Systematic Engineering to Enhance ß-Myrcene Production in Yeast.

ß-Myrcene is an important monoterpene compound widely used in the fragrance, agricultural, and food industries. The microbial production of ß-myrcene conforms to the trend of green biological manufacturing, which has great potential for development. The poor catalytic activity of ß-myrcene synthase (MS) and the insufficient supply of precursors are considered to be the bottlenecks of ß-myrcene production. Here, source screening, subcellular localization, enzyme fusion, and precursor-enhancing strategies were integrated for ß-myrcene biosynthesis with Saccharomyces cerevisiae. The ß-myrcene titer gradually increased by 218-fold (up to 63.59 mg/L) compared to that of the initial titer of the shake flask. Moreover, the titer reached 66.82 mg/L after the addition of antioxidants (1 mM glutathione, GSH, and 1% butylated hydroxytoluene, BHT). Ultimately, 142.64 mg/L ß-myrcene in S. cerevisiae was achieved in 5.0 L of fed-batch fermentation under a carbon restriction strategy, which was the highest reported titer in yeast thus far. This study not only established a platform for ß-myrcene production but also provided a reference for the efficient biosynthesis of other monoterpene compounds.

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.

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.