Enhanced Biosynthesis of d-Allulose from a d-Xylose-Methanol Mixture and Its Self-Inductive Detoxification by Using Antisense RNAs in Escherichia coli.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

Transporter mining and metabolic engineering of Escherichia coli for high-level D-allulose production from D-fructose by thermo-swing fermentation.

D-Allulose is an ultra-low-calorie sweetener with broad market prospects in the fields of food, beverage, health care, and medicine. The fermentative synthesis of D-allulose is still under development and considered as an ideal route to replace enzymatic approaches for large-scale production of D-allulose in the future. Generally, D-allulose is synthesized from D-fructose through Izumoring epimerization. This biological reaction is reversible, and a high temperature is beneficial to the conversion of D-fructose. Mild cell growth conditions seriously limit the efficiency of producing D-allulose through fermentation. FryABC permease was identified to be responsible for the transport of D-allulose in Escherichia coli by comparative transcriptomic analysis. A cell factory was then developed by expression of ptsG-F, dpe, and deletion of fryA, fruA, manXYZ, mak, and galE. The results show that the newly engineered E. coli was able to produce 32.33 ± 1.33 g L-1 of D-allulose through a unique thermo-swing fermentation process, with a yield of 0.94 ± 0.01 g g-1 on D-fructose.

Engineering of Escherichia coli for D-allose fermentative synthesis from D-glucose through izumoring cascade epimerization.

D-Allose is a potential alternative to sucrose in the food industries and a useful additive for the healthcare products in the future. At present, the methods for large-scale production of D-allose are still under investigation, most of which are based on in vitro enzyme-catalyzed Izumoring epimerization. In contrast, fermentative synthesis of D-allose has never been reported, probably due to the absence of available natural microorganisms. In this work, we co-expressed D-galactose: H+ symporter (GalP), D-glucose isomerase (DGI), D-allulose 3-epimerase (DAE), and ribose-5-phosphate isomerase (RPI) in Escherichia coli, thereby constructing an in vivo Izumoring pathway for yielding D-allose from D-glucose. The carbon fluxes and carbon catabolite repression (CCR) were rationally regulated by knockout of FruA, PtsG, Glk, Mak, PfkA, and PfkB involved in the pathways capable of phosphorylating D-fructose, D-glucose, and fructose-6-phosphate. Moreover, the native D-allose transporter was damaged by inactivation of AlsB, thus driving the reversible Izumoring reactions towards the target product. Fermentation was performed in the M9 medium supplemented with glycerol as a carbon source and D-glucose as a substrate. The results show that the engineered E. coli cell factory was able to produce approximately 127.35 mg/L of D-allose after 84 h. Our achievements in the fermentative production of D-allose in this work may further promote the green manufacturing of rare sugars.

Methanol-Dependent Carbon Fixation for Irreversible Synthesis of d-Allulose from d-Xylose by Engineered Escherichia coli.

d-Allulose is a rare hexose with great application potential, owing to its moderate sweetness, low energy, and unique physiological functions. The current strategies for d-allulose production, whether industrialized or under development, utilize six-carbon sugars such as d-glucose or d-fructose as a substrate and are usually based on the principle of reversible Izumoring epimerization. In this work, we designed a novel route that coupled the pathways of methanol reduction, pentose phosphate (PP), ribulose monophosphate (RuMP), and allulose monophosphate (AuMP) for Escherichia coli to irreversibly synthesize d-allulose from d-xylose and methanol. After improving the expression of AlsE by SUMO fusion and regulating the carbon fluxes by knockout of FrmRAB, RpiA, PfkA, and PfkB, the titer of d-allulose in fed-batch fermentation reached ≈70.7 mM, with a yield of ≈0.471 mM/mM on d-xylose or ≈0.512 mM/mM on methanol.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Intelligent self-control of carbon metabolic flux in SecY-engineered Escherichia coli for xylitol biosynthesis from xylose-glucose mixtures.

Xylitol is a salutary sugar substitute that has been widely used in the food, pharmaceutical, and chemical industries. Co-fermentation of xylose and glucose by metabolically engineered cell factories is a promising alternative to chemical hydrogenation of xylose for commercial production of xylitol. Here, we engineered a mutant of SecY protein-translocation channel (SecY [ΔP]) in xylitol-producing Escherichia coli JM109 (DE3) as a passageway for xylose uptake. It was found that SecY (ΔP) channel could rapidly transport xylose without being interfered by XylB-catalyzed synthesis of xylitol-phosphate, which is impossible for native XylFGH and XylE transporters. More importantly, with the coaction of SecY (ΔP) channel and carbon catabolite repression (CCR), the flux of xylose to the pentose phosphate (PP) pathway and the xylitol synthesis pathway in E. coli could be automatically controlled in response to glucose, thereby ensuring that the mutant cells were able to fully utilize sugars with high xylitol yields. The E. coli cell factory developed in this study has been proven to be applicable to a broad range of xylose-glucose mixtures, which is conducive to simplifying the mixed-sugar fermentation process for efficient and economical production of xylitol.

