Topological Magneto-optical Effect from Skyrmions in Two-Dimensional Ferromagnets.

Skyrmions in two-dimensional (2D) magnetic materials are considered as ideal candidates for information carriers in next-generation spintronic devices. However, conventional methods for elucidating the physical properties of skyrmions have limited the development of skyrmions in diverse 2D magnetic material systems due to their requirements for electrical conductivity. To overcome this limitation, we propose to utilize an optical method (magneto-optical Kerr technique) to detect the skyrmions in 2D magnetic materials. Herein, the graphene/Fe3GeTe2/graphene vertical van der Waals (vdW) heterostructure devices are fabricated to generate stabilized skyrmions by applying out-of-plane current. In combination with magnetic circular dichroism measurements, we observe topological-reflective magnetic circular dichroism (T-RMCD) effects in Fe3GeTe2 flakes and attribute the peak-shaped component in T-RMCD to the annihilation of skyrmion magnetic domains. Notably, the T-RMCD signal can maintain up to a temperature as high as the Curie temperature of Fe3GeTe2 flakes (∼200 K). Our work provides a universal, contactless, and nondestructive approach for studying the physical properties of skyrmions in 2D vdW magnetic materials while adding another degree of freedom to the modulation of skyrmions.

Programmable Biosynthesis of Plant-Derived 4'-Deoxyflavone Glycosides by an Unconventional Yeast Consortium.

Previous data established 4'-deoxyflavone glycosides (4'-DFGs) as important pharmaceutical components in the roots of rare medical plants like Scutellaria baicalensis Georgi. Extracting these compounds from plants involves land occupation and is environmentally unfriendly. Therefore, a modular ("plug-and-play") yeast-consortium platform is developed to synthesize diverse 4'-DFGs de novo. By codon-optimizing glycosyltransferase genes from different organisms for Pichia pastoris, six site-specific glycosylation chassis are generated to be capable of biosynthesizing 18 different 4'-DFGs. Cellular factories showed increased 4'-DFG production (up to 18.6-fold) due to strengthened synthesis of UDP-sugar precursors and blocked hydrolysis of endogenous glycosides. Co-culturing upstream flavone-synthesis-module cells with downstream glycoside-transformation-module cells alleviated the toxicity of 4'-deoxyflavones and enabled high-level de novo synthesis of 4'-DFGs. Baicalin is produced at the highest level (1290.0 mg L-1 ) in a bioreactor by controlling the consortium through carbon-source shifting. These results provide a valuable reference for biosynthesizing plant-derived 4'-DFGs and other glycosides with potential therapeutic applications.

[Biosynthesis of natural products by non-conventional yeasts].

Non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides and Hansenula polymorpha have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.

Manipulation and Optical Detection of Artificial Topological Phenomena in 2D Van der Waals Fe5 GeTe2 /MnPS3 Heterostructures.

2D ferromagnet is a good platform to investigate topological effects and spintronic devices owing to its rich spin structures and excellent external-field tunability. The appearance of the topological Hall Effect (THE) is often regarded as an important sign of the generation of chiral spin textures, like magnetic vortexes or skyrmions. Here, interface engineering and an in-plane current are used to modulate the magnetic properties of the nearly room-temperature 2D ferromagnet Fe5 GeTe2 . An artificial topology phenomenon is observed in the Fe5 GeTe2 /MnPS3 heterostructure by using both anomalous Hall Effect and reflective magnetic circular dichroism (RMCD) measurements. Through tuning the applied current and the RMCD laser wavelength, the amplitude of the humps and dips observed in the hysteresis loops can be modulated accordingly. Magnetic field-dependent hysteresis loops demonstrate that the observed artificial topological phenomena are induced by the generation and annihilation of the magnetic domains. This work provides an optical method for investigating the topological-like effects in magnetic structures and proposes an effective way to modulate the magnetic properties of magnetic materials, which is important for developing magnetic and spintronic devices in van der Waals magnetic materials.

Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol.

