High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit.

Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

Combinatorial metabolic engineering enables high yield production of α-arbutin from sucrose by biocatalysis.

α-Arbutin has been widely used as a skin-whitening ingredient. Previously, we successfully produced α-arbutin via whole-cell biocatalysis and found that the conversion rate of sucrose to α-arbutin was low (~45%). To overcome this issue, herein, we knocked out the genes of enzymes related to the sucrose hydrolysis, including sacB, sacC, levB, and sacA. The sucrose consumption was reduced by 17.4% in 24 h, and the sucrose conversion rate was increased to 51.5%. Furthermore, we developed an inducible protein degradation system with Lon protease isolated from Mesoplasma florum (MfLon) and proteolytic tag to control the PfkA activity, so that more fructose-6-phosphate (F6P) can be converted into glucose-1-phosphate (Glc1P) for α-arbutin synthesis, which can reduce the addition of sucrose and increase the sucrose conversion efficiency. Finally, the pathway of F6P to Glc1P was enhanced by integrating another copy of glucose 6-phosphate isomerase (Pgi) and phosphoglucomutase (PgcA); a high α-arbutin titer (~120 g/L) was obtained. The sucrose conversion rate was increased to 60.4% (mol/mol). In this study, the substrate utilization rate was boosted due to the attenuation of its hydrolysis and the assistance of the intracellular enzymes that converted the side product back into the substrate for α-arbutin synthesis. This strategy provides a new idea for the whole-cell biocatalytic synthesis of other products using sucrose as substrate, especially valuable glycosides.Key points The genes of sucrose metabolic pathway were knocked out to reduce the sucrose consumption. The by-product fructose was reused to synthesize α-arbutin. The optimized whole-cell system improved sucrose conversion by 15.3%.

Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli.

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

Creating an in vivo bifunctional gene expression circuit through an aptamer-based regulatory mechanism for dynamic metabolic engineering in Bacillus subtilis.

Aptamer-based regulatory biosensors can dynamically regulate the expression of target genes in response to ligands and could be used in dynamic metabolic engineering for pathway optimization. However, the existing aptamer-ligand biosensors can only function with non-complementary DNA elements that cannot replicate in growing cells. Here, we construct an aptamer-based synthetic regulatory circuit that can dynamically upregulate and downregulate the expression of target genes in response to the ligand thrombin at transcriptional and translational levels, respectively, and further used this system to dynamically engineer the synthesis of 2'-fucosyllactose (2'-FL) in Bacillus subtilis. First, we demonstrated the binding of ligand molecule thrombin with the aptamer can induce the unwinding of fully complementary double-stranded DNA. Based on this finding, we constructed a bifunctional gene expression regulatory circuit using ligand thrombin-bound aptamers. The expression of the reporter gene ranged from 0.084- to 48.1-fold. Finally, by using the bifunctional regulatory circuit, we dynamically upregulated the expression of key genes fkp and futC and downregulated the expression of gene purR, resulting in the significant increase of 2'-FL titer from 24.7 to 674 mg/L. Compared with the other pathway-specific dynamic engineering systems, here the constructed aptamer-based regulatory circuit is independent of pathways, and can be generally used to fine-tune gene expression in other microbes.

Effect of quercetin and oil water separation system on formation of ß-carboline heterocyclic amines during frying process of braised chicken drumsticks.

The effect of quercetin and oil water separation system on the formation of heterocyclic amines (HAs) was investigated during the frying process of braised chicken drumsticks. The results showed that two ß-carboline HAs (ß-CHAs) were detected in the chicken samples: 9H-pyrido [3,4-b] indole (Norharman) and1-methyl-9H-pyrido [3,4-b] indole (Harman). ß-CHAs content in the chicken samples increased as the cycle times of the frying oil rose (P < 0.05). The addition of quercetin and use of oil water separation system significantly inhibited the formation of Harman (27.3% and 28.2%; P < 0.05) and Norharman (28.7% and 64.1%; P < 0.05), respectively, and the combined use of the two treatments had a better effect (47.0% and 80.2%; P < 0.05). It can be attributed to the lower consumption of ß-CHAs precursors (tryptophan) and reduced generation of the intermediates (carbonyl compounds and 1,2,3,4-Tetrahydro-ß-carboline-3-carboxylic acid). This provides a promising way for reducing ß-CHAs during the frying process of braised chicken products.

The mechanism of Leuconostoc mesenteroides subsp. IMAU:80679 in improving meat color: Myoglobin oxidation inhibition and myoglobin derivatives formation based on multi enzyme-like activities.

