Identification of BMAL1-Regulated circadian genes in mouse liver and their potential association with hepatocellular carcinoma: Gys2 and Upp2 as promising candidates.

Identification and functional analysis of key genes regulated by the circadian clock system will provide a comprehensive understanding of the underlying mechanisms through which circadian clock disruption impairs the health of living organisms. The initial phase involved bioinformatics analysis, drawing insights from three RNA-seq datasets (GSE184303, GSE114400, and GSE199061) derived from wild-type mouse liver tissues, which encompassed six distinct time points across a day. As expected, 536 overlapping genes exhibiting rhythmic expression patterns were identified. By intersecting these genes with differentially expressed genes (DEGs) originating from liver RNA-seq data at two representative time points (circadian time, CT: CT2 and CT14) in global Bmal1 knockout mice (Bmal1-/-), hepatocyte-specific Bmal1 knockout mice (L-Bmal1-/-), and their corresponding control groups, 80 genes potentially regulated by BMAL1 (referred to as BMAL1-regulated genes, BRGs) were identified. These genes were significantly enriched in glycolipid metabolism, immune response, and tumorigenesis pathways. Eight BRGs (Nr1d1, Cry1, Gys2, Homer2, Serpina6, Slc2a2, Nmrk1, and Upp2) were selected to validate their expression patterns in both control and L-Bmal1-/- mice livers over 24 h. Real-time quantitative polymerase chain reaction results demonstrated a comprehensive loss of rhythmic expression patterns in the eight selected BRGs in L-Bmal1-/- mice, in contrast to the discernible rhythmic patterns observed in the livers of control mice. Additionally, significant reductions in the expression levels of these selected BRGs, excluding Cry1, were also observed in L-Bmal1-/- mice livers. Chromatin immunoprecipitation (ChIP)-seq (GSE13505 and GSE39860) and JASPAR analyses validated the rhythmic binding of BMAL1 to the promoter and intron regions of these genes. Moreover, the progression of conditions, from basic steatosis to non-alcoholic fatty liver disease, and eventual malignancy, demonstrated a continuous gradual decline in Bmal1 transcripts in the human liver. Combining the aforementioned BRGs with DEGs derived from human liver cancer datasets identified Gys2 and Upp2 as potential node genes bridging the circadian clock system and hepatocellular carcinoma (HCC). In addition, CCK8 and wound healing assays demonstrated that the overexpression of human GYS2 and UPP2 proteins inhibited the proliferation and migration of HepG2 cells, accompanied by elevated expression of p53, a tumor suppressor protein. In summary, this study systematically identified rhythmic genes in the mouse liver, and a subset of circadian genes potentially regulated by BMAL1. Two circadian genes, Gys2 and Upp2, have been proposed and validated as potential candidates for advancing the prevention and treatment of HCC.

Short-term transplantation effect of a tissue-engineered meniscus constructed using drilled allogeneic acellular meniscus and BMSCs.

During the construction of tissue-engineered meniscus, the low porosity of extracellular matrix restricts the flow of nutrient solution and the migration and proliferation of cells, thus affecting the tissue remodeling after transplantation. In this study, the canine allogeneic meniscus was drilled first and then decellularized. The drilled tissue-engineered menisci (Drilled Allogeneic Acellular Meniscus + Bone Marrow Mesenchymal Stem Cells, BMSCs) were transplanted into the knee joints of model dogs. On the basis of ensuring the mechanical properties, the number of the porosity and the cells implanted in allogeneic acellular meniscus was significantly increased. The expression levels of glycosaminoglycans and type II collagen in the drilled tissue-engineered meniscus were also improved. It was determined that the animals in the experimental group recovered well-compared with those in the control group. The graft surface was covered with new cartilage, the retraction degree was small, and the tissue remodeling was good. The surface wear of the femoral condyle and tibial plateau cartilage was light. The results of this study showed that increasing the porosity of allogeneic meniscus by drilling could not only maintain the mechanical properties of the meniscus and increase the number of implanted cells but also promote cell proliferation and differentiation. After transplantation, the drilled tissue-engineered meniscus provided a good remodeling effect in vivo and played a positive role in repairing meniscal injury, protecting articular cartilage and restoring knee joint function.

[Advances in enzymatic production of L-homophenylalanine].

L-homophenylalanine (L-HPA) is an important non-natural amino acid that has been used as a key intermediate for the synthesis of Puli drugs for the treatment of hypertension. At present, L-HPA is synthesized using chemical methods, which has the disadvantages of expensive raw materials, tedious steps and serious pollution. Therefore, researchers have conducted in-depth research on the enzymatic production of L-HPA. This review summarizes the research progress on the enzymatic synthesis of L-HPA, including the dehydrogenase process, the transaminase process, the hydantoinase process, and the decarboxylase process, with the hope to facilitate the industrial production of L-HPA.

Autophagy activation attenuates the circadian clock oscillators in U2OS cells via the ATG5 pathway.

