Improving the activity of an inulosucrase by rational engineering for the efficient biosynthesis of low-molecular-weight inulin.

Inulin, a widely recognized prebiotic, has diverse applications across various industrial sectors. Although inulin is primarily produced through plant extraction, there is growing interest in enzymatic synthesis as an alternative. The enzymatic production of inulin from sucrose, which yields polymers with degrees of polymerization similar to those of plant-derived inulin, shows potential as a viable replacement for traditional extraction methods. In this study, an inulosucrase from Neobacillus bataviensis was identified, demonstrating a non-processive mechanism specifically tailored for synthesizing inulin with polymerization degrees ranging from 3 to approximately 40. The enzyme exhibited optimal activity at pH 6.5 and 55 °C, efficiently producing inulin with a yield of 50.6%. Ca2+ can improve the activity and thermostability of this enzyme. To enhance catalytic total activity, site-directed and truncated mutagenesis techniques were applied, resulting in the identification of a mutant, T149S, displaying a significant 57% increase in catalytic total activity. Molecular dynamics simulations unveiled that the heightened flexibility observed in three surface regions positively influenced enzymatic activity. This study not only contributes to the theoretical foundation for inulosucrase engineering but also presents a potential avenue for the production of inulin.

Regulation of exogenous sugars on the biosynthesis of key secondary metabolites in Cyclocarya paliurus.

The biosynthesis and accumulation of secondary metabolites play a vital role in determining the quality of medicinal plants, with carbohydrate metabolism often influencing secondary metabolism. To understand the potential regulatory mechanism, exogenous sugars (sucrose, glucose/fructose) were applied to the leaves of Cyclocarya paliurus, a highly valued and multiple function tree species. The results showed that exogenous sugars enhanced the accumulation of soluble sugar and starch while increasing the enzyme activity related to carbohydrate metabolism. In addition, the plant height was increased by a mixture of exogenous mixed sugars, the addition of sucrose promoted the net photosynthetic rate, while all types of exogenous sugars facilitated the accumulation of flavonoids and terpenoids. Based on weighted gene co-expression network analysis (WGCNA), two key gene modules and four candidate transcription factors (TFs) related to carbohydrate metabolism and secondary metabolite biosynthesis were identified. A correlation analysis between transcriptome and metabolome data showed that exogenous sugar up-regulated the expression of key structural genes in the flavonoid and terpenoid biosynthetic pathway. The expression levels of the four candidate TFs, TIFY 10A, WRKY 7, EIL 3 and RF2a, were induced by exogenous sugar and were strongly correlated with the key structural genes, which enhanced the synthesis of specific secondary metabolites and some plant hormone signal pathways. Our results provide a comprehensive understanding of key factors in the quality formation of medicinal plants and a potential approach to improve the quality.

Arbuscular mycorrhizal fungi alter carbon metabolism and invertase genes expressions of Populus simonii × P. nigra under drought stress.

Arbuscular mycorrhizal fungi (AMF) play a crucial role in regulating the allocation of carbon between source and sink tissues in plants and in regulating their stress responses by changing the sucrose biosynthesis, transportation, and catabolism in plants. Invertase, a key enzyme for plant development, participates in the response of plants to drought stress by regulating sucrose metabolism. However, the detailed mechanisms by which INV genes respond to drought stress in mycorrhizal plants remain unclear. This study examined the sugar content, enzyme activity, and expression profiles of INV genes of Populus simonii × P. nigra (PsnINVs) under two inoculation treatments (inoculation or non-inoculation) and two water conditions (well-watered or drought stress). Results showed that under drought stress, AMF up-regulated the expressions of PsnA/NINV1, PsnA/NINV2, PsnA/NINV3, and PsnA/NINV5 in leaves, which may be related to the enhancement of photosynthetic capacity. Additionally, AMF up-regulated the expressions of PsnA/NINV6, PsnA/NINV10, and PsnA/NINV12 in leaves, which may be related to enhancing osmotic regulation ability and drought tolerance.

Metabolome and Transcriptome Joint Analysis Reveals That Different Sucrose Levels Regulate the Production of Flavonoids and Stilbenes in Grape Callus Culture.

To reveal the effect of sucrose concentration on the production of secondary metabolites, a metabolome and transcriptome joint analysis was carried out using callus induced from grape variety Mio Red cambial meristematic cells. We identified 559 metabolites-mainly flavonoids, phenolic acids, and stilbenoids-as differential content metabolites (fold change ≥2 or ≤0.5) in at least one pairwise comparison of treatments with 7.5, 15, or 30 g/L sucrose in the growing media for 15 or 30 days (d). Resveratrol, viniferin, and amurensin contents were highest at 15 d of subculture; piceid, ampelopsin, and pterostilbene had higher contents at 30 d. A transcriptome analysis identified 1310 and 498 (at 15 d) and 1696 and 2211 (at 30 d) differentially expressed genes (DEGs; log2(fold change) ≥ 1, p < 0.05) in 7.5 vs. 15 g/L and 15 vs. 30 g/L sucrose treatments, respectively. In phenylpropane and isoflavone pathways, DEGs encoding cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, and flavanone 3-hydroxylase were more highly expressed at 15 d than at 30 d, while other DEGs showed different regulation patterns corresponding to sucrose concentrations and cultivation times. For all three sucrose concentrations, the stilbene synthase (STS) gene exhibited significantly higher expression at 15 vs. 30 d, while two resveratrol O-methyltransferase (ROMT) genes related to pterostilbene synthesis showed significantly higher expression at 30 vs. 15 d. In addition, a total of 481 DEGs were annotated as transcription factors in pairwise comparisons; an integrative analysis suggested MYB59, WRKY20, and MADS8 as potential regulators responding to sucrose levels in flavonoid and stilbene biosynthesis in grape callus. Our results provide valuable information for high-efficiency production of flavonoids and stilbenes using grape callus.

Nitric oxide participates in sucrose-TOR signaling during meristem activation in Arabidopsis thaliana.

MAIN CONCLUSION: This study provides evidence about the relationship between Target of Rapamycin (TOR) kinase and the signal molecule nitric oxide (NO) in plants. We showed that sucrose (SUC)-mediated TOR activation of root apical meristem (RAM) requires NO and that NO, in turn, participates in the regulation of TOR signaling. Nitric oxide (NO) constitutes a signal molecule that regulates important target proteins related to growth and development and also contributes to metabolic reprogramming that occurs under adverse conditions. Taking into account the important role of NO and its relationship with Target of Rapamycin (TOR) signaling in animals, we wondered about the putative link between both pathways in plants. With this aim, we studied a TOR-dependent process which is the reactivation of the root apical meristem (RAM) in Arabidopsis thaliana. We used pharmacological and genetic tools to evaluate the relationship between NO and TOR on the sugar induction of RAM, using SNP as NO donor, cPTIO as NO scavenger and the nitrate reductase (NR) mutant nia2. The results showed that sucrose (SUC)-mediated TOR activation of the RAM requires NO and that NO, in turn, participates in the regulation of TOR signaling. Interestingly, TOR activation induced by sugar increased the NO levels. We also observed that NO could mediate the repression of SnRK1 activity by SUC. By computational prediction we found putative S-nitrosylation sites in the TOR complex proteins and the catalytic subunit of SnRK1, SnRK1.1. The present work demonstrates for the first time a link between NO and TOR revealing the complex interplay between the two pathways in plants.

Effect of different exogenous sugars on the growth and nutrient uptake of Malus baccata Borkh. under sub-low root zone temperature.

In northern China, soil temperature slowly rises in spring, often subjecting apple roots to sub-low-temperature stress. Sugar acts as both a nutrient and signaling molecule in roots in response to low-temperature stress. To explore the effects of exogenous sugars on the growth and nutrient absorption of Malus baccata Borkh., we analyzed growth parameters, photosynthetic characteristics of leaves, and mineral element content in different tissues of M. baccata seedlings under five treatments, including control (CK), sub-low root zone temperature (L), sub-low root zone temperature + sucrose (LS), sub-low root zone temperature + fructose (LF), and sub-low root zone temperature + glucose (LG). The results showed that compared to CK, plant height, root growth parameters, aboveground biomass, leaf photosynthesis, fluorescence parameters, chlorophyll content, and the contents of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) in M. baccata seedlings were significantly decreased under the L treatment, and the content of Ca in roots was significantly increased. Compared to the L treatment without exogenous sugar, photosynthesis, functional parameters, chlorophyll content, and growth parameters increased to different degrees after exogenous sucrose, fructose, and glucose application. The N and P contents in roots were significantly increased. The N, P, and K contents significantly increased in stems while only the Ca content significantly increased in stems treated with sucrose. Leaf N, P, K, Ca, and Mg contents significantly increased after being treated with the three exogenous sugars. In conclusion, exogenous sugars can improve photosynthetic efficiency, promote mineral element absorption, and alleviate the inhibition of growth and development of M. baccata at sub-low root zone temperatures, and the effect of sucrose treatment was better than that of fructose and glucose treatments.

Sucrose contributed to the biofilm formation of Tetragenococcus halophilus and changed the biofilm structure.

Based on the previous research results that the addition of sucrose in the medium improved the biofilm formation of Tetragenococcus halophilus, the influence of sucrose on biofilm formation was explored. Moreover, the influence of exogenous expression of related genes sacA and galE from T. halophilus on the biofilm formation of L. lactis NZ9000 was investigated. The results showed that the addition of sucrose in the medium improved the biofilm formation, the resistance of biofilm cells to freeze-drying stress, and the contents of exopolysaccharides (EPS) and eDNA in the T. halophilus biofilms. Meanwhile, the addition of sucrose in the medium changed the monosaccharide composition of EPS and increased the proportion of glucose and galactose in the monosaccharide composition. Under 2.5% (m/v) salt stress condition, the expression of gene sacA promoted the biofilm formation and the EPS production of L. lactis NZ9000 with the sucrose addition in the medium and changed the EPS monosaccharide composition. The expression of gene galE up-regulated the proportion of rhamnose, galactose, and arabinose in the monosaccharide composition of EPS, and down-regulated the proportion of glucose and mannose. This study will provide a theoretical basis for regulating the biofilm formation of T. halophilus, and provide a reference for the subsequent research on lactic acid bacteria biofilms.

A sucrose ferulate cycle linchpin for ferulyolation of arabinoxylans in plant commelinids.

A transformation in plant cell wall evolution marked the emergence of grasses, grains and related species that now cover much of the globe. Their tough, less digestible cell walls arose from a new pattern of cross-linking between arabinoxylan polymers with distinctive ferulic acid residues. Despite extensive study, the biochemical mechanism of ferulic acid incorporation into cell walls remains unknown. Here we show that ferulic acid is transferred to arabinoxylans via an unexpected sucrose derivative, 3,6-O-diferuloyl sucrose (2-feruloyl-O-α-D-glucopyranosyl-(1'→2)-3,6-O-feruloyl-ß-D-fructofuranoside), formed by a sucrose ferulate cycle. Sucrose gains ferulate units through sequential transfers from feruloyl-CoA, initially at the O-3 position of sucrose catalysed by a family of BAHD-type sucrose ferulic acid transferases (SFT1 to SFT4 in maize), then at the O-6 position by a feruloyl sucrose feruloyl transferase (FSFT), which creates 3,6-O-diferuloyl sucrose. An FSFT-deficient mutant of maize, disorganized wall 1 (dow1), sharply decreases cell wall arabinoxylan ferulic acid content, causes accumulation of 3-O-feruloyl sucrose (α-D-glucopyranosyl-(1'→2)-3-O-feruloyl-ß-D-fructofuranoside) and leads to the abortion of embryos with defective cell walls. In vivo, isotope-labelled ferulic acid residues are transferred from 3,6-O-diferuloyl sucrose onto cell wall arabinoxylans. This previously unrecognized sucrose ferulate cycle resolves a long-standing mystery surrounding the evolution of the distinctive cell wall characteristics of cereal grains, biofuel crops and related commelinid species; identifies an unexpected role for sucrose as a ferulate group carrier in cell wall biosynthesis; and reveals a new paradigm for modifying cell wall polymers through ferulic acid incorporation.

Temperature-regulated cascade reaction for homogeneous oligo-dextran synthesis using a fusion enzyme.

Based on the principle of cascade reaction, a fusion enzyme of dextransucrase and dextranase was designed without linker to catalyze the production of oligo-dextran with homogeneous molecular weight from sucrose in one catalytic step. Due to the different effects of temperature on the two components of the fusion enzyme, temperature served as the "toggle switch" for the catalytic efficiency of the two-level fusion enzyme, regulating the catalytic products of the fusion enzyme. Under optimal conditions, the fusion enzyme efficiently utilized 100 % of the sucrose, and the yield of oligo-dextran with a homogeneous molecular weight reached 70 %. The product has been purified and characterized. The probiotic potential of the product was evaluated by analyzing the growth of 10 probiotic species. Its cytotoxic and anti-inflammatory activities were also determined. The results showed that the long-chain oligo-dextran in this study had significantly better probiotic potential and anti-inflammatory activity compared to other oligosaccharides. This study provides a strategy for the application of oligo-dextran in the food and pharmaceutical industries.

An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.

Synthetic redesign of Escherichia coli W for faster metabolism of sugarcane molasses.

BACKGROUND: Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes. RESULTS: In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP. CONCLUSIONS: Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications.

Sugar signaling modulates SHOOT MERISTEMLESS expression and meristem function in Arabidopsis.

In plants, development of all above-ground tissues relies on the shoot apical meristem (SAM) which balances cell proliferation and differentiation to allow life-long growth. To maximize fitness and survival, meristem activity is adjusted to the prevailing conditions through a poorly understood integration of developmental signals with environmental and nutritional information. Here, we show that sugar signals influence SAM function by altering the protein levels of SHOOT MERISTEMLESS (STM), a key regulator of meristem maintenance. STM is less abundant in inflorescence meristems with lower sugar content, resulting from plants being grown or treated under limiting light conditions. Additionally, sucrose but not light is sufficient to sustain STM accumulation in excised inflorescences. Plants overexpressing the α1-subunit of SUCROSE-NON-FERMENTING1-RELATED KINASE 1 (SnRK1) accumulate less STM protein under optimal light conditions, despite higher sugar accumulation in the meristem. Furthermore, SnRK1α1 interacts physically with STM and inhibits its activity in reporter assays, suggesting that SnRK1 represses STM protein function. Contrasting the absence of growth defects in SnRK1α1 overexpressors, silencing SnRK1α in the SAM leads to meristem dysfunction and severe developmental phenotypes. This is accompanied by reduced STM transcript levels, suggesting indirect effects on STM. Altogether, we demonstrate that sugars promote STM accumulation and that the SnRK1 sugar sensor plays a dual role in the SAM, limiting STM function under unfavorable conditions but being required for overall meristem organization and integrity under favorable conditions. This highlights the importance of sugars and SnRK1 signaling for the proper coordination of meristem activities.

Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade.

Arabinoside and derived nucleoside analogs, a family of nucleoside analogs, exhibit diverse typically biological activities and are widely used as antibacterial, antiviral, anti-inflammatory, antitumor, and other drugs in clinical and preclinical trials. Although with a long and rich history in the field of medicinal chemistry, the biosynthesis of arabinoside has only been sporadically designed and studied, and it remains a challenge. Here, we constructed an in vitro multi-enzymatic cascade for the biosynthesis of arabinosides. This artificial biosystem was systematically optimized, involving an exquisite pathway design, NADP+ regeneration, meticulous enzyme selection, optimization of the key enzyme dosage, and the concentration of inorganic phosphate. Under the optimized conditions, we achieved 0.37 mM of vidarabine from 5 mM of sucrose and 2 mM of adenine, representing 18.7% of the theoretical yield. Furthermore, this biosystem also has the capability to produce other arabinosides, such as spongouridine, arabinofuranosylguanine, hypoxanthine arabinofuranoside, fludarabine, and 2-methoxyadenine arabinofuranoside, from sucrose, and corresponding nucleobase by introducing different nucleoside phosphorylases. Overall, our biosynthesis approach provides a pathway for the biosynthesis of arabinose-derived nucleoside analogs, offering potential applications in the pharmaceutical industry.

Experimental evolution of yeast shows that public-goods upregulation can evolve despite challenges from exploitative non-producers.

Microbial secretions, such as metabolic enzymes, are often considered to be cooperative public goods as they are costly to produce but can be exploited by others. They create incentives for the evolution of non-producers, which can drive producer and population productivity declines. In response, producers can adjust production levels. Past studies suggest that while producers lower production to reduce costs and exploitation opportunities when under strong selection pressure from non-producers, they overproduce secretions when these pressures are weak. We challenge the universality of this trend with the production of a metabolic enzyme, invertase, by Saccharomyces cerevisiae, which catalyses sucrose hydrolysis into two hexose molecules. Contrary to past studies, overproducers evolve during evolutionary experiments even when under strong selection pressure from non-producers. Phenotypic and competition assays with a collection of synthetic strains - engineered to have modified metabolic attributes - identify two mechanisms for suppressing the benefits of invertase to those who exploit it. Invertase overproduction increases extracellular hexose concentrations that suppresses the metabolic efficiency of competitors, due to the rate-efficiency trade-off, and also enhances overproducers' hexose capture rate by inducing transporter expression. Thus, overproducers are maintained in the environment originally thought to not support public goods production.

Sugar transporters PpSWEET9a and PpSWEET14 synergistically mediate peach sucrose allocation from source leaves to fruit.

Sugar content is a critical indicator of fruit quality and is mainly controlled by sugar transporters. Sugars will eventually be exported transporters (SWEET) proteins play an indispensable role in sugar allocation between and within plant organs. Sucrose is the major sugar in many fruits and the predominant form of sugar translocated in peach (Prunus persica). However, the role of the multiple peach SWEET genes in sucrose allocation to fruit remains elusive. In this study, a total of 19 SWEET candidates have been identified in the peach genome, and two Clade III SWEET genes, PpSWEET9a and PpSWEET14, are found to be highly expressed in mature source leaves and branches. Complementation assays, transgene manipulations, and protein interaction studies reveal that PpSWEET9a and PpSWEET14 serve as sucrose efflux proteins and form a heterooligomer that synergistically directs sucrose allocation from source leaves to fruits. Our findings provide insights into the effect of SWEETs on sugar accumulation in peach fruit and identify genetic candidates for improving fruit quality.

Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable ß-caryophyllene production.

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce ß-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum ß-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO2 and light through co-culture systems.

Synergistic Effect Mechanism of Binary Sweet Taste Compounds.

In our previous study, phloridzin, sucrose, l-alanine, and dulcitol presented synergistic effects in Camellia nanchuanica black tea (NCBT). This study aims to verify the synergistic effects of the aforementioned sweet taste compounds and the mechanism involved. By conducting σ-τ plot analysis, phloridzin at the recognition threshold concentration (phl) exhibited synergistic effects with different concentrations of sucrose (Lsuc-6suc). Various concentrations of sucrose, phloridzin, and their combinations were selected to investigate the impact on sweet taste receptor cells. The results revealed that sucrose/phloridzin significantly increased the calcium signal compared to phloridzin and sucrose alone, attributed to the greater stability of the sucrose/phloridzin combination when binding to Taste 1 Receptor Member 3 (TAS1R3; one subunit of sweet taste receptor proteins). Ultimately, the sweet taste signal of sucrose/phloridzin was transmitted to the brain, triggering the activation of more brain regions associated with sweet taste perception (right insular, postcentral, and amygdala).

Optimizing pollen germination and subcellular dynamics in pollen tube of Torreya grandis.

Torreya grandis, a dioecious Taxaceae species of significant economic value in southeast China, presents challenges for natural pollination due to asynchronous maturation of its sex organs and low pollen vitality. In order to enhance fertilization success through artificial pollination of T. grandis, this study investigated the optimal conditions for in vitro pollen germination and pollen tube growth of T. grandis. The optimal in vitro growth medium was found to contain 29 mM sucrose, 0.8 mM H3BO3, 0.72 mM CaCl2, and 0.32 mM MgSO4, supplemented with 4 µM NAA, 2 µM GA3, and 5 µM 2,4-D at pH=5.6. Under these conditions, we achieved a maximum pollen germination ratio of 69.99 ± 5.17 % and a pollen tube length of 34.38 ± 6.04 µm after 6 days germination at 28°C. FM4-64 dye and Mitotracker Red staining revealed highly dynamics of vesicles and mitochondria during germination, which were accumulated at the tip of pollen tube and exhibited biphasic movement patterns. The total number, motion rate, and movement velocity of vesicles as well as mitochondria showed an initially increase followed by a gradual decrease pattern. The presence of sucrose in the medium significantly increased the dynamics and metabolic activity of both vesicles and mitochondria, which may relate with higher pollen germination ratio and faster pollen tube growth compared to sucrose-depleted conditions. Thus, these findings shed light on the physiological characteristics of Torreya pollen germination and provide scientific information for improving Torreya fruit yield through artificial pollination.

Inferior spikelet filling is affected by T6P/SnRK1-mediated NSC remobilization in large-panicle rice (Oryza sativa L.).

Poor grain filling in inferior spikelets (IS), which is influenced by the remobilization of nonstructural carbohydrates (NSC) stored in the sheath and internode of rice plants, limits the expected high yield of large-panicle rice. NSC remobilization from the sheath to the panicle is regulated by the T6P/SnRK1 pathway. However, in large-panicle rice, it is unclear whether IS grain filling is related to the NSC remobilization mediated by T6P/SnRK1 signaling. In this study, two large-panicle cultivars-W1844 and CJ03-with distinct differences in IS grain filling were used to explore the physiological mechanism mediating IS development. Compared to W1844, CJ03 IS showed lower expression of the genes related to sucrose uploading, later sucrose peaking, and delayed starch accumulation. In the CJ03, low OsSUTs expression and NSC output, transport rate, and contribution rate were detected in the sheaths and internodes. These results suggest that poor NSC remobilization results in insufficient assimilate supply for the IS, and consequently, poor IS grain filling. Furthermore, poor NSC remobilization coincided with the increased T6P content and decreased SnRK1 activity during grain filling in CJ03 IS. The expression levels of genes related to T6P metabolism and those encoding the catalytic subunit of SnRK1 were consistent with the observed T6P content and SnRK1 activity in the sheaths and internodes. Therefore, IS grain filling is potentially affected by T6P/SnRK1 signaling-mediated NSC remobilization in large-panicle rice.

Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase.

BACKGROUND: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon. RESULTS: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm. CONCLUSIONS: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.