Natural methylotrophs are attractive methanol utilization hosts, but lack flexible expression tools. In this study, we developed yeast transcriptional device libraries for precise synthesis of value-added chemicals from methanol. We synthesized transcriptional devices by fusing bacterial DNA-binding proteins (DBPs) with yeast transactivation domains, and linking bacterial binding sequences (BSs) with the yeast core promoter. Three DBP-BS pairs showed good activity when working with transactivation domains and the core promoter of PAOX1 in the methylotrophic yeast, Pichia pastoris. Fine-tuning of the tandem BSs, spacers and differentiated input promoters further enabled a constitutive transcriptional device library (cTRDL) composed of 126 transcriptional devices with an expression strength of 16-520% and an inducible TRDL (iTRDL) composed of 162 methanol-inducible transcriptional devices with an expression strength of 30-500%, compared with PAOX1. Selected devices from iTRDL were adapted to the dihydromonacolin L biosynthetic pathway by orthogonal experimental design, reaching 5.5-fold the production from the PAOX1-driven pathway. The full factorial design of the selected devices from the cTRDL was adapted to the downstream pathway of dihydromonacolin L to monacolin J. Monacolin J production from methanol reached 3.0-fold the production from the PAOX1-driven pathway. Our engineered toolsets ensured multilevel pathway control of chemical synthesis in methylotrophic yeasts.

Nonlocal Manipulation of Magnetism in an Itinerant Two-Dimensional Ferromagnet.

Two-dimensional (2D) magnets are crucial in the construction of 2D magnetic and spintronic devices. Many devices, including spin valves and multiple tunneling junctions, have been developed by vertically stacking 2D magnets with other functional blocks. However, owing to limited local interactions at the interfaces, the device structures are typically extremely complex. To solve this problem, the nonlocal manipulation of magnetism may be a good solution. In this study, we use the magneto-optical Kerr effect technique to demonstrate the nonlocal manipulation of magnetism in an itinerant 2D ferromagnet, Fe3GeTe2 (FGT), whose magnetism can be manipulated via an antiferromagnet/ferromagnet interface or a current-induced spin-orbital torque placed distant from the local site. It is discovered that the coupling of a small piece of MnPS3 (∼40 µm2) with FGT can significantly enhance the coercive field and emergence of exchange bias in the entire FGT flake (∼2000 µm2). Moreover, FGT flakes with different thicknesses have the same coercive field at low temperatures if they are coupled together. Our study provides an understanding of the basic magnetism of 2D itinerant ferromagnets as well as opportunities for engineering magnetism with an additional degree of freedom.

Investigating Fungal Biosynthetic Pathways Using Pichia pastoris as a Heterologous Host.

Fungal natural products have extensive biological activities, and thus have been largely commercialized in the pharmaceutical, agricultural, and food industries. Recently, heterologous expression has become an irreplaceable technique to functionalize fungal biosynthetic gene clusters and synthesize fungal natural products in various chassis organisms. This chapter describes the general method of using Pichia pastoris as a chassis host to investigate fungal biosynthetic pathways.

De Novo Production of Plant 4'-Deoxyflavones Baicalein and Oroxylin A from Ethanol in Crabtree-Negative Yeast.

Baicalein and oroxylin A are well-known medicinal 4'-deoxyflavones found mainly in the roots of traditional medicinal plant Scutellaria baicalensis Georgi. However, extraction from plants is time-consuming, environmentally unfriendly, and insufficient. Although microbial synthesis of flavonoids has been extensively reported, synthesis of downstream modified 4'-deoxyflavones has not, and their yields are extremely low. Here, we reassembled the S. baicalensis 4'-deoxyflavone biosynthetic pathway in a Crabtree-negative yeast, Pichia pastoris, with activity analysis and combinatorial expression of eight biosynthetic genes, allowing production of 4'-deoxyflavones like baicalein, oroxylin A, wogonin, norwogonin, 6-methoxywogonin, and the novel 6-methoxynorwogonin. De novo baicalein synthesis was then achieved by complete pathway assembly. Toxic intermediates highly impaired the cell production capacity; hence, we alleviated cinnamic acid growth inhibition by culturing the cells at near-neutral pH and using alcoholic carbon sources. To achieve pathway balance and improve baicalein and oroxylin A synthesis, we further divided the pathway into five modules. A series of ethanol-induced and constitutive transcriptional amplification devices were constructed to adapt to the modules. This fine-tuning pathway control considerably reduced byproduct and intermediate accumulation and achieved high-level de novo baicalein (401.9 mg/L with a total increase of 1182-fold, the highest titer reported) and oroxylin A (339.5 mg/L, for the first time) production from ethanol. This study provides new strategies for the microbial synthesis of 4'-deoxyflavones and other flavonoids.