The circadian clock and autophagy are essential biological mechanisms involved in regulating many physiological processes. Accumulating evidence has revealed that autophagic activity is regulated by the circadian clock system. However, whether autophagy regulates the circadian clock system remains unclear. In this study, rapamycin and AICAR, two classical activators of autophagy, were used to create autophagy activation models in BMAL1-dLuc U2OS cell line. The results showed that the mRNA expression of MAP1LC3B and ATG5 were significantly upregulated after autophagy activation, whereas the mRNA expression of circadian clock genes (BMAL1, PER2, REV-ERBα, and DBP) were significantly decreased. Consistent with these data, the relative ratio of LC3-II/LC3-I and the protein level of ATG5 were increased after rapamycin or AICAR treatment. In contrast, BMAL1 and REV-ERBα levels were decreased. Notably, the mRNA expression of circadian clock genes (BMAL1, PER2, REV-ERBα, and DBP) and autophagy-related genes (MAP1LC3B and ATG5) showed rhythmic expression patterns in both untreated and rapamycin/AICAR-treated U2OS cells. Moreover, the autophagy inhibitor 3-methyladenine partially reversed the inhibitory effects of autophagy on circadian clock genes expression and BMAL1-Luc oscillations. Another critical finding was that ATG5 knockout alleviates the inhibitory effect of rapamycin-mediated autophagy activation on the circadian clock oscillators in U2OS cells. Collectively, our data indicate that autophagy activation attenuates the circadian clock oscillators in U2OS cells via the ATG5 pathway.

Age-related endoplasmic reticulum stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

Senile animals exhibit a high risk of elevated endoplasmic reticulum (ER) stress, attenuated circadian clock, and impaired steroidogenesis in testes. However, how these three processes are intertwined in mouse Leydig cells remains unclear. In this study, a mouse model of aging and hydrogen peroxide (H2O2)-induced senescent TM3 Leydig cells were used to dissect the connections among ER stress, circadian oscillators, and steroidogenesis in Leydig cells. Additionally, thapsigargin (Tg, 60 nM)/tunicamycin (Tm, 60 ng/mL)-induced ER stress were established to investigate the underlying mechanisms by which ER stress regulated testosterone synthesis via circadian clock-related signaling pathways in TM3 cells and primary Leydig cells. Elevated ER stress, attenuated circadian clock, and diminished steroidogenesis were detected in the testes of aged mice (24-month-old) and H2O2-induced (200 µM) senescent TM3 cells in comparison with their control groups. Tg/Tm-induced ER stress reduced the transcription of the circadian clock and steroidogenic genes in TM3 cells and LH-treated (100 ng/mL) primary Leydig cells. Furthermore, 4-phenylbutyric acid (4-PBA, 1 µM), an inhibitor of ER stress, alleviated the inhibitory effect of Tg-mediated ER stress on Per2:Luc oscillations in primary Leydig cells isolated from mPer2Luc knock-in mice, and attenuated the repressive effect of H2O2-induced or Tg-mediated ER stress on the transcription of circadian clock and steroidogenic genes expression and testosterone synthesis in TM3 cells. Collectively, these data indicate that age-related ER stress represses testosterone synthesis via attenuation of the circadian clock in Leydig cells.

Efficient Production of L-Homophenylalanine by Enzymatic-Chemical Cascade Catalysis.

L-Homophenylalanine (L-HPA) is a vital building block for the synthesis of numerous chiral drugs. However, the high cost of starting materials limits the industrial production of L-HPA. In this study, an enzymatic-spontaneous chemical cascade route for L-HPA production was designed based on retrosynthetic analysis. This route, using simple benzaldehyde and pyruvate as starting materials, is extremely cost-effective. The enzymes were screened and further assembled in E. coli, and TipheDH was identified as the rate-limiting enzyme. Therefore, TipheDH was engineered to improve its specific activity (by 82 %) and expression level (by 254 %), thus generating the best strain (W14). W14 exhibited the optimum enzyme activity ratio (1.7 : 1.1 : 1 : 1.8) and demonstrated production of 100.9 g L-1 of L-HPA (with 94 % conversion, >99 % ee) in a 5-L reactor. This route effectively exploits the power of cascades and offers insight into avenues for synthesizing other valuable chemicals from inexpensive building blocks.

Transcriptional Feedback Loops in the Caprine Circadian Clock System.

The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

The Roles of Different Multigene Combinations of Pdx1, Ngn3, Sox9, Pax4, and Nkx2.2 in the Reprogramming of Canine ADSCs Into IPCs.

Adipose-derived mesenchymal stem cells (ADSCs) are ideal sources for the treatment of diabetes, and the differentiation of ADSCs into insulin-producing cells (IPCs) through transfection of exogenous regulatory genes in vitro has been studied in depth. The differentiation of ADSCs is strictly regulated by a variety of transcription factors such as Pdx1, Ngn3, Pax4, Nkx2.2, and Sox9. However, whether these genes can coordinately regulate the differentiation of ADSCs into IPCs is still unknown. In this study, five multigene coexpressing adenovirus vectors (pAdTrack-Pdx1-Ngn3-AdEasy, pAdTrack-Pdx1-Ngn3-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Pax4-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Nkx2.2-Sox9-AdEasy, and pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) were constructed, and then the stocks of the packaged adenoviruses were used to infect the canine ADSCs (cADSCs). Based on results of morphological observation, dithizone staining, sugar-stimulated insulin secretion test, cellular insulin immunofluorescence assays, and the detection of pancreatic ß-cell development-related genes in the induced cells, the best induction combination (pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) was identified after comparative screening. This study provides a theoretical reference and an experimental basis for further research on stem cell replacement therapy for diabetes.

Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5.

Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes (Bmal1, Per2, Nr1d1, and Dbp) and an autophagy-related gene (Atg5) exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of Bmal1, Per2, and Dbp in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, Nr1d1 knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of Bmal1-/- mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