Molecular Insights into Converting Hydroxide Adenosyltransferase into Halogenase.

Halogenation plays a unique role in the design of agrochemicals. Enzymatic halogenation reactions have attracted great attention due to their excellent specificity and mild reaction conditions. S-adenosyl-l-methionine (SAM)-dependent halogenases mediate the nucleophilic attack of halide ions (X-) to SAM to produce 5'-XDA. However, only 11 SAM-dependent fluorinases and 3 chlorinases have been reported, highlighting the desire for additional halogenases. SAM-dependent hydroxide adenosyltransferase (HATase) has a similar reaction mechanism as halogenases but uses water as a substrate instead of halide ions. Here, we explored a HATase from the thermophile Thermotoga maritima MSB8 and transformed it into a halogenase. We identified a key dyad W8L/V71T for the halogenation reaction. We also obtained the best performing mutants for each halogenation reaction: M1, M2 and M4 for Cl-, Br- and I-, respectively. The M4 mutant retained the thermostability of HATase in the iodination reaction at 80 °C, which surpasses the natural halogenase SalL. QM/MM revealed that these mutants bind halide ions with more suitable angles for nucleophilic attack of C5' of SAM, thus conferring halogenation capabilities. Our work achieved the halide ion specificity of halogenases and generated thermostable halogenases for the first time, which provides new opportunities to expand the halogenase repertoire from hydroxylase.

One-step Solid-State Synthesis of V1.13Se2/V2O3 Heterostructure as a High Pseudocapacitance Anode for Fast-Charging Sodium-Ion Batteries.

Sodium-ion batteries (SIBs) offer several benefits, including cost-efficiency and fast-charging characteristics, positioning them as attractive substitutes for lithium-ion batteries in energy storage applications. However, the inferior capacity and cycling stability of electrodes in SIBs necessitate further enhancement due to sluggish reaction kinetics. In this respect, the utilization of heterostructures, which can provide an inherent electric field and abundant active sites on the surface, has emerged as a promising strategy for augmenting the cycling stability and rate features of the electrodes. This work delves into the utilization of V1.13Se2/V2O3 heterostructure materials as anodes, initially fabricated via a simplified one-step solid-state sintering technique. The high pseudocapacitance and low characteristic relaxation time constant give the V1.13Se2/V2O3 heterostructure impressive properties, such as a high capacity of 328.5 mAh g-1 even after 1500 cycles at a high current density of 2 A g-1 and rate capability of 278.9 mAh g-1 at 5 A g-1. Moreover, the assembled sodium-ion full battery delivers a capacity of 118.5 mAh g-1 after 1000 cycles at 1 A g-1. These findings provide novel insight and guidance for the rapid synthesis of heterojunction materials and the advancement of SIBs.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

Enzyme Engineering Renders Chlorinase the Activity of Fluorinase.

Organofluorine compounds have attracted substantial attention owing to their wide application in agrochemistry. Fluorinase (FlA) is a unique enzyme in nature that can incorporate fluorine into an organic molecule. Chlorinase (SalL) has a similar mechanism as fluorinase and can use chloride but not fluoride as a substrate to generate 5'-chloro-deoxyadenosine (5'-ClDA) from S-adenosyl-l-methionine (SAM). Therefore, identifying the features that lead to this selectivity for halide ions is highly important. Here, we engineered SalL to gain the function of FlA. We found that residue Tyr70 plays a key role in this conversion through alanine scanning. Site-saturation mutagenesis experiments demonstrated that Y70A/C/S/T/G all exhibited obvious fluorinase activity. The G131S mutant of SalL, in which the previously thought crucial residue Ser158 for fluoride binding in FlA was introduced, did not exhibit fluorination activity. Compared with the Y70T single mutant, the double mutant Y70T/W129F increased 5'-fluoro-5-deoxyadenosine (5'-FDA) production by 76%. The quantum mechanics (QM)/molecular mechanics (MM) calculations suggested that the lower energy barriers and shorter nucleophilic distance from F- to SAM in the mutants than in the SalL wild-type may contribute to the activity. Therefore, our study not only renders SalL the activity of FlA but also sheds light on the enzyme selectivity between fluoride versus chloride.

[Advances in abscisic acid biosynthesis].