A programmable high-expression yeast platform responsive to user-defined signals.

Rapidly growing yeasts with appropriate posttranslational modifications are favored hosts for protein production in the biopharmaceutical industry. However, limited production capacity and intricate transcription regulation restrict their application and adaptability. Here, we describe a programmable high-expression yeast platform, SynPic-X, which responds to defined signals and is broadly applicable. We demonstrated that a synthetic improved transcriptional signal amplification device (iTSAD) with a bacterial-yeast transactivator and bacterial-yeast promoter markedly increased expression capacity in Pichia pastoris. CRISPR activation and interference devices were designed to strictly regulate iTSAD in response to defined signals. Engineered switches were then constructed to exemplify the response of SynPic-X to exogenous signals. Expression of α-amylase by SynPic-R, a specific SynPic-X, in a bioreactor proved a methanol-free high-production process of recombinant protein. Our SynPic-X platform provides opportunities for protein production in customizable yeast hosts with high expression and regulatory flexibility.

Transposon insertion mutation of Antarctic psychrotrophic fungus for red pigment production adaptive to normal temperature.

Polar regions are rich in microbial and product resources. Geomyces sp. WNF-15A is an Antarctic psy chrotrophic filamentous fungus producing high quality red pigment with potential for industrial use. However, efficient biosynthesis of red pigment can only realize at low temperature, which brings difficult control and high cost for the large-scale fermentation. This study aims to develop transposon insertion mutation method to improve cell growth and red pigment production adaptive to normal temperature. Genetic manipulation system of this fungus was firstly developed by antibiotic marker screening, protoplast preparation and transformation optimization, by which transformation efficiency of ∼50% was finally achieved. Then transposable insertion systems were established using Helitron, Fot1, and Impala transposons. The transposition efficiency reached 11.9%, 9.4%, and 4.6%, respectively. Mutant MP1 achieved the highest red pigment production (OD520 of 39) at 14°C, which was 40% higher than the wild-type strain. Mutant MP14 reached a maximum red pigment production (OD520 of 14.8) at 20°C, which was about twofold of the wild-type strain. Mutants MP2 and MP10 broke the repression mechanism of red pigment biosynthesis in the wild-type and allowed production at 25°C. For cell growth, eight mutants grew remarkably better (12%∼30% biomass higher) than the wild-type at 25°C. This study established an efficient genetic manipulation and transposon insertion mutation platform for polar filamentous fungus. It provides reference for genetic breeding of psychrotrophic fungi from polar and other regions.

Minos and Restless transposon insertion mutagenesis of psychrotrophic fungus for red pigment synthesis adaptive to normal temperature.

The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD520 of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD520 of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.

High-level living cell production of cytidine-5'-diphosphocholine in metabolically engineered yeast.

Industrial production of neuroprotective drug CDP-choline is accomplished via permeabilized or lysed cell biotransformation because of the inefficient penetration of substrates into intact cells. We previously proposed a novel one-step living cell method for CDP-choline production by engineered yeast, but obtained low titer and molar yield. This study develops a high-production strain with improved molar yield by metabolic engineering strategies. The selective markers previously integrated into host cell were recovered for facilitating genetic modification, which however resulted a strain with improved CDP-choline titer and molar yield to CMP. Knockout of 5'-NT or CDA in CMP sinking pathway but not APY in CTP sinking pathway further improved CDP-choline titer and molar yield to CMP. However, overexpression of seven enzymes in CTP synthetic pathway showed no positive functions. Finally, optimization of CMP and choline phosphate levels for the optimized recombinant strains achieved a high-level CDP-choline of ~30 g/L, which was enhanced by 400% compared to the previous work. Also, the molar yield of CDP-choline to CMP increased from 40% to 84.7%. The titer and molar yield are comparable to the reported permeabilized or lysed cell based biotransformation methods. It represents a novel and competitive paradigm for the potential industrial production of CDP-choline.