Engineering Escherichia coli for d-Allulose Production from d-Fructose by Fermentation.

d-Allulose is considered an ideal alternative to sucrose and has shown tremendous application potential in many fields. Recently, most efforts on production of d-allulose have focused on in vitro enzyme-catalyzed epimerization of cheap hexoses. Here, we proposed an approach to efficiently produce d-allulose through fermentation using metabolically engineered Escherichia coli JM109 (DE3), in which a SecY (ΔP) channel and a d-allulose 3-epimerase (DPEase) were co-expressed, ensuring that d-fructose could be transported in its nonphosphorylated form and then converted into d-allulose by cells. Further deletion of fruA, manXYZ, mak, galE, and fruK and the use of Ni2+ in a medium limited the carbon flux flowing into the byproduct-generating pathways and the Embden-Meyerhof-Parnas (EMP) pathway, achieving a ≈ 0.95 g/g yield of d-allulose on d-fructose using E. coli (DPEase, SecY [ΔP], ΔFruA, ΔManXYZ, ΔMak, ΔGalE, ΔFruK) and 8 µM Ni2+. In fed-batch fermentation, the titer of d-allulose reached ≈23.3 g/L.

Relationship between polymorphisms of the lipid metabolism-related gene PLA2G16 and risk of colorectal cancer in the Chinese population.

This study aimed to investigate the relationship between polymorphisms in the lipid metabolism-related gene PLA2G16 encoding Group XVI phospholipase A2 and the risk of colorectal cancer (CRC) in the Chinese population. A total of 185 patients with CRC and 313 healthy controls were enrolled. Thirteen single nucleotide polymorphisms (SNPs) of PLA2G16 were genotyped with SNPscan™. Linkage disequilibrium and haplotypes were analysed using Haploview software. Multivariate logistic regression was used to determine the association between the various genotypes and CRC risk. We identified five PLA2G16 SNPs (rs11600655, rs3809072, rs3809073, rs640908 and rs66475048) that were associated with CRC risk after adjusting for age, sex and body mass index. Two haplotypes (CTC and GGA) of rs11600655, rs3809073 and rs3809072, were relevant to CRC risk. The rs11600655 polymorphism was also associated with lymph node metastasis and CRC staging, while rs3809073 and rs3809072 may affect transcriptional regulation of PLA2G16 by altering transcription factor binding. These findings suggest that PLA2G16 polymorphisms-especially CTC and GGA haplotypes-increase CRC susceptibility. Importantly, we showed that the rs11600655 CC, rs640908 CT and rs66475048 GA genotypes are independent risk factors for CRC in the Chinese population.

Clinical application and importance of one-step human CYP2C19 genotype detection.

BACKGROUND: To directly achieve cytochrome P450 2C19 gene ( CYP2C19) classification using one-step real-time fluorescent PCR detection and to verify the capabilities of this method with nucleic acid extracted from whole blood samples. METHODS: A human CYP2C19 genotyping kit based on one-step real-time fluorescent PCR detection was used to analyze whole blood or genomic DNA samples. This method was compared with pyrosequencing and another quantitative (q)PCR kit for its accuracy, repeatability, detection range analysis, sensitivity, specificity, and anti-interference analysis. RESULTS: The one-step real-time PCR method achieved a 100% accuracy rate compared with pyrosequencing and the other qPCR kit. When detecting different concentrations of known genes, concentrations of each sample ranging from 0.2 to 125 ng/µL could be correctly detected. The genotypes of samples treated with anticoagulants, including EDTA and sodium citrate, and chyle blood samples could be correctly detected. CONCLUSION: The one-step detection method demonstrated high accuracy and a wide detection range. It also had high levels of repeatability, sensitivity, and specificity for the assessment of genomic DNA test samples.

Gene expression profiles in an hepatitis B virus transfected hepatoblastoma cell line and differentially regulated gene expression by interferon-alpha.

AIM: To study interactions between hepatitis B virus (HBV) and interferon-alpha in liver- derived cells. METHODS: mRNAs were separately isolated from an HBV-transfected cell line (HepG(2)2.2.15) and its parental cell line (HepG(2)) pre- and post-interferon-alpha (IFN-alpha) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis. RESULTS: Compared to HepG(2) cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG(2)2.2.15 cells prior to IFN-alpha treatment. After interferon-alpha treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG(2)2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-alpha treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions. CONCLUSION: The up-regulated genes in HepG(2)2.2.15 cell line suggested that under IFN-alpha treatment, these repressed cellular genes in HBV infected hepatocytes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.

Analysis of gene expression in hepatitis B virus transfected cell line induced by interferon.

Infection of hepatitis B virus (HBV) continues to be a significant health problem. alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma) have been proven to be effective in inhibiting HBV replication. To study the global effect of HBV persistent existence on IFN induced cellular gene expression, cDNA microarrays dotted with 14 112 human genes were used to examine the transcriptional changes between an HBV DNA transfected cell line (HepG2.2.15) and its parental cell line (HepG2) after the treatment of IFN-alpha or IFN-gamma for 6 h. The results showed that many genes related to cell cycle, proliferation, apoptosis and new ESTs were regulated by IFN-alpha and/or IFN-gamma. Many genes involved in kinase and signal transduction, transcription regulation, antigen presentation and processing were differentially regulated between these two cell lines post IFN-alpha or IFN-gamma treatment. Interestingly, several IFN-differentially regulated genes, such as MyD88 and Diubiquitin, were found to inhibit HBV gene expression, and MyD88 was proved to inhibit HBV replication. Taken together, our results revealed the global effects of HBV persistent existence on IFN-induced cellular gene expression. The novel antiviral genes identified by microarray could be potentially developed as new anti-HBV drugs or for novel therapies.