Abscisic acid, a plant hormone that inhibits growth, is a key factor in balancing plant endogenous hormones and regulating growth and metabolism. Abscisic acid can improve the drought resistance and salt tolerance of crops, reduce fruit browning, reduce the incidence rate of malaria and stimulate insulin secretion, so it has a broad application potential in agriculture and medicine. Compared with traditional plant extraction and chemical synthesis, abscisic acid synthesis by microorganisms is an economic and sustainable route. At present, a lot of progress has been made in the synthesis of abscisic acid by natural microorganisms such as Botrytis cinerea and Cercospora rosea, while the research on the synthesis of abscisic acid by engineered microorganisms is rarely reported. Saccharomyces cerevisiae, Yarrowia lipolytica and Escherichia coli are common hosts for heterologous synthesis of natural products due to their advantages of clear genetic background, easy operation and friendliness for industrial production. Therefore, the heterologous synthesis of abscisic acid by microorganisms is a more promising production method. The author reviews the research on the heterologous synthesis of abscisic acid by microorganisms from five aspects: selection of chassis cells, screening and expression enhancement of key enzymes, regulation of cofactors, enhancement of precursor supply and promotion of abscisic acid efflux. Finally, the future development direction of this field is prospected.

Elimination of enzymes catalysis compartmentalization enhancing taxadiene production in Saccharomyces cerevisiae.

Taxadiene is an important precursor in taxol biosynthesis pathway, but its biosynthesis in eukaryotic cell factories is limited, which seriously hinders the biosynthesis of taxol. In this study, it is found that there was the catalysis compartmentalization between two key exogenous enzymes of geranylgeranyl pyrophosphate synthase and taxadiene synthase (TS) for taxadiene synthesis progress, due to their different subcellular localization. Firstly, the enzyme-catalysis compartmentalization was overcome by means of the intracellular relocation strategies of taxadiene synthase, including N-terminal truncation of taxadiene synthase and enzyme fusion of GGPPS-TS. With the help of two strategies for enzyme relocation, the taxadiene yield was increased by 21% and 54% respectively, among them the GGPPS-TS fusion enzyme is more effective. Further, the expression of GGPPS-TS fusion enzyme was improved via the multi-copy plasmid, resulting that the taxadiene titer was increased by 38% to 21.8 mg/L at shake-flask level. Finally, the maximum taxadiene titer of 184.2 mg/L was achieved by optimization of the fed-batch fermentation conditions in 3 L bioreactor, which is the highest reported titer of taxadiene biosynthesis accomplished in eukaryotic microbes. This study provides a successful example for improving biosynthesis of complex natural products by solving the critical problem of multistep enzymes catalysis compartmentalization.

Systematic Engineering to Enhance 8-Hydroxygeraniol Production in Yeast.

8-Hydroxygeraniol, an important component of insect sex pheromones and defensive secretions, can be used as a potential biological insect repellent in agriculture. Microbial production provides sustainable and green means to efficiently gain 8-hydroxygeraniol. The conversion of geraniol to 8-hydroxygeraniol by P450 geraniol-8-hydroxylase (G8H) was regarded as the bottleneck for 8-hydroxygeraniol production. Herein, an integrated strategy consisting of the fitness between G8H and cytochrome P450 reductase (CPR), endoplasmic reticulum (ER) engineering, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) supply is implemented to enhance the production of 8-hydroxygeraniol in Saccharomyces cerevisiae. The titer of 8-hydroxygeraniol was gradually increased by 2.1-fold (up to 158.1 mg/L). Moreover, dehydrogenase ADH6 and reductase ARI1 responsible for the reduction of 8-hydroxygeraniol toward shunt products were also deleted, elevating 8-hydroxygeraniol production to 238.9 mg/L at the shake flask level. Consequently, more than 1.0 g/L 8-hydroxygeraniol in S. cerevisiae was achieved in 5.0 L fed-batch fermentation by a carbon restriction strategy, which was the highest-reported titer in microbes so far. Our work not only provides a sustainable way for de novo biosynthesis of 8-hydroxygeraniol but also sets a good reference in P450 engineering in microbes.

Modular Coculture to Reduce Substrate Competition and Off-Target Intermediates in Androstenedione Biosynthesis.