Enhancement of the Coercive Field and Exchange Bias Effect in Fe3GeTe2/MnPX3 (X = S and Se) van der Waals Heterostructures.

Fe3GeTe2/MnPS3 and Fe3GeTe2/MnPSe3 van der Waals heterostructures were fabricated by mechanical exfoliation. Via the magneto-optical Kerr effect and reflected magnetic circular dichroism measurements, we have observed nearly three times enhancement of the coercive field, improvement of Curie temperature, and exchange bias effect in both heterostructures. These observations may provide new insights into the emergent heterostructure devices between itinerant ferromagnets and metal thio- and selenophosphates for both applied and fundamental research studies in magnetic correlations.

Structure and physicochemical properties of amphiphilic agar modified with octenyl succinic anhydride.

A novel amphiphilic agar with high transparency and freeze-thaw stability was prepared using octenyl succinic anhydride (OSA). Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy confirmed that the hydrophobic OS groups were successfully introduced in OSA-modified agar (OSAR) backbone. The OSAR showed higher emulsion stability and oil loading capacity than the native agar (NA). Compared with gel transparency (47.1 %), syneresis (42.1 %) of NA, OSAR exhibited high gel transparency (80 %) and low syneresis (3.3 %) when the degree of substitution (DS) was 0.06 and 0.12, respectively. Meanwhile, the OSAR showed a decreased interface tension and average molecular weight after modification. Thermogravimetric analysis indicated the thermal stability of OSAR was decreased, while texture profile analysis showed the springiness of the OSAR gel was enhanced. Dynamic rheology measurements revealed the OSAR with low gel strength displayed more liquid-like properties. Moreover, the OSAR exhibited lower turbidity and melting temperatures than the NA.

Evaluation of a novel self-emulsifiable dodecenyl succinylated agarose in microencapsulation of docosahexaenoic acid (DHA) through spray-chilling process.

Agarose is a potential wall material for encapsulation owing to its high oxygen barrier, but its high hydrophilia and low emulsifying activity restrict its application as a wall material for hydrophobic actives. The aim of this study was to evaluate the potential of a novel self-emulsifiable dodecenyl succinic anhydride-esterified agarose (DSAG) as a single emulsifier and a wall material to encapsulate DHA. Results showed that DSAG was suitable for DHA encapsulation via spray-chilling process, and DHA microcapsules with different sizes (100-400 µm) could be easily prepared by controlling the spray pressure. The encapsulation efficiency of DSAG reached as high as 65-85%. The wet microcapsules were nearly spherical but they showed cavities or wrinkles on the surface after freeze drying. The DHA microcapsules, whether dry or wet or of different sizes, showed excellent oxidative stability, and exhibited good release characteristics under the simulated gastric and intestinal fluid conditions.

Combinatorial strategies for production improvement of red pigments from Antarctic fungus Geomyces sp.

Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF-15A, isolated from Antarctica. A high-production medium was developed by statistical experimental design, which was further simplified for industrial use by single-factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF-15A-M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild-type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6 mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5-L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11-fold of the control and 1.95-fold of the precursor regulation group, respectively. This study provides high-production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future. PRACTICAL APPLICATION: Antarctic Geomyces red pigments showed high color value, nontoxic characteristic, and good water solubility. It holds potential for industrial use and is under development for food additive in China currently. This study provides an optional manufacturing process for this new kind of red pigments.

Engineered dynamic distribution of malonyl-CoA flux for improving polyketide biosynthesis in Komagataella phaffii.

