The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.
Amino Acids ; Biocatalysis ; Amino Alcohols ; Coenzymes/metabolism* ; Diamines

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.
Benzyl Alcohols ; Escherichia coli/metabolism* ; Gastrodia/chemistry* ; Metabolic Engineering ; Tyrosine/metabolism*

This study investigated the effects and mechanisms of 6-gingerol on adipose tissue insulin resistance in naturally aging rats with glycolipid metabolism disorders. Twenty-seven aging male SD rats were randomly divided into a model group(aged, n=9) and two groups treated with 6-gingerol at 0.05 mg·kg~(-1)(G-L, n=9) and 0.2 mg·kg~(-1)(G-H, n=9). Six young rats were randomly assigned to a normal control group(NC). Rats were treated for seven weeks by gavage. Non-esterified fatty acid(NEFA) and insulin content was determined by enzyme-linked immunosorbent assay(ELISA), and adipose tissue insulin resistance index(Adipo-IR) was calculated. HE staining was used to observe the size of adipocytes in epididymal white adipose tissue(eWAT). The gene and protein expression levels of adiponectin receptor 1(AdipoR1), AMP-activated protein kinase α(AMPKα), phosphorylated AMPK(p-AMPKα~(Thr172)), peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α), phosphatidylinositol 3-kinase(PI3 K), protein kinase B(Akt), phosphorylated Akt(p-Akt~(Ser473)), tumor necrosis factor-α(TNF-α), c-Jun N-terminal kinase 1/2(JNK1/2), phosphorylated JNK1/2(p-JNK~(Thr183/Tyr185)), interleukin-1β(IL-1β), and interleukin-6(IL-6) in adiponectin(APN), insulin, and inflammatory factor signaling pathways were detected by Western blot and real-time RCR, respectively. The results showed that 6-gingerol at a high dose could significantly decrease the fasting plasma content of NEFA and insulin and reduce Adipo-IR. Additionally, 6-gingerol at a high dose significantly increased the protein and mRNA expression of APN, AdipoR1, PGC-1α, and PI3 K in eWAT, elevated the relative expression of p-AMPK~(Thr172) and p-Akt~(Ser 473), reduced the protein and mRNA expression of TNF-α, IL-1, and IL-6 in eWAT, and decreased the relative expression of p-JNK1 and p-JNK2. This study reveals that 6-gingerol can improve insulin sensitivity of adipose tissues in aging rats with glycolipid metabolism disorders, and this effect is presumedly achieved by enhancing the PI3 K/Akt signaling pathway, inhibiting adipose tissue inflammation, increasing APN synthesis, enhancing AdipoR1 expression, and activating its downstream AMPK/PGC-1α signaling pathway.
Adipose Tissue ; Aging ; Animals ; Catechols ; Fatty Alcohols ; Insulin Resistance ; Male ; Rats ; Rats, Sprague-Dawley