The Leuconostoc mesenteroides subsp. IMAU:80679 (LM) was chosen for its superior capability in enhancing redness, and was incubated in a broth system containing metmyoglobin (MetMb) to investigate its mechanisms for color improvement. The a* value of LM group reached its highest level of 52.75 ± 1.04 at 24 h, significantly higher than control of 19.75 ± 0.6 (p < 0.05). The addition of LM could inhibit myoglobin oxidation to some extent. Meanwhile, higher content of nitrosylmyoglobin (NOMb) and Zn-protoporphyrin (Znpp) were observed in LM samples during the whole incubation period. Furthermore, enzymatic activity and encoded genes related to MetMb reduction and pigment formation were determined to explain its possible mechanism on color enhancement. Finally, by extracting crude enzymes and adding them to meat batters, the redness of crude enzyme group was comparable to that achieved with 20 ppm nitrite, providing a potential method on compensating for nitrite/nitrate substitution in meat products.

Zinc protoporphyrin IX generation by Leuconostoc strains isolated from bulged pasteurized vacuum sliced hams.

The colour of meat typically fades as it decays. However, it has been observed that certain vacuum-packaged spoiled hams can maintain a pink colour even when the packaging is bulged. A large amount of Zinc protoporphyrin IX (ZnPP) was found in these hams, compared to fresh red hams or spoiled and grey hams. Combined with high-throughput sequencing and cultural isolation, the potential cultures of Leuconostoc mesenteroides S-13 (LM), Leuconostoc citreum OCLC11 (LC), and Leuconostoc mesenteroides subsp. IMAU:80679 (LS) were selected based on their ability to produce ZnPP. Subsequently, these cultures were introduced into a fermented sausage model to assess their effect on colour conversion. The analysis of absorption and fluorescent spectra showed that Nitrite sausages contained nitrosyl heme pigment, while bacteria-inoculated sausages were predominantly composed of ZnPP. In addition, the a* value of the LS sausage was close to the Nitrite group at the end of fermentation, significantly higher than control, indicating the effect of bacterial metabolism on the redness. Meanwhile, the Ferrochelatase (FECH) activity of LM, LC and LS groups were 140 ± 13, 113 ± 16 and 201 ± 20 U/g sausage, respectively, providing a potential method on compensating for nitrite/nitrate substitution based on the presence of ZnPP in meat products.

Z-scheme CoAl-layereddoublehydroxide/indium vanadate heterojunction for enhanced and highly selective photocatalytic reduction of carbon dioxide to carbon monoxide.

Development of efficient photocatalysts is essential for carbon dioxide (CO2) photocatalytic reduction. In this study, Z-scheme CoAl-layered double hydroxide (LDH)/indium vanadate (InVO4) heterojunction photocatalysts were synthesized using hydrothermal method, and their performance toward CO2 reduction and mechanism were determined. Results of characterizations showed that the CoAl-LDH/InVO4-30 exhibited desired morphology, the most efficient photogenerated carriers separation and charge transfer, and the highest photocurrent response. X-ray photoelectron spectroscopy (XPS) and electron spin resonance (ESR) manifested that charge transfer of the CoAl-LDH/InVO4 conformed to Z-scheme mechanism. The CoAl-LDH/InVO4-30 exhibited the highest carbon monoxide (CO) yield of 174.4 µmol g-1 within 2 h of reaction, which was 2.46 and 9.79 times of pure CoAl-LDH and InVO4, respectively. The CO selectivity was up to nearly 100%. Moreover, in-situ fourier transform infrared spectroscopy (ISFT-IR) demonstrated that bicarbonate (HCO3*) and carboxylate (COOH*) were the main intermediates during the CO2 reduction process, and possible CO2 reduction pathways were proposed. This work provides a reference for construction of Z-scheme LDH-based heterojunctions for efficient CO2 photoreduction.

Constructing a methanol-dependent Bacillus subtilis by engineering the methanol metabolism.

Methanol is a promising green feedstock for producing fuels and chemicals because it is inexpensive, clean, environmentally friendly, and easily prepared. Thus, many studies have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. This study adopted a series of strategies to develop a synthetic methylotrophic Bacillus subtilis that can use methanol as the carbon source, including the heterologous expression of methanol dehydrogenase (Mdh), enhancement of the expressions of 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi), regulation of the expressions of key enzymes at both the translational and transcriptional levels, stabilization of the key enzyme expression through a dual-system for expressing the target genes on both the plasmid and genome, and improvement of the catalytic activity of Mdh with a recycling strategy for NAD+. As a result, the methanol consumption of the synthetic methylotrophic B. subtilis reached 4.09 g/L, with the maximum OD600 showing a 2.21-fold increase compared with the wild-type B. subtilis, which cannot use methanol. We further deleted the phosphoglucose isomerase (Pgi) and added co-substrates to increase the supply of ribulose-5-phosphate (Ru-5-P), and the specific methanol consumption rate increased by an additional 27.54%. Finally, we successfully constructed two strains that cannot grow in M9 medium with xylose or ribose unless methanol is utilized. The strategies used in this study are generally applicable to other studies on synthetic methylotrophy.

Insight into the correlation between microbial diversity and flavor profiles of traditional dry-cured duck from the metabolomic perspective.

The purpose of this work was to reveal the relationship between the microbial diversity and flavor profiles of traditional dry-cured duck from a metabolomic perspective. Enterococcus, Psychrobacter, Macrococcus, Salinivbrio, and Staphylococcus were the dominant bacterial genera, while Trichophyton, Kurtzmaniella, Blumeria, Cladosporium, Lysurus, Aspergillus, Starmerella and Debaryomyces were the dominant fungal genera of dry-cured duck. The results showed that aldehydes, alcohols, furan, and ketone compounds were the main volatile flavor compounds of dry-cured duck. Moreover, the identified metabolites of dry-cured duck were classified and included amino acids, amines, polypeptides, amino acid derivatives, polyols, fatty acids, organic acids, flavonoids and isoflavones. Heatmap analysis was used to illuminate the relationships between the microbial diversity and flavor profiles, as well as metabolites. These results will provide an effective theoretical reference for the standardization and modernization of dry-cured duck production.

Combined Effect of High-Pressure Processing with Spice Extracts on Quality of Low-Salt Sausage during Refrigerated Storage.

The study evaluated the combined effect of high-pressure processing (HPP) and spice extracts on low-salt sausages during refrigerated storage. Physicochemical and microbiological characteristics of the sausages were determined. HPP treatment increased the thiobarbituric acid reactive substances (TBARS) value and the carbonyl content of the samples (p < 0.05), which meant lipid and protein oxidation was accelerated. Adding clove and cinnamon extracts can retard the oxidation caused by HPP (p < 0.05). The pH of the sausages treated with both the spice extracts and HPP maintained a higher pH value during the storage (p > 0.05). Compared with the samples treated with HPP or with the spice extracts alone, the combined treatment observably inhibited the growth of spoilage bacteria (p < 0.05) and improved the microbial community. The results demonstrated that the use of clove and cinnamon extracts in conjunction with HPP improved the storage quality and prolonged the shelf-life of the low-salt sausages. Thus, the combined use of spice extracts and HPP can be developed as a promising way to preserve low-salt meat products.

Improving the functionality of chitosan-based packaging films by crosslinking with nanoencapsulated clove essential oil.

The study aimed to obtain chitosan composite films with gratifying physical and functional properties. First, we developed a Pickering emulsion containing clove essential oil (CEO)-loaded nanoparticles with 1:2 (w/w) zein and sodium caseinate (NaCas). We found that in this ratio, the CEO-loaded zein-NaCas (C/ZN) nanoparticles had smaller particle size, proper polydispersity index (PDI) and zeta potential as well as higher encapsulation efficiency. Then, the acquired C/ZN nanoparticles were incorporated into chitosan film at three levels (0.2%, 0.4% and 0.6%), reducing the water vapor permeability to 4.62 × 10-6 g·s-1·m-1·Pa. Also, the tensile strength and break elongation of chitosan films were increased, reaching 38.67 MPa and 1.56%, respectively. The infrared spectroscopy verified that the intermolecular hydrogen bonds exist between chitosan and C/ZN nanoparticles. The chitosan composite films showed a controlled-release property of CEO in 96 h. Finally, the chitosan composite films showed the improved antibacterial property by creating larger inhibition zones against Escherichia coli (3.29 mm) and Staphylococcus aureus (6.15 mm). In general, we improved the water resistance, light blocking, mechanical strength, controlled-release and antibacterial properties of chitosan film with C/ZN nanoparticles. The current edible antibacterial films have great potential on applications for food preservation and food delivery system.

Fatty acid synthase and hormone-sensitive lipase expression in liver are involved in zinc-alpha2-glycoprotein-induced body fat loss in obese mice.

OBJECTIVE: To explore the effects of zinc-alpha2-glycoprotein (ZAG) on body weight and body fat in high-fat-diet (HFD)-induced obesity in mice and the possible mechanism. METHODS: Thirty-six male mice were fed with standard food (SF) (n = 9) and HFD (n = 27), respectively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) in liver tissue were determined by reverse transcription-polymerase chain reaction. RESULTS: Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice (0.51 +/- 0.10 AU vs. 0.75 +/- 0.07 AU, P < 0.01). Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r = -0.56, P < 0.001), epididymal fat mass (r = -0.67, P < 0.001), percentage of epididymal fat (r = -0.65, P < 0.001), and increased weight (r = -0.57, P < 0.001) in simple SF- and HFD-fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididymal fat. Furthermore, FAS mRNA expression decreased (P < 0.01) and HSL mRNA expression increased (P < 0.001) in the liver in ZAG over-expressing mice. CONCLUSIONS: ZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.

[Activin inhibits the promoter activity of human growth hormone gene in rat pituitary GH3 cells].

The present study was aimed at investigating the effect of activin on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the underlying molecular mechanism. The method of luciferase reporter gene was used. We firstly established a stable GH3 cell line which contains hGH gene promoter (-484 to 30 bp) and luciferase reporter gene by transfecting pGL3-484-Luc2 luciferase expression plasmid into GH3 cells using Lipofectamine transfection reagent. After treating these cells with activin or activin plus various signaling transduction activators, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured. The results showed that activin (5 nmol/L, 50 nmol/L) decreased the secretion and synthesis of GH. The amounts of GH content in GH3 lysate and medium treated with 50 nmol/L activin were 82% and 59% of the control, respectively. Furthermore, activin (5, 50 nmol/L) reduced the luciferase expression in stable GH3 cells, with the expression being 77% and 69% of the control (P<0.001). Among the activators of intracellular signaling transduction pathways, mitogen-activated protein kinases kinase (MAPKK/MEK) activators C(6) ceramide (1 micromol/L) abolished completely the inhibitory effect of activin. Western blot analysis further confirmed the inhibition of phosphorylated MEK in GH3 cells. The inhibitory effect of activin was abrogated following the deletion of the fragment from -132 to -66 bp within the hGH gene promoter. These results indicate that activin decreases the activity of hGH gene promoter in rat pituitary GH3 cells. The intracellular MEK dependent signaling pathway and the promoter sequence that spans the -132 to -66 bp fragment of hGH gene are involved in the inhibitory effect of activin.

[Construction of Zinc-alpha2-glycoprotein expression plasmid and its expression in 3t3-l1 preadipocytes].

OBJECTIVE: To construct mouse Zinc-alpha2-glycoprotein (mZAG) eucaryotic expression plasmid and identify its expression in 3T3-L1 preadipocytes. METHODS: The total RNA from mouse liver tissue was extracted. The reverse-transcript(RT)-PCR method was used to amplify the complete domain sequence of mZAG, and the confirmed PCR products was inserted into expression plasmid by DNA ligation. The mZAG expression plasmids with various concentrations (0, 0.4, 0.8, and 1.6 microg) were transfected into 3T3-L1 preadipocytes, and ZAG expression in mRNA and protein level was determined by real-time fluorescence quantitative PCR and Western blot, respectively. RESULTS: DNA sequencing confirmed the right sequence of mZAG expression plasmid pcDNA3.1(-)-mZAG. After the mZAG expression plasmid with different concentrations were transfected into 3T3-L1 preadipocytes, mZAG mRNA level significantly increased and reached 2.58 folds (P=0.002), 3.67 folds (P=0.000 and 5.19 folds (P=0.001) of that in the control group (no mZAG transfection). mZAG protein level also significantly increased and reached 2.75 folds of that in the control group (P=0.017). Treating 3T3-L1 cells with small interfering RNA (siRNA) sequence siRNA 1 and siRNA 4 resulted in a decrease of mZAG mRNA to 49% and 41% of those in the control group(no siRNA sequence transfection) (P=0.002P=0.000)and a decrease of mZAG protein to 55% and 62% of that in the control group (P=0.004,P=0.025). CONCLUSIONS: mZAG expression plasmid pcDNA3.1(-)-mZAG was successfully established in this study. This plasmid can be well expressed in 3T3-L1 preadipocytes. siRNA 1 and siRNA 4 can effectively inhibit the expression of mZAG in these cells.

[Effect of bodyweight on the onset of puberty of female children and adolescents].

OBJECTIVE: To investigate the possible effects and roles of bodyweight on the puberty onset in adolescent girls. METHODS: Totally 288 Chinese female children and adolescent girls aged 5 to 16 were followed up yearly for four consecutive years. The height, bodyweight, fat percentage, sexual characteristics, and the serum levels of leptin and insulin-like growth factor-1 (IGF-1) were studied to analyze the influential factors of puberty onset and age of menarche. RESULTS: The serum level of leptin elevated significantly from age 13 [(9.23 +/- 1.25) microg/L] and reached peak at age 16 [(13.19 +/- 1.45) microg/L]. IGF-1 significantly correlated with the timing of puberty onset (r = 0.292, P = 0.016). BMI and fat percentage had no significant effects on the onset of puberty, but were negatively correlated with the age of menarche (r = -0.323, P = 0.037, r = -0.298, P = 0.038 respectively). CONCLUSION: Bodyweight may have effect on puberty onset in female adolescents.

Biocatalytic synthesis of lactosucrose using a recombinant thermostable ß-fructofuranosidase from Arthrobacter sp. 10138.

As a prebiotics, lactosucrose plays an important role in maintaining human gastrointestinal homeostasis. In this study, a thermostable enzyme from Arthrobacter sp. 10138 was screened from six ß-fructofuranosidase-producing strains for the lactosucrose production and the coding gene was heterologously expressed in Escherichia coli for efficient expression. Recombinant ß-fructofuranosidase was purified and biochemically characterized by MALDI-TOFMS spectrometry. The transfructosylation product by this recombinant enzyme was determined to be lactosucrose rather than other oligosaccharides or polysaccharides by HPLC and LC-MS. Efficient extracellular secretion of ß-fructofuranosidase was achieved by the optimization of signal peptide and induction conditions. It was found that with the signal peptide torT, the highest extracellular activity reached 111.01 U/mL, which was 38.4-fold higher than that with the OmpA signal peptide. Under the optimal conditions (pH 6.0, temperature 50°C, enzyme amount 40 µg/ml, sucrose 150 g/L and lactose 150 g/L), 109 g/L lactosucrose was produced with a molar conversion ratio of 49.3%. Here the thermostable ß-fructofuranosidase from Arthrobacter sp. 10138 can be used for efficient synthesis of lactosucrose, and this provides a good startpoint for the industrial production of lactosucrose in the future.

A fluorescence polarization aptasensor coupled with polymerase chain reaction and streptavidin for chloramphenicol detection.

The authors describe a fluorescence polarization (FP) aptasensor based on the polymerase chain reaction (PCR) and streptavidin as dual FP amplifiers to detect chloramphenicol residues in food. Briefly, label-free aptamer was incubated with chloramphenicol and the aptamer-chloramphenicol conjugate was used as a template. Subsequently, the FAM-labeled forward primer and biotin-labeled reverse primer were added for PCR to amplify the template and the FAM-labeled primer. The molecular weight of FAM-labeled primer increased rapidly and the corresponding FP also enhanced. Finally, with the introduction of streptavidin, the PCR products and streptavidin were combined with the biotin-streptavidin interactions, resulting in much larger molecular weight. Thus, a dual amplified FP signal was obtained. Under optimal conditions, we were able to achieve a wide linear detection range of 0.001-200 nM. In addition, the designed strategy was applied to detect chloramphenicol in honey samples with high accuracy. Moreover, the strategy can be easily extended to detect other small molecules by changing the corresponding aptamers, which provide a promising avenue for the detection of small molecules by FP.

Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2'-Fucosyllactose Synthesis in Bacillus subtilis.

Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants' intestinal health and immune systems. 2'-Fucosyllactose (2'-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2'-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2'-FL producer by engineering the substrate transport and cofactor guanosine 5'-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2'-FL precursor guanosine 5'-diphosphate-l-fucose (GDP-l-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2'-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2'-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous ß-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2'-FL and GDP-l-fucose was improved by fine-tuning the expression of cofactor guanosine 5'-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykfN, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2'-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2'-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2'-FL production in the future.

Mek and p38 MAPK-dependant pathways are involoved in the positive effect of interleukin-6 on human growth hormone gene expression in rat MtT/S somatotroph cells.

OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S. METHODS: The plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed. Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3. 1(+) with DMRIE-C transfection reagent After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways, the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism. RESULTS: The 10(3) U/mL IL-6 stimulated GH secretion and synthesis, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-6. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6. The results showed that the stimulatory effect of IL-6 was abolished following deletion of the -196 to - 132 bp fragment. CONCLUSIONS: IL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells. The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the - 196 to - 132 bp of the gene, but may be unlinked with Pit-1 protein.