Malonyl-CoA is a basic but limited precursor for the biosynthesis of various bioactive compounds and life-supporting fatty acids in cells. This study develops a biosynthetic system to dynamically redirect malonyl-CoA flux and improve production of malonyl-CoA derived polyketide (6-MSA) in Komagataella phaffii. A synthetic regulatory protein fusing a yeast activator Prm1 with a bacterial repressor FapR was proved to work with a hybrid promoter (-7)fapO-cPAOX1 and activate gene expression. Expression mode by the Prm1-FapR/(-7)fapO-cPAOX1 device was not affected by intracellular malonyl-CoA levels. Further, 9 promoter variants of PGAP with insertion of fapO at various sites were tested with the Prm1-FapR. It generated a biosensor of Prm1-FapR/PGAP-(+2)fapO with regulation behavior of malonyl-CoA-low-level repression/high-level derepression. Both devices were subsequently integrated into a single cell, for which fatty acid synthesis module was driven by Prm1-FapR/PGAP-(+2)fapO but 6-MSA synthesis module was expressed by Prm1-FapR/(-7)fapO-cPAOX1. The integrated system allowed continuous polyketide synthesis but malonyl-CoA-high-level "on"/low-level "off" fatty acid synthesis. This design finally increased 6-MSA production capacity by 260 %, proving the positive effects of dynamic malonyl-CoA distribution to the target compounds. It provides a new strategy for synthesis of malonyl-CoA derived compounds in eukaryotic chassis hosts.

A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii.

Malonyl-CoA is a key metabolic molecule that participates in a diverse range of physiological responses and can act as a building block for a variety of value-added pharmaceuticals and chemicals. The cytosolic malonyl-CoA concentration is usually very low, and thus dynamic metabolic control of malonyl-CoA variation will aid its stable formation and efficient consumption. We developed a synthetic malonyl-CoA metabolic oscillator in yeast. A synthetic regulatory protein, Prm1-FapR, was constructed by fusing a yeast transcriptional activator, Prm1, with a bacterial malonyl-CoA-sensitive transcription repressor, FapR. Two oppositely regulated biosensors were then engineered. A total of 18 hybrid promoter variants were designed, each carrying the operator sequence (fapO) of FapR and the core promoter of PAOX1 (cPAOX1), which is naturally regulated by Prm1. The promoter activities of these variants, regulated by Prm1-FapR, were tested. Through this process, a sensor for Prm1-FapR/(-52)fapO-PAOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of PGAP with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/PGAP-(+22) fapO that contained a repression/derepression regulation module. Both sensors were subsequently integrated into a single cell, which exhibited correct metabolic switching of eGFP and mCherry reporters following manipulation of cytosolic malonyl-CoA levels. The Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were also used to control the malonyl-CoA source and sink pathways, respectively, for the synthesis of 6-methylsalicylic acid. This finally led to an oscillatory metabolic mode of cytosolic malonyl-CoA. Such a metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.

Engineering substrate and energy metabolism for living cell production of cytidine-5'-diphosphocholine.

Cytidine-5'-diphosphocholine (CDP-choline) is a widely used neuroprotective drug for multiple indications. In industry, CDP-choline is synthesized by a two-step cell culture/permeabilized cell biotransformation method because substrates often do not enter cells in an efficient manner. This study develops a novel one-step living cell fermentation method for CDP-choline production. For this purpose, the feasibility of Pichia pastoris as a chassis was demonstrated by substrate feeding and CDP-choline production. Overexpression of choline phosphate cytidylyltransferase and choline kinase enhanced the choline transformation pathway and improved the biosynthesis of CDP-choline. Furthermore, co-overexpression of ScHnm1, which is a heterologous choline transporter, highly improved the utilization of choline substrates, despite its easy degradation in cells. This strategy increased CDP-choline titer by 55-folds comparing with the wild-type (WT). Overexpression of cytidine-5'-monophosphate (CMP) kinase and CDP kinase in the CMP transformation pathway showed no positive effects. An increase in the ATP production by citrate stimulation or metabolic pathway modification further improved CDP-choline biosynthesis by 120%. Finally, the orthogonal optimization of key substrates and pH was carried out, and the resulting CDP-choline titer (6.0 g/L) at optimum conditions increased 88 times the original titer in the WT. This study provides a new paradigm for CDP-choline bioproduction by living cells.