have great potential application in screening cell efflux and influx pumps for small molecules. First, a vanillin whole-cell biosensor was developed by altering the specificity of a TRP, VanR, and strains with improved vanillin productions that were selected from a random genome mutagenesis library by using this biosensor as a high-throughput screening tool. A high intracellular vanillin concentration was found to accumulate due to the inactivation of the AcrA protein, indicating the involvement of this protein in vanillin efflux. Then, the application of this biosensor was extended to explore efflux and influx pumps, combined with directed genome evolution. Elevated intracellular vanillin levels resulting from efflux pump inactivation or influx pump overexpression could be rapidly detected by the whole-cell biosensor, markedly facilitating the identification of genome targets related to small-molecule transmembrane transport, which is of great importance in metabolic engineering.

De Novo Biosynthesis of Chlorogenic Acid Using an Artificial Microbial Community.

Engineering an artificial microbial community for natural product production is a promising strategy. As mono- and dual-culture systems only gave non-detectable or minimal chlorogenic acid (CGA) biosynthesis, here, a polyculture of three recombinant Escherichia coli strains, acting as biosynthetic modules of caffeic acid (CA), quinic acid (QA), and CGA, was designed and used for de novo CGA biosynthesis. An influx transporter of 3-dehydroshikimic acid (DHS)/shikimic acid (SA), ShiA, was introduced into the QA module-a DHS auxotroph. The QA module proportion in the polyculture and CGA production were found to be dependent on ShiA expression, providing an alternative approach for controlling microbial community composition. The polyculture strategy avoids metabolic flux competition in the biosynthesis of two CGA precursors, CA and QA, and allows production improvement by balancing module proportions. The performance of this polyculture approach was superior to that of previously reported approaches of de novo CGA production.

Metabolic Engineering for Improved Curcumin Biosynthesis in Escherichia coli.

The biosynthetic efficiency of curcumin, a highly bioactive compound from the plant Curcuma longa, needs to be improved. In this study, we performed host cell and biosynthetic pathway engineering to improve curcumin biosynthesis. Using in vivo-directed evolution, the expression level of curcuminoid synthase (CUS), the rate-limiting enzyme in the curcumin biosynthetic pathway, was significantly improved. Furthermore, as curcumin is a highly hydrophobic compound, two cell membrane engineering strategies were applied to optimize the biosynthetic efficiency. Curcumin storage was increased by overexpression of monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii, which optimized the cell membrane morphology. Furthermore, unsaturated fatty acid supplementation was used to enhance membrane fluidity, which greatly ameliorated the damaging effect of curcumin on the cell membrane. These two strategies enhanced curcumin biosynthesis and demonstrated an additive effect.

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps.

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

[New thoughts on material basis and mechanism of Yin-tonifying traditional Chinese medicine in treatment of diabetes mellitus and its complications].

Diabetes mellitus is a serious chronic metabolic disease, and the patient's hyperglycemia is often accompanied by complications. In the circles of medical science, traditional Chinese medicine(TCM) has the earliest knowledge and research about diabetes. According to TCM, the clinical characteristics of diabetes mellitus were basically the same as "Xiaoke". TCM also believes that "Yin deficiency and dryness heat" was the main pathogenesis of diabetes. Therefore, Yin-tonifying TCMs is widely used in clinical treatment of diabetes mellitus, including Dendrobii Caulis, Lilii Bulbus, Ophiopogonis Radix, Polygonati Rhizome. The effective component for treating diabetes in the above Chinese materia medica is polysaccharides, which is used to treat complications of diabetes mellitus, like vascular disease, nephropathy, retinopathy, peripheral neuropathy. According to literature reports, except for specific some Yin-tonifying TCMs with effective ingredients for preventing and treating diabetes, other Yin-tonifying TCMs only contain water, alcohol extracts or polysaccharides in the treatment of diabetes. However, due to unclear material basis, dose-effect relationship and mechanism target of hypoglycemic drugs, Yin-tonifying TCMs are restricted in clinical application, with certain difficulties in in-depth studies. In this paper, the literatures related to the treatment of diabetes mellitus and its complications with Yin-tonifying TCM are analyzed and summarized, the existing problems are analyzed, and the research ideas and methods based on chromatographic technology and metabonomics are put forward, in order to provide reference for the application and development of Yin-tonifying TCM.