Substrate competition within a metabolic network constitutes a common challenge in microbial biosynthesis system engineering, especially if indispensable enzymes can produce multiple intermediates from a single substrate. Androstenedione (4AD) is a central intermediate in the production of a series of steroidal pharmaceuticals; however, its yield via the coexpression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17α-hydroxylase/17,20-lyase (CYP17A1) in a microbial chassis affords a nonlinear pathway in which these enzymes compete for substrates and produce structurally similar unwanted intermediates, thereby reducing 4AD yields. To avoid substrate competition, we split the competing 3ß-HSD and CYP17A1 pathway components into two separate Yarrowia lipolytica strains to linearize the pathway. This spatial segregation increased substrate availability for 3ß-HSD in the upstream strain, consequently decreasing the accumulation of the unwanted intermediate 17-hydroxypregnenolone (17OHP5) from 94.8 ± 4.4% in single-chassis monocultures to 24.8 ± 12.6% in cocultures of strains expressing 3ß-HSD and CYP17A1 separately. Orthologue screening to increase CYP17A1 catalytic efficiency and the preferential production of desired intermediates increased the biotransformation capacity in the downstream pathway, further decreasing 17OHP5 accumulation to 3.9%. Furthermore, nitrogen limitation induced early 4AD accumulation (final titer, 7.71 mg/L). This study provides a framework for reducing intrapathway competition between essential enzymes during natural product biosynthesis as well as a proof-of-concept platform for linear steroid production.

Compartmentalization engineering of yeasts to overcome precursor limitations and cytotoxicity in terpenoid production.

Metabolic engineering strategies for terpenoid production have mainly focused on bottlenecks in the supply of precursor molecules and cytotoxicity to terpenoids. In recent years, the strategies involving compartmentalization in eukaryotic cells has rapidly developed and have provided several advantages in the supply of precursors, cofactors and a suitable physiochemical environment for product storage. In this review, we provide a comprehensive analysis of organelle compartmentalization for terpenoid production, which can guide the rewiring of subcellular metabolism to make full use of precursors, reduce metabolite toxicity, as well as provide suitable storage capacity and environment. Additionally, the strategies that can enhance the efficiency of a relocated pathway by increasing the number and size of organelles, expanding the cell membrane and targeting metabolic pathways in several organelles are also discussed. Finally, the challenges and future perspectives of this approach for the terpenoid biosynthesis are also discussed.

Modular Pathway Compartmentalization for Agroclavine Overproduction in Saccharomyces cerevisiae.

Agroclavine, which has anti-depressant activity and anti-Alzheimer effects, is the raw material used to synthesize ergo-based drugs. Although the production of agroclavine from Saccharomyces cerevisiae is possible, its yield is exceptionally low. The current study proposes a modular compartmentalization strategy for identifying and modifying the bottleneck step in agroclavine overproduction. The agroclavine synthetic pathway was reconstituted in yeast, and the best combination of Claviceps fusiformis EasA with Claviceps purpurea EasD/EasG was identified. According to the data on the expression and subcellular localization of agroclavine pathway proteins, the whole pathway was divided into two modules by chanoclavine-I. Separate enzyme distribution within the downstream module and low expression of DmaW and EasE in the upstream module were identified as the bottleneck steps in the pathway. The pathway efficiency was enhanced 2.06-fold when the downstream module was entirely anchored to the endoplasmic reticulum compartment. Increasing NADPH supply by overexpressing POS5 further improved the agroclavine yield by 27.4%. Altering the intracellular localization of DmaW from the peroxisome to the endoplasmic reticulum (ER) not only improved protein expression but also accelerated the accumulation of agroclavine by 59.9%. Integration of all modified modules into the host chromosome resulted in an improved yield of agroclavine at 101.6 mg/L with flask fermentation (a 241-fold improvement over the initial strain) and ultimately produced 152.8 mg/L of agroclavine on fed-batch fermentation. The current study unlocked the potential of S. cerevisiae in the advanced biosynthesis of ergot alkaloids. It also provides a promising strategy to reconstitute compartmentalized pathways.

Adaptive Evolution and Metabolic Engineering Boost Lycopene Production in Saccharomyces cerevisiae via Enhanced Precursors Supply and Utilization.

Lycopene is a red carotenoid with remarkable antioxidant activity, which has been widely used in food, cosmetics, medicine, and other industries. Production of lycopene in Saccharomyces cerevisiae provides an economic and sustainable means. Many efforts have been done in recent years, but the titer of lycopene seems to reach a ceiling. Enhancing the supply and utilization of farnesyl diphosphate (FPP) is generally regarded as an efficient strategy for terpenoid production. Herein, an integrated strategy by means of atmospheric and room-temperature plasma (ARTP) mutagenesis combined with H2O2-induced adaptive laboratory evolution (ALE) was proposed to improve the supply of upstream metabolic flux toward FPP. Enhancing the expression of CrtE and introducing an engineered CrtI mutant (Y160F&N576S) increased the utilization of FPP toward lycopene. Consequently, the titer of lycopene in the strain harboring the Ura3 marker was increased by 60% to 703 mg/L (89.3 mg/g DCW) at the shake-flask level. Eventually, the highest reported titer of 8.15 g/L of lycopene in S. cerevisiae was achieved in a 7 L bioreactor. The study highlights an effective strategy that the synergistic complementarity of metabolic engineering and adaptive evolution facilitates natural product synthesis.