Objective: To study the effect of gastrodin on oxidative stress and bone tissue around implants in type 2 diabetes mellitus rats (T2DM), so as to provide therapeutic basis for the problems of poor osseointegration and long repair period of implants in type 2 diabetic patients, to provide the basis for the development of new therapeutic drugs. Methods: Forty SD rats aged 5 weeks were divided into control group (n=10) and model group (n=30). The model group was fed with high glycolipid diet for 4 weeks and injected with 35 mg/kg streptozocin. The T2DM were divided into diabetic group (n=10) and gastrodin group (n=10). Pure screw titanium implants were placed in the metaphysis of both tibia. Rats in gastrodin group were given gastrodin 13.6 mg/kg everyday, while the control group and diabetic group were given the same dose of saline solution. Blood glucose was measured every two weeks after operation in all rats. After 4 and 8 weeks, the contents of serum malondialdehyde (MDA) and superoxide dismutase (SOD) were measured to analyze the oxidative stress level in rats. The expression of anti-apoptosis protein [B cell lymphoma/lewkmia-2 (bcl-2)]and apoptosis protein [bcl-2-associated X protein (bax)] was analyzed by immunohistochemistry. HE staining and micro-CT scanning were used to analyze the osseointegration around the implants. Results: The blood glucose of gastrodin [(12.98±2.53), (13.64±1.96), (14.58±3.40) and (12.84±2.82) mmol/L] were significantly lower than that of diabetic group [(20.97±2.27), (17.94±3.76), (23.66±2.90) and (21.22±2.67) mmol/L] at corresponding time point (P<0.017). Four and 8 weeks following surgery, the MDA concentration in gastrodin group [(5.21±1.60) and (3.47±1.17) nmol/ml] was significantly lower than that in diabetic group [(11.42±5.11) and (7.31±1.37) nmol/ml] at the corresponding time points (P<0.017), while SOD activity in gastrodin group [(42.89±6.00) and (53.12±9.73) U/ml] was significantly higher than those in diabetic group [(27.09±6.11) and (32.08±2.97) U/ml] at the corresponding time points (P<0.017). Immunohistochemistry showed that the bcl-2 expression in gastrodin group was significantly higher than that in diabetic group at 4 and 8 weeks following surgery (P<0.017), while the bax expression in gastrodin group was significantly lower than that in diabetic group (P<0.017). Four weeks after surgery, the bone volume/tissue volume (BV/TV), connection density (Conn.D), trabecular number (Tb.N), trabecular thickness (Tb.Th) in gastrodin group were significantly higher than those in diabetic group (P<0.017). There was no significant difference in trabecular separation/spacing (Tb.sp) between gastrodin group and diabetic group (P<0.017). Eight weeks after surgery, the BV/TV, Tb.N in gastrodin group were significantly higher than those in the diabetic group (P<0.017). There was no significant difference in Conn.D between gastrodin group and diabetic group (P<0.017). There was no significant difference in Tb.Th and Tb.sp in three groups (P<0.017). The trabecular structure in gastrodin group was better than that in diabetic group by HE staining. Conclusions: Gastrodin can decrease blood glucose, improve oxidative stress, protect bone tissue from apoptosis and promote bone formation around implants in T2DM.
Animals ; Benzyl Alcohols ; Blood Glucose ; Dental Implants ; Diabetes Mellitus, Experimental ; Diabetes Mellitus, Type 2 ; Glucosides ; Proto-Oncogene Proteins c-bcl-2 ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; Tibia ; bcl-2-Associated X Protein

This paper aims to study the difference in the intestinal absorption kinetics of main active components of Sini decoction and its separated recipes and explain the scientificity and rationality of the compatibility of Sini Decoction. A in situ intestinal perfusion rat model was established to evaluate the differences in the absorption of benzoylmesaconine, benzoylaconine, benzoylhypacoitine, mesaconitine, hypaconitine, glycyrrhizic acid, liquiritin and 6-gingerol from Sini Decoction and its separated recipes in the duodenum, jejunum and ileum by high performance liquid chromatography(HPLC). The results indicated that the Sini Decoction group was superior to the Aconiti Lateralis Radix Praeparata group in terms of absorption degree and rate for aconitum alkaloids. The absorption of benzoylmesaconine and hypaconitine in the duodenum, jejunum and ileum was faster and stronger in the Sini Decoction group(P<0.05). The absorption degree of glycyrrhizic acid in the duodenum was significantly higher in the Sini Decoction group than in the Glycyrrhizae Radix et Rhizoma group and the Glycyrrhizae Radix et Rhizoma-Zingiberis Rhizoma group(P<0.05). The absorption rate and degree of 6-gingerol in the ileum in the Sini Decoction group were significantly higher than those in the Zingiberis Rhizoma group(P<0.05). In short, Zingiberis Rhizoma and Glycyrrhizae Radix et Rhizoma can promote the absorption of aconitum alkaloids in different intestinal segments, which reflects the scientific composition of Sini Decoction.
Aconitine/analogs & derivatives* ; Aconitum ; Alkaloids ; Animals ; Catechols ; Drugs, Chinese Herbal ; Fatty Alcohols ; Glycyrrhizic Acid ; Intestinal Absorption ; Kinetics ; Rats