Development of a highly efficient and specific L-theanine synthase.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Dynamic control of toxic natural product biosynthesis by an artificial regulatory circuit.

To mimic the delicately regulated metabolism in nature for improved efficiency, artificial and customized regulatory components for dynamically controlling metabolic networks in multiple layers are essential in laboratory engineering. For this purpose, a novel regulatory component for controlling vanillin biosynthetic pathway was developed through directed evolution, which was responsive to both the product vanillin and substrate ferulic acid, with different capacities. This regulatory component facilitated pathway expression via dynamic control of the intracellular substrate and product concentrations. As vanillin is an antimicrobial compound, low pathway expression and vanillin formation levels enabled better cell growth at an early stage, and the product feedback-activated pathway expression at later stages significantly improved biosynthesis efficiency. This novel multiple-layer dynamic control was demonstrated effective in managing the trade-off between cell growth and production, leading to improved cell growth and vanillin production compared to the conventional or quorum-sensing promoter-controlled pathway. The multiple-layer dynamic control enabled by designed regulatory components responsive to multiple signals shows potential for wide applications in addition to the dynamic controls based on biosynthetic intermediate sensing and quorum sensing reported to date.

Sensing Sub-10 nm Wide Perturbations in Background Nanopatterns Using Optical Pseudoelectrodynamics Microscopy (OPEM).

Using light as a probe to investigate perturbations with deep subwavelength dimensions in large-scale wafers is challenging because of the diffraction limit and the weak Rayleigh scattering. In this Letter, we report on a nondestructive noninterference far-field imaging method, which is built upon electrodynamic principles (mechanical work and force) of the light-matter interaction, rather than the intrinsic properties of light. We demonstrate sensing of nanoscale perturbations with sub-10 nm features in semiconductor nanopatterns. This framework is implemented using a visible-light bright-field microscope with a broadband source and a through-focus scanning apparatus. This work creates a new paradigm for exploring light-matter interactions at the nanoscale using microscopy that can potentially be extended to many other problems, for example, bioimaging, material analysis, and nanometrology.

Electrostatic and magnetostatic properties of random materials.

Scale dependence of electrostatic and magnetostatic properties is investigated in the setting of spatially random linear lossless materials with statistically homogeneous and spatially ergodic random microstructures. First, from the Hill-Mandel homogenization conditions adapted to electric and magnetic fields, uniform boundary conditions are formulated for a statistical volume element (SVE). From these conditions, there follow upper and lower mesoscale bounds on the macroscale (effective) electrical permittivity and magnetic permeability. Using computational electromagnetics methods, these bounds are obtained through numerical simulations for composites of two types: (i) two-dimensional (2D) random checkerboard (two-phase) microstructures and (ii) analogous 3D random (two-phase) media. The simulation results demonstrate a scale-dependent trend of these bounds toward the properties of a representative volume element (RVE). This transition from SVE to RVE is described using a scaling function dependent on the mesoscale δ, the volume fraction v_{f}, and the property contrast k between two phases. The scaling function is calibrated through fitting the data obtained from extensive simulations (∼10000) conducted over the aforementioned parameter space. The RVE size of a given microstructure can be estimated down to within any desired accuracy using this scaling function as parametrized by the contrast and the volume fraction of two phases.

Promiscuous enzymatic activity-aided multiple-pathway network design for metabolic flux rearrangement in hydroxytyrosol biosynthesis.

Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate. We show that the synthetic capacity is significantly improved when two pathways work simultaneously comparing to each individual pathway. Next, we engineer flavin-dependent monooxygenase HpaBC for tyrosol hydroxylase, tyramine hydroxylase, and promiscuous hydroxylase active on both tyrosol and tyramine using directed divergent evolution strategy. Then, the mutant HpaBCs are employed to catalyze two missing steps in the hydroxytyrosol biosynthetic pathways designed above. Our results demonstrate that the promiscuous tyrosol/tyramine hydroxylase can minimize the cell metabolic burden induced by protein overexpression and allow the biosynthetic carbon flow to be divided between two pathways. Thus, the efficiency of the hydroxytyrosol biosynthesis is significantly improved by rearranging the metabolic flux among multiple pathways.

Engineering the effector specificity of regulatory proteins for the in vitro detection of biomarkers and pesticide residues.

Transcriptional regulatory proteins (TRPs)-based whole-cell biosensors are promising owing to their specificity and sensitivity, but their applications are currently limited. Herein, TRPs were adapted for the extracellular detection of a disease biomarker, uric acid, and a typical pesticide residue, carbaryl. A mutant regulatory protein that specifically recognizes carbaryl as its non-natural effector and activates transcription upon carbaryl binding was developed by engineering the regulatory protein TtgR from Pseudomonas putida. The TtgR mutant responsive to carbaryl and a regulatory protein responsive to uric acid were used for in vitro detection, based on their allosteric binding of operator DNA and inducer molecules. Based on the quantitative polymerase chain reactions (qPCRs) output, the minimum detectable concentration was between 1 nM-1 µM and 1-10 nM for uric acid and carbaryl, respectively. Our results demonstrated that engineering the effector specificity of regulatory proteins is a potential technique for generating molecular recognition elements for not only in vivo but also in vitro applications.

[Characterization of sediment of water extract of Guizhi decoction and its effect on relevant compound (fractions) in decoction].

In order to study the effect of sediment of water extract of Guizhi decoction on the stability, clarity and peaks area, and characterize the chemical composition of the sediments, HPLC and MS methods were established. Through comparison of the common peak areas and the turbidity value of water extract and filtrate, the sediments could greatly change the common peak areas of the decoction (for more than 5 times of the study standard); at the same time, the turbidity value of the decoction could increase by （38.66±1.57）% in 48 h [particularly by （24.54±1.68）% in 6 h]. The test indicated that the sediments had an effect on the stability and clarity under the test conditions in Guizhi decoction. The study confirmed that the sediments were mainly derived from Cassia twig, Paeonia lactiflora and Glycyrrhiza uralensis. On the basis of the reference information, the accurate molecular weight and fragment ion information provided by LC-MS were analyzed, the molecular formula of sediments components A-F were determined, and the possible structural information of components B, C, D and F were deduced. It was suggested that the multi-index, multi-target and multi-angle analysis could ensure the quality of traditional Chinese medicine and the effect of clinical medication. The study also suggested the effect of the sediments on clinical application and the preparation of traditional Chinese medicine.

Towards the construction of high-quality mutagenesis libraries.

OBJECTIVES: To improve the quality of mutagenesis libraries in directed evolution strategy. RESULTS: In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells. CONCLUSIONS: This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.

Biosensor-aided high-throughput screening of hyper-producing cells for malonyl-CoA-derived products.

BACKGROUND: Malonyl-coenzyme A (CoA) is an important biosynthetic precursor in vivo. Although Escherichia coli is a useful organism for biosynthetic applications, its malonyl-CoA level is too low. RESULTS: To identify strains with the best potential for enhanced malonyl-CoA production, we developed a whole-cell biosensor for rapidly reporting intracellular malonyl-CoA concentrations. The biosensor was successfully applied as a high-throughput screening tool for identifying targets at a genome-wide scale that could be critical for improving the malonyl-CoA biosynthesis in vivo. The mutant strains selected synthesized significantly higher titers of the type III polyketide triacetic acid lactone (TAL), phloroglucinol, and free fatty acids compared to the wild-type strain, using malonyl-CoA as a precursor. CONCLUSION: These results validated this novel whole-cell biosensor as a rapid and sensitive malonyl-CoA high-throughput screening tool. Further analysis of the mutant strains showed that the iron ion concentration is closely related to the intracellular malonyl-CoA biosynthesis.