Structural Variations and Adaptations of Synthetic Chromosome Ends Driven by SCRaMbLE in Haploid and Diploid Yeasts.

Variations and adaptations of chromosome ends play an important role in eukaryotic karyotype evolution. Traditional experimental studies of the adaptations of chromosome ends mainly rely on the strategy of introducing defects; thus, the adaptation methods of survivors may vary depending on the initial defects. Here, using the SCRaMbLE strategy, we obtained a library of haploid and diploid synthetic strains with variations in chromosome ends. Analysis of the SCRaMbLEd survivors revealed four routes of adaptation: homologous recombination between nonhomologous chromosome arms (haploids) or homologous chromosome arms (diploids), site-specific recombination between intra- or interchromosomal ends, circularization of chromosomes, and loss of whole chromosomes (diploids). We also found that circularization of synthetic chromosomes can be generated by SCRaMbLE. Our study of various adaptation routes of chromosome ends provides insight into eukaryotic karyotype evolution from the viewpoint of synthetic genomics.

Investigation of Genome Biology by Synthetic Genome Engineering.

Synthetic genomes were designed based on an understanding of natural genomic information, offering an opportunity to engineer and investigate biological systems on a genome-wide scale. Currently, the designer version of the M. mycoides genome and the E. coli genome, as well as most of the S. cerevisiae genome, have been synthesized, and through the cycles of design-build-test and the following engineering of synthetic genomes, many fundamental questions of genome biology have been investigated. In this review, we summarize the use of synthetic genome engineering to explore the structure and function of genomes, and highlight the unique values of synthetic genomics.

[Performance Evaluation of a New Microfluidic Platelet Function Test Platform].

OBJECTIVE: To evaluate the performance of a microfluidic platelet function test platform (MPFTP) previously established by our research group. METHODS: The effects of flow shear rate and storage time on platelet function test were analyzed taking the MPFTP as the object. The intra-assay variability of the MPFTP was evaluated. The function of platelet in peripheral venous blood from 24 healthy volunteers was assessed using the MPFTP and light transmission turbidity, either in the presence of 20 µmol/L acetylsalicylic acid (AS, an inhibitor of cyclooxygenase 1) or 50 µmol/L 5-phospho-2-methylthioadenosine (2-MeSAMP, a P2Y12 receptor inhibitor). The diagnostic performance of both methods in assaying platelet function inhibition by AS and 2-MeSAMP was analyzed by using receiver operating characteristic (ROC) curve. RESULTS: Under the flow shear rate of 1 500 s-1, our MPFTP could dynamically monitor platelet adhesion and aggregation, as well as quantify platelet function. Platelet aggregation increased with the increase of flow shear rate, while sample storage at room temperature for up to 5 h did not affect results of platelet function test. The intra-assay variability coefficient of variation of the MPFTP was <15%. The area under the curve of ROC showed that this platform had good diagnostic performance in the identification of platelet function inhibition by AS and 2-MeSAMP. CONCLUSION: This MPFTP shows good analytical performance for the assay of platelet function and can be developed into a new clinical platelet function test device in the future.

VO2·0.26H2O nanobelts@reduced graphene oxides as cathode materials for high-performance aqueous zinc ion batteries.

A simple microwave-assisted hydrothermal approach is adopted to synthesize VO2·0.26H2O nanobelts@reduced graphene oxide (VO2·0.26H2O@rGO), which is regarded as a promising cathode material for ZIBs. Compared to VO2·0.26H2O, VO2·0.26H2O@rGO has a large specific surface area and low charge transfer resistance, improving its electrochemical properties. The large lattice spacing, high capacitive Zn2+ storage behavior, fast Zn2+ transfer rate and stable structure are also responsible for the performance.

Ion Mobility-Derived Collision Cross-Sections Add Extra Capability in Distinguishing Isomers and Compounds with Similar Retention Times: The Case of Aphidicolanes.

The hyphenation of ion mobility spectrometry with high-resolution mass spectrometry has been widely used in the characterization of various metabolites. Nevertheless, such a powerful tool remains largely unexplored in natural products research, possibly mainly due to the lack of available compounds. To evaluate the ability of collision cross-sections (CCSs) in characterizing compounds, especially isomeric natural products, here we measured and compared the traveling-wave IMS-derived nitrogen CCS values for 75 marine-derived aphidicolanes. We established a CCS database for these compounds which contained 227 CCS values of different adducts. When comparing the CCS differences, 36 of 57 pairs (over 60%) of chromatographically neighboring compounds showed a ΔCCS over 2%. What is more, 64 of 104 isomeric pairs (over 60%) of aphidicolanes can be distinguished by their CCS values, and 13 of 18 pairs (over 70%) of chromatographically indistinguishable isomers can be differentiated from the mobility dimension. Our results strongly supported CCS as an important parameter with good orthogonality and complementarity with retention time. CCS is expected to play an important role in distinguishing complex and diverse marine natural products.

Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory.

Parthenolide, a kind of sesquiterpene lactone, is the direct precursor for the promising anti-glioblastoma drug ACT001. Compared with traditional parthenolide source from plant extraction, de novo biosynthesis of parthenolide in microorganisms has the potential to make a sustainable supply. Herein, an integrated strategy was designed with P450 source screening, nicotinamide adenine dinucleotide phosphate (NADPH) supply, and endoplasmic reticulum (ER) size rewiring to manipulate three P450s regarded as the bottleneck for parthenolide production. Germacrene A oxidase from Cichorium intybus, costunolide synthase from Lactuca sativa, and parthenolide synthase from Tanacetum parthenium have the best efficiency, resulting in a parthenolide titer of 2.19 mg/L, which was first achieved in yeast. The parthenolide titer was further increased by 300% with NADPH supplementation and ER expanding stepwise. Finally, the highest titers of 31.0 mg/L parthenolide and 648.5 mg/L costunolide in microbes were achieved in 2.0 L fed-batch fermentation. This study not only provides an alternative microbial platform for producing sesquiterpene lactones in a sustainable way but also highlights a general strategy for manipulating multiple plant-derived P450s in microbes.

Development of Organogels for Live Yarrowia lipolytica Encapsulation.

Encapsulation of cells/microorganisms attracts great attention in many applications, but current studies mainly focus on hydrophilic encapsulation materials. Herein, we develop a new class of hydrophobic and lipophilic organogels for highly efficient encapsulation of Yarrowia lipolytica, an oleaginous yeast, by a mild and nonsolvent photopolymerization method. The organogels allow free diffusion of hydrophobic molecules that oleaginous yeasts require to survive and function. Moreover, they are mechanically robust and possess favorable biocompatibility, thus providing a free-standing platform and an ideal survival environment for oleaginous Y. lipolytica encapsulation. By tuning monomer structures and cross-linking densities, the optimized organogel, Gel12-1.5%, achieves the highest viability of ∼96%. Furthermore, organogels can inhibit the cryoinjuries to oleaginous yeasts in cryopreservation, exhibiting the potential for long-term storage. It is also found that with varying alkyl lengths, the organogels show different temperature-dependent phase transition properties, which enable the rapid selection of targeted yeasts for steganography. Findings in this work provide guidance for designing biocompatible, hydrophobic, and lipophilic encapsulation materials.

Enhancement and mapping of tolerance to salt stress and 5-fluorocytosine in synthetic yeast strains via SCRaMbLE.

Varied environmental stress can affect cell growth and activity of the cellular catalyst. Traditional path of adaptive evolution generally takes a long time to achieve a tolerance phenotype, meanwhile, it is a challenge to dissect the underlying genetic mechanism. Here, using SCRaMbLE, a genome scale tool to generate random structural variations, a total of 222 evolved yeast strains with enhanced environmental tolerances were obtained in haploid or diploid yeasts containing six synthetic chromosomes. Whole genome sequencing of the evolved strains revealed that these strains generated different structural variants. Notably, by phenotypic-genotypic analysis of the SCRaMbLEd strains, we find that a deletion of gene YFR009W (GCN20) can improve salt tolerance of Saccharomyces cerevisiae, and a deletion of gene YER056C can improve 5-flucytosine tolerance of Saccharomyces cerevisiae. This study shows applications of SCRaMbLE to accelerate phenotypic evolution for varied environmental stress and to explore relationships between structural variations and evolved phenotypes.

CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production.

Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the ß-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (kcat/Km) toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.