OBJECTIVE: To investigate the therapeutic mechanism of gastrodin injection for alleviating lung injury caused by focal cerebral ischemia in rats and the role of the NGF-TrkA pathway in mediating this effect. METHODS: Forty SD rats were equally randomized into normal group, sham-operated group, model group and gastrodin group, and in the latter two groups, rat models of focal cerebral ischemia were established by embolization of the right middle cerebral artery. After successful modeling, the rats were treated with intraperitoneal injection of gastrodin injection at the daily dose of 10 mg/kg for 14 days. After the treatment, the wet/dry weight ratio of the lung tissue was determined, the pathological changes in the lung tissue were observed using HE staining, and the levels of IL-10 and TNF-α in the arterial blood were detected with ELISA. The expressions of NF-κB p65 and TNF-α in the lung tissue were detected with Western blotting, and the expressions of NGF and TrkA were detected using immunohistochemical staining and Western blotting. RESULTS: Compared with the normal control and sham-operated groups, the rats in the model group showed obvious inflammatory lung injury, significantly increased wet/ dry weight ratio of the lungs (P < 0.01), increased TNF-α level in arterial blood (P < 0.01), and significantly up-regulated protein expressions of NF-κB p65 (P < 0.01), TNF-α (P < 0.01), NGF (P < 0.05) and TrkA(P < 0.05) in the lung tissue. Treatment with gastrodin injection obviously alleviated lung inflammation, decreased the wet/dry weight ratio of the lungs (P < 0.05), and significantly lowered TNF-α level (P < 0.01) and increased IL-10 level in the arterial blood in the rat models (P < 0.01); gastrodin injection also significantly decreased the protein expressions of NF-κB p65 and TNF-α (P < 0.05) and up-regulated the expressions of NGF and TrkA in the lung tissue of the rats (P < 0.05). CONCLUSION The NGF/TrkA pathway may participate in cerebral ischemia-induced inflammatory lung injury, which can be obviously alleviated by gastrodin through the activation of the anti-inflammatory pathway mediated by the NGF/TrkA pathway.
Animals ; Anti-Inflammatory Agents ; Benzyl Alcohols ; Brain Ischemia ; Glucosides ; Lung/metabolism* ; Lung Injury ; NF-kappa B ; Nerve Growth Factor ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha

Higher alcohols are one of the main by-products of Saccharomyces cerevisiae in brewing. High concentration of higher alcohols in alcoholic beverages easily causes headache, thirst and other symptoms after drinking. It is also the main reason for chronic drunkenness and difficulty in sobering up after intoxication. The main objective of this review is to present an overview of the flavor characteristics and metabolic pathways of higher alcohols as well as the application of mutagenesis breeding techniques in the regulation of higher alcohol metabolism in S. cerevisiae. In particular, we review the application of metabolic engineering technology in genetic modification of amino transferase, α-keto acid metabolism, acetate metabolism and carbon-nitrogen metabolism. Moreover, key challenges and future perspectives of realizing optimization of higher alcohols metabolism are discussed. This review is intended to provide a comprehensive understanding of metabolic regulation system of higher alcohols in S. cerevisiae and to provide insights into the rational development of the excellent industrial S. cerevisiae strains producing higher alcohols.
Alcoholic Beverages ; Alcohols/analysis* ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Higher alcohols that contain more than two carbon atoms have better fuel properties than ethanol, making them important supplements and alternatives to fossil fuels. Using microbes to produce higher alcohols from renewable biomass can alleviate the current energy and environmental crises, and has become a major future direction for green biomanufacturing. Since natural microbes can only produce a few higher alcohols in small amounts, it is necessary to reconstruct the synthetic pathways for higher alcohols in model industrial strains through metabolic engineering and synthetic biology to overcome the metabolic bottlenecks. A series of milestones have been accomplished in past decades. The authors of this review have witnessed the entire journey of this field from its first success to the leaping development. On the 30th anniversary of the founding of the discipline of metabolic engineering, this review dates back to the great milestones in achieving heterologous production of higher alcohols in non-native strains. The design and optimization of high alcohol biosynthetic pathways, the expansion of feedstock, the engineering of host strains and the industrialization process are summarized. This review aims to draw further attention to microbial synthesis of higher alcohols, inspire the development of novel techniques and strategies of metabolic engineering, and promote the innovation and upgrade of China's biofuel industry.
Alcohols ; Biofuels ; Biosynthetic Pathways ; Ethanol ; Metabolic Engineering ; Synthetic Biology

Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis is the most frequently used herbal pair in the treatment of Parkinson's disease(PD). Gastrodin and isorhynchophylline are important components of Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis herb pair with anti-Parkinson mechanism. This study aimed to investigate the effect of gastrodin combined with isorhynchophylline on 1-methyl-4-phenylpyridinium(MPP~+)-induced apoptosis of PC12 cells and their antioxidant mechanism. The leakage of lactate dehydrogenase(LDH) from cells to media was analyzed by spectrophotometry. Apoptotic cells were labeled with Annexin V-fluorescein isothiocyanate(FITC) and propidium iodide(PI) and analyzed by flow cytometry. The cell cycle was analyzed using propidium iodide(PI) staining. Lipid peroxidation(LPO) level was analyzed by spectrophotometry. The mRNA expression of caspase-3 was examined by Real-time RT-PCR. The protein expressions of heme oxygenase 1(HO-1) and NADPH: quinoneoxidore-ductase 1(NQO-1) were determined by Western blot. Gastrodin combined with isorhynchophylline reduced the percentage of Annexin V-positive cells and cell cycle arrest in MPP~+-induced PC12 cells. Gastrodin combined with isorhynchophylline down-regulated the mRNA expression of caspase-3, up-regulated the protein expressions of HO-1 and NQO-1, and reduced LPO content in MPP~+-induced PC12 cells. PD98059, LY294002 or LiCl could partially reverse these changes pretreated with gastrodin combined with isorhynchophylline, suggesting that gastrodin combined with isorhynchophylline inhibited MPP~+-induced apoptosis of PC12 cells and oxidative stress through ERK1/2 and PI3 K/GSK-3β signal pathways. Our experiments showed that gastrodin combined with isorhynchophylline could down-re-gulate the mRNA expression of caspase-3 and up-regulate the protein expressions of HO-1 and NQO-1, so as to reduce oxidative stress and inhibit apoptosis.
1-Methyl-4-phenylpyridinium/toxicity* ; Animals ; Antioxidants ; Apoptosis ; Benzyl Alcohols ; Cell Survival ; Glucosides ; Glycogen Synthase Kinase 3 beta ; Oxindoles ; PC12 Cells ; Rats

The efficacy of gastrodin as a Chinese herbal medicine extract in the treatment of tension-type headache has been confirmed. This paper systematically reviewed the efficacy and safety of gastrodin in the treatment of tension-type headache, aiming to provide a new choice for the treatment of this disease. In this study, four Chinese databases, four English databases and two trial registries were searched from the date of establishment to September 2020. The related randomized controlled trials(RCTs) were screened out according to the predetermined criteria. The bias risk assessment tool developed by Cochrane collaboration was used to evaluate the quality of the reports. RevMan 5.4.1 was used for Meta-analysis, and GRADE system for the evidence-based evaluation on the quality of outcome indicators. A total of 177 articles were retrieved and 8 articles were finally included for analysis, with a total sample size of 1 091 cases, which included 565 cases in the treatment group and 526 cases in the control group. The overall quality of included stu-dies was not high. The results of Meta-analysis are as follows:(1)In terms of headache frequency, gastrodin group was better than wes-tern medicine group(MD=-2.90, 95%CI[-3.76,-2.03], P<0.000 01).(2)In terms of number of abnormal blood vessels in TCD, gastrodin group was better than western medicine group(MD=-88.96, 95%CI[-102.36,-75.55], P<0.000 01).(3)In terms of effective rate, gastrodin group was better than western medicine group(RR=1.47, 95%CI[1.29, 1.68], P<0.000 01). The results of subgroup analysis are as follows:(1)Effective rate based on age, for the patients upper age limit 40-46 years old, gastro-din group was better than western medicine group(RR=1.69, 95%CI[1.50, 1.90], P<0.000 01); for the patients upper age limit 55-60 years old, gastrodin group was better than western medicine group(RR=1.27, 95%CI[1.16, 1.38], P<0.000 01).(2)Effective rate based on dosage form, both the gastrodin capsules and injection groups were better than western medicine group(RR_(capsules)=1.42, 95%CI[1.08, 1.88], P=0.01; RR_(injection)=1.50, 95%CI[1.26, 1.77], P<0.000 01). GRADE evaluation showed that the above outcomes had low quality of evidence. Only one article detailed the occurrence of adverse reactions and thus the present study cannot make a positive conclusion on the safety of gastrodin in the treatment of tension-type headache. The small number and low quality of the included reports affected the reliability of the results. In the future, more high-quality randomized controlled trails are needed to improve the evaluation on the efficacy and safety of gastrodin in the treatment of tension-type headache.
Adult ; Benzyl Alcohols/therapeutic use* ; Drugs, Chinese Herbal/adverse effects* ; Glucosides ; Humans ; Middle Aged ; Reproducibility of Results ; Tension-Type Headache