Gastrodia elata is an edible plant whose phenolic compounds show alpha-glucosidase inhibitory activity. Through screening, it was found that 4-hydroxybenzyl alcohol (HBA) is the most potent inhibitor (IC50 = 0.0406 mg/mL). However, HBA occurs at very low levels in native G. elata, making it difficult to acquire sufficient active components via routine approaches. Different from conventional organic solvent extraction and microbial fermentation, the (3-glucosidase-catalyzed strategy developed here has the advantages of short processing time, mild reaction conditions and environmental friendliness. Compared with water extraction, this method increased HBA content by 9.76 fold and improved alpha-glucosidase inhibitory activity by 5.31 fold. Enzymolysis conditions were optimized via response surface methodology at 51.5 degrees C, 6 U/mL and 4.7 h, yielding 832 mu g/mL HBA with an inhibition rate of 71.4%. Pearson correlation confirmed HBA as an important contributor to the bioactivity (r = 0.6669). Mechanistic analyses via kinetics, spectroscopy, molecular docking, and 100 ns molecular dynamics simulations revealed HBA acts as a non-competitive inhibitor binding to the allosteric site at Ser236, Asn317, and His423. The optimized extract (5% v/v, 322 mu M HBA) significantly promoted glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells without cytotoxicity. It also dose-dependently reduced glucose, triglyceride, and total cholesterol in high-glucose-stimulated zebrafish larvae. This efficient catalytic approach achieves high HBA enrichment and clarifies the underlying inhibitory mechanism, supporting G. elata extract as a promising functional food for glycemic and lipid regulation.

D-chiro-inositol (DCI), a key signaling molecule in glucose and lipid metabolism and steroid synthesis, plays important roles in ameliorating human metabolic disorders. To achieve de novo DCI biosynthesis in Corynebacterium glutamicum, the inositol catabolism-blocked chassis strain DCI‑1 was engineered by dynamically downregulating pgi, pfkA, and zwf genes. The most effective strain DPa‑1 produced 2.89 g/L myo-inositol (MI), the direct precursor of DCI, and 0.83 g/L DCI, with the total inositol over four times the control. Subsequently, to promote the conversion of MI to DCI and the supply of precursor glucose-6-phosphate, key enzymes for DCI synthesis (ino1, iolI2, and iolG) and glucose transport‑phosphorylation were overexpressed. In combination with the knockout of ndnR to enhance NAD(H) supply, the resulting strain DPaZbN‑3 accumulated 2.82 g/L DCI and 2.93 g/L MI. Further regulation of the mycothiol synthetic pathway by σᴮ-recognized promoter in the final engineered strain DPaZbNMh‑3 enabled efficient accumulation of 3.39 g/L DCI and 4.12 g/L MI, representing the highest reported level of microbial DCI production from glucose to date. This research provided effective metabolic engineering strategies and a robust microbial platform for the green biomanufacturing of DCI.

4-Hydroxyisoleucine (4-HIL) holds potential value in the treatment of diabetes. It can be produced by expressing the exogenous isoleucine dioxygenase gene ido in L-isoleucine (Ile) producing Corynebacterium glutamicum strains. But the stable expression of ido on plasmids relies on the usage of antibiotics. To make the harboring of ido independent of plasmid, this study developed a chromosome-engineered strain for synthesizing 4-HIL directly from glucose. First, the ido-cat-ido expressing cassette was inserted into the chromosome of C. glutamicum, and the copy number of ido was increased through chemically inducible chromosome evolution (CIChE). After successive rounds of CIChE by increasing chloramphenicol concentration, 7 copies of ido were integrated in the chromosome of C. glutamicum SE04, and the 4-HIL production reached 20.3 ± 4.99 g/L, 3.5-fold higher than the initial strain SC12 harboring two-copies of ido. To cease further homologous recombination, recA was deleted in CIChE strains, but cell growth and 4-HIL production were damaged. Notably, the stability of chromosomally inserted genes in the evolved strain SE04 was confirmed. Ultimately, the evolved C. glutamicum SE04 strain produced 30.3 g/L of 4-HIL in a 2-L bioreactor. This study established a plasmid-free strain of C. glutamicum for 4-HIL production, offering new insights into utilizing multi-copy integration methods for producing other valuable biochemical substances in C. glutamicum.

gamma-Aminobutyric acid (GABA) is widely applied in pharmaceuticals, foods and feeds. Corynebacterium glutamicum that expresses exogenous glutamate decarboxylase (GAD) gene gad can produce GABA from glucose using self-produced L-glutamate. However, the incongruity between optimal pH for cell growth (7.0-7.5) and GAD (4.0-5.0) severely restricts the production of GABA. In this study, several GADs active at near-neutral pH were separately expressed in C. glutamicum by plasmid, LsGAD derived from Lactobacillus senmaizukei performed better and generated 10.9 g/L GABA. Subsequently, to perform the GAD reaction at the more acidic extracellular environment, LsGAD was displayed on cell surface by several anchoring motifs, and displaying by PorH and NCgl1307 motifs produced 9.9 g/L and 1.3 g/L GABA, respectively. To further improve GABA production, the metabolic pathways were modified and accompanied by integrating several gad genes in the chromosome, the best strain GSL-6 could produce 15.6 g/L GABA. Finally, the surface display plasmid of LsGAD was introduced into the chromosomally modified strain GSL-6 to catalyze GAD reaction both intracellularly and extracellularly, and 25.3 g/L and 42.3 g/L GABA was finally produced by shake flask and fed-batch fermentation, respectively. Thereby, this synergistic strategy is beneficial for GABA production in C. glutamicum.

Mechanical pressing in Daqu production has introduced quality-affecting variations. Up to now, clear elucidation has not yet been applied to the mechanisms behind this phenomenon, and the determinants of Daqu quality are not yet completely excavated. For this reason, the physicochemical factors, enzyme activity, metabolites, and microbial communities were compared between the mechanical Daqu (MDQ) and traditional Daqu (TDQ) in this paper. The results showed significant differences in amino acids between MDQ and TDQ, with arginine being the key differentiator. High temperature, high moisture, and low acidity were crucial to Daqu's amino acid richness. Additionally, mechanical pressing affected core community stability. Bacillus was the primary biological factor for the discrepancy in amino acids. Further investigation indicated that arginine contributed to the reduction of lactic acid and higher alcohols in Jiupei, confirming that amino acids were potential novel markers affecting the quality of Daqu.

Conventional rice cooking methods often induce excessive starch gelatinization and rapid digestibility owing to continuous contact with water. To address this issue, we developed a noncontinuous rice-water contact (NRWC) method in which rice alternates contact with water. In this study, the moisture content, texture, microstructure, and in vitro starch digestibility of cooked rice were investigated and compared with those of cooked rice subjected to continuous rice-water contact (CRWC). The in vitro starch digestibility indicated that the NRWC method increased the content of slowly digestible and resistant starches in cooked rice. Meanwhile, the kinetic constant of NRWC cooked rice (k, 1.4 x 10- 2-1.8 x 10- 2 min- 1) and in vitro starch digestibility were lower than those of CRWC cooked rice (k = 2.0 x 10- 2 min- 1). NRWC samples with low moisture content (45.1 %-59.3 %) and high hardness (26.5-10.2 N) have the potential to reduce starch digestibility. From the perspective of morphological properties, the NRWC method was beneficial to maintaining the surface smoothness, internal compactness, starch-protein framework, and integrity of rice. Collectively, this study provides a new perspective on the cooking method of preparing slowly digestible rice.

Hypertension is very harmful to health. Tartary buckwheat (TB) is a healthy food, and its peptides have been proven to have anti-hypertensive effects. However, about 70% of TB is starch, which is unnecessary for its antihypertensive function. In order to degrade starch in TB, amylolytic lactic acid bacteria (ALABs) were screened from food raw materials and explored to ferment TB in this study. Forty-eight ALABs capable of degrading soluble starch were obtained, among which Lactobacillus pentosus CF55, Lactiplantibacillus plantarum CF60, Lp. plantarum CF66, and Lb. sp. CF80 all exhibited degradation rate of over 88% and were safe strains. Then TB was used as the sole carbon source and fermented by each of these 4 strains. After fermentation, lots of pores and voids were presented in TB. The chemical bonds and crystal structure of TB starch did not change, but the shortrange orderliness of TB starch decreased, while the relative crystallization increased from 2.53% to 4.52%- 5.79%, and the gelatinization enthalpy increased by 0.20-1.21 J/g. Importantly, TB starch was significantly degraded after fermentation, with the degradation rate reaching 43.68%-78.36%, and the remaining starch could be digested easier during gastrointestinal simulation. In addition, similar total amount of TB protein could be extracted and the derived TB peptide (TBP) showed strong hypotensive activity, among which the angiotensin converting enzyme inhibition rate of CF80-fermented TBP increased from 81.56% to 91.84%. Furthermore, the polyphenols and flavonoids contents increased by 49%-201%. This study provides novel strategy for improving the function of TB.

In contrast to the inhibition of angiotensin-converting enzyme (ACE), inhibition of renin activity controls increases in blood pressure at the source. Few renin inhibitory peptides have been reported. In this study, novel renin inhibitory peptides were identified from tartary buckwheat albumin hydrolysates (TBAHs). The structural characteristics of the peptide fractions with renin inhibitory activity in TBAHs were investigated by column separation. The potential renin inhibitory peptides were screened and the interactions of the peptides with renin were investigated by molecular docking. The peptides fractions with more charge and hydrophobicity were found to have a greater inhibitory effect on renin. After the screening, seven peptides were selected and synthesized, all of which showed renin inhibitory activity. The IC50 values of the peptides LFFR and LGLLPYFR were 5.00mM and 10.19mM，which both contained leucine (L) at the N-terminus and arginine (R) at the C-terminus. The molecular docking analysis indicated that the peptides interact with the active center of renin mainly by hydrophobic interactions. This was the first study to isolate renin inhibitory peptides from tartary buckwheat.

Corynebacterium glutamicum is widely used in the production of amino acids. C. glutamicum possesses seven sigma factors, among which SigD is responsible for the transcription of genes involved in the synthesis of mycolic acid (MA) and its derivatives, the unique cell envelope of C. glutamicum. To understand the influence of MA synthesis on amino acid production and membrane phenotype of C. glutamicum, the expression of sigD gene and some mycolyltransferase genes, i.e., cmt1, cop1 and cmt2, were regulated by several growth-regulated promoters in this study. Except for 2 mutant strains of Pcg3096-sigD and Pcg1633-cop1, the growth and 4-hydroxyisoleucine (4-HIL) titer of most modified strains did not change significantly. But the 4-HIL titer of PodhI-sigD strain increased by 20.73

D-chiro-inositol (DCI) is a potential drug for the treatment of type II diabetes and polycystic ovary syndrome. In order to effectively synthesize DCI in Corynebacterium glutamicum, the genes related to inositol catabolism in clusters iol1 and iol2 were knocked out in C. glutamicum SN01 to generate the chassis strain DCI-1. DCI-1 did not grow in and catabolize myo-inositol (MI). Subsequently, different exogenous and endogenous inosose isomerases were expressed in DCI-1 and their conversion ability of DCI from MI were compared. After fermentation, the strain DCI-7 co-expressing inosose isomerase IolI2 and inositol dehydrogenase IolG was identified as the optimal strain. Its DCI titer reached 3.21 g/L in the presence of 20 g/L MI. On this basis, the pH, temperature and MI concentration during whole-cell conversion of DCI by strain DCI-7 were optimized. Finally, the optimal condition that achieved the highest DCI titer of 6.96 g/L were obtained at pH 8.0, 37 °C and addition of 40 g/L MI. To our knowledge, it is the highest DCI titer ever reported.

Accurately identifying life-threatening prostate cancer (PCa) at time of diagnosis remains an unsolved problem. We evaluated whether DNA methylation status of selected candidate genes can predict the risk of metastasis beyond clinical risk factors in men with untreated PCa. A nested case-control study was conducted among men diagnosed with localized PCa at Kaiser Permanente California between 01/01/1997-12/31/2006 who did not receive curative treatments. Cases were those who developed metastasis within 10 years from diagnosis. Controls were selected using density sampling. Ninety-eight candidate genes were selected from functional categories of cell cycle control, metastasis/tumour suppressors, cell signalling, cell adhesion/motility/invasion, angiogenesis, and immune function, and 41 from pluripotency genes. Cancer DNA from diagnostic biopsy blocks were extracted and analysed. Associations of methylation status were assessed using CpG site level and principal components-based analysis in conditional logistic regressions. In 215 cases and 404 controls, 27 candidate genes were found to be statistically significant in at least one of the two analytical approaches. The agreement between the methods was 25.9% (7 candidate genes, including 2 pluripotency markers). The DNA methylation status of several candidate genes was significantly associated with risk of metastasis in untreated localized PCa patients. These findings may inform future risk prediction models for PCa metastasis beyond clinical characteristics.

BACKGROUNDTartary buckwheat protein peptides have been shown to be able to inhibit angiotensin-converting enzyme (ACE), but the exact protein type has been less studied for ACE activity inhibition, and only a few types of ACE inhibitory peptides have been reported. In this study, we purified and identified ACE inhibitory peptides from albumin hydrolysate (AH). RESULTSAlbumin, globulin, prolamin and glutelin were extracted from Tartary buckwheat, and their ACE active peptides were obtained by a pepsin-trypsin sequential hydrolysis process. All four hydrolysates exhibited ACE inhibitory activity, and AH displayed the strongest ACE inhibition activity and the highest peptide yield (82.28%). At 0.2 mg mL(-1), the inhibition rate of AH was 79.89%, followed by globulin hydrolysate at 71.84%, while prolamin hydrolysate and glutelin hydrolysate showed lower inhibition rates. The peptides with the highest inhibition rate were then isolated from AH using gel filtration chromatography and reversed-phase high-performance liquid chromatography, and identified using nanoscale high-performance liquid chromatography-tandem mass spectrometry. After isolation and purification, 42 ACE inhibitory peptides were identified in the fraction with the highest inhibition rate, 14 of which were completely novel discoveries in this study. These 14 peptides showed potent ACE inhibitory effects through computer analysis. CONCLUSIONTartary buckwheat albumin can be used as a good source of ACE inhibitory peptides and can be further developed and utilized as edible supplements or drugs. (c) 2023 Society of Chemical Industry.

To explore an l-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01. Four Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying PtacM-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L−1) were similar with control strain S-D5I (15.73 ± 2.66 g 4-HIL L−1). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated l-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-D5I and KIRSA-3-9I, and Ile concentration was maintained less than 35 mmol L−1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-9I produced 22.46 ± 0.96 g 4-HIL L−1. The screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.

Corynebacterium glutamicum, an important industrial producer, is a model microorganism. However, the limited gene editing methods and their defects limit the efficient genome editing of C. glutamicum. To improve the screening efficiency of second-cross-over strains of traditional SacB editing system, a universal pCS plasmid which harbors CRISPR-Cpf1 system targeting kan gene of SacB system was designed and established to kill the false positive single-cross-over strains remained abundantly after the second-cross-over events. The lethality of pCS plasmid to C. glutamicum carrying kan gene on its genome was as high as 98.6

With the development of synthetic biology, some quorum sensing (QS) systems have been studied and applied to coordinate growth and production. Recently, a novel ComQXPA-PsrfA system with different response strengths was constructed in Corynebacterium glutamicum. However, the plasmid-harbored ComQXPA-PsrfA system lacks genetic stability, which restricts the application of this QS system. In this study, the comQXPA expression cassette was integrated into the chromosome of C. glutamicum SN01, resulting in QSc chassis strain. The green fluorescence protein (GFP) was expressed by the natural and mutant PsrfA promoters (PsrfAM) with various strengths in QSc. All the expressions of gfp were activated to the related level in a cell density-dependent manner. Therefore, ComQXPA-PsrfAM circuit was applied for modulating the dynamic biosynthesis of 4-hydroxyisoleucine (4-HIL). First, the expression of ido encoding α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase was dynamically regulated by PsrfAM promoters, resulting in QSc/NI. The 4-HIL titer (125.18 ± 11.26 mM) increased by 45.1

4-hydroxyisoleucine (4-HIL) has a potential value in treating diabetes. The α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase (IDO) can catalyze the hydroxylation of L-isoleucine (Ile) to form 4-HIL by consuming O2. In our previous study, the ido gene was overexpressed in an Ile-producing Corynebacterium glutamicum strain to synthesize 4-HIL from glucose. Here, a triple-functional dynamic control system was designed to regulate the activity of IDO, the supply of α-KG, O2, and Ile and the synthesis of by-product L-lysine (Lys) for promoting 4-HIL synthesis. Firstly, the codon-optimized ido was positively regulated by seven Ile biosensors Lrp-PbrnFEN with different intensities, and the resulting seven D-NI strains produced 38.7–111.1 mM 4-HIL. Then on the basis of D-NI, odhI and vgb were simultaneously regulated by three PbrnFEN with different intensities to synergistically control α-KG and O2 supply. The 4-HIL titer of twelve D-NINONV strains was more than 90 mM, with D-0I7O7V generating the highest titer of 141.1 ± 15.5 mM. Thirdly, ilvA was negatively regulated by an Ile attenuator PilvBNC on the basis of D-NI strains and some D-NINONV strains to balance the synthesis and conversion of Ile. The resulting D-NIPA strains produced 73.6–123.2 mM 4-HIL, while D-7I7O1VPA accumulated 127.1 ± 20.2 mM 4-HIL. Finally, dapA was negatively regulated by a Lys-OFF riboswitch and Lys content decreased by approximately 70% in most D-RS-NIPA strains. A strain D-RS-5IPA with the highest 4-HIL titer (177.3 ± 8.9 mM) and the lowest Lys concentration (6.1 ± 0.6 mM) was successfully obtained. Therefore, dynamic regulation of main and branch pathway by three functional biosensors can effectively promote 4-HIL biosynthesis in C. glutamicum. • Three biosensors were coordinated for dynamic 4-HIL biosynthesis in C. glutamicum • Bidirectional regulation of Ile synthesis and conversion promoted 4-HIL synthesis • Negative regulation of Lys synthesis further increased 4-HIL production

4-hydroxyisoleucine (4-HIL) is a potential drug for diabetes and weight control. 4-HIL was produced by expressing ido gene in L-isoleucine (Ile)-producing Corynebacterium glutamicum . But L-lysine (Lys) was also accumulated as the main by-product in this recombinant strain SN02. To attenuate Lys synthesis, two genes in Lys synthetic pathway, i.e., ddh encoding the diaminopimelic acid dehydrogenase and lysE encoding the specific Lys exporter were deleted in SN02. However, the deletion of ddh increased 4-HIL titer by 28.1%, but did not decrease Lys content; while the deletion of lysE significantly reduced Lys content by 66.7%, but 4-HIL titer also decreased by 19.3%. Therefore, we carried out transcriptome analysis to reveal the global variation in these mutants. Deletion of ddh and lysE (especially lysE ) enhanced the transcription of key enzymes in succinylase branch of Lys synthesis pathway (DapD and DapC) and several enzymes involved in succinyl-CoA accessibility (SucC, SucD and OdhI), suggesting the compensatory synthesis of Lys via succinylase branch. In addition, the transcription of ilvBN in Ile synthesis pathway was improved, while the transcription of some genes in the 2-methylcitrate cycle and inositol metabolism pathway was weakened in these mutants. Mere deletion of ddh enhanced the transcription of aceA , ppc and pck , thus promoting oxaloacetate supply and 4-HIL synthesis. Deletion of lysE affected the transcription of some stress-related genes and transporter genes, suggesting that this mutant would be under stress, thus attenuating its 4-HIL synthesis. These findings will be helpful for systematic microbiology and bio-manufacturing of C. glutamicum .

4-羟基异亮氨酸(4-hydroxyisoleucine,4-HIL)在治疗Ⅱ型糖尿病方面极具潜力.异亮氨酸双加氧酶(isoleucine dioxygenase,IDO)能够将L-异亮氨酸(L-isoleucine,Ile)转变成4-HIL.为提高重组谷氨酸棒杆菌的4-HIL产量同时降低副产物L-赖氨酸(L-lysine,Lys)的合成,首先将Lys-OFF核糖体开关整合到Lys合成途径的关键基因dapA序列上游,得到D-RS菌株.该菌株能够根据胞内外Lys的浓度,动态弱化Lys合成,使Lys的含量降低了46.7％.其次,在该菌株中再利用Ile激活型传感器Lrp-PbrnFE N去控制密码子优化后的ido基因的表达,使4-HIL的产量提高至116.3 mmol/L.最后,为了进一步提高4-HIL产量,利用强启动子PbrnFE7动态控制odhI和vgb的表达,增强α-酮戊二酸和O2的供应.最终得到了1株摇瓶发酵4-HIL产量高达166.0 mmol/L,Lys含量降至6.5 mmol/L的重组菌株D-RS-0 I7 O7 V.研究所采用的动态调控策略为4-HIL的高效合成提供了一种新思路.

采用高温流化技术处理青稞米,改善其蒸煮品质.考察流化温度、进料速度、处理时间及补水量4个因素对青稞米蒸煮硬度的影响,正交实验得到最优工艺参数为:流化温度175℃、进料速度60 kg/h、流化时间80 s.当青稞米与大米同煮时,青稞米的蒸煮硬度为1 409.75 g,与大米蒸煮硬度相近;淀粉糊化度升高了17.6％,高温流化青稞米与大米同煮能够同煮同熟,且其咀嚼性降低、黏着性增加,食味品质得到明显改善.高温流化使得青稞米淀粉颗粒间隙增大、结晶度下降、吸水率增加,吸水性能提升,这是青稞米蒸煮品质改善的主要原因.

Highland barley (HB) is prone to deterioration due to the lipid rancidity induced by lipase (LA) activity. Therefore radio frequency (RF) heating was applied to inactivate the LA of HB. The objectives of this study were to reveal the critical reason for triggering lipid deterioration during RF heating and to establish effective RF treatment protocols by controlling this trigger. After RF stabilization treatment, HB was packaged by three methods: Ziplock bags (ZB), vacuum (Va) and vacuum combined with an oxygen absorber (Va-OA). The changes in the free fatty acid value (FFAV), peroxide value (POV), free radical intensity, hexanal content and free fatty acid (FFA) composition in RF-treated HB samples were also compared. The optimal operating conditions of RF heating to inactivate LA and control free radical intensity were target temperature of 95 degrees C with holding for 0 min, electrode gap of 120 mm and water supplementation of 2%. Under these conditions, the free radical intensity of RF-stabilized HB decreased to the lowest level of 0.1367, and the inactivation rate of LA reached 44.19%. After 30 d of storage at 37 degrees C, the hexanal content showed the lowest level of 2.103 mu g/g, and LA activity did not increase significantly during the whole storage period. During the 6-month storage under ambient temperature, the FFAV, POV and free radical intensity of RF-stabilized HB packaged by ZB, Va and Va-OA were all significantly lower than those of untreated HB, while their FFA composition changed more slowly. The lowest hexanal content was observed in Va-OA packaged RF-treated samples highlighting the importance of free radical control during RF heating and the subsequent control of oxygen accessibility during storage. In summary, this study suggested the great potential of RF heating technology for stabilizing HB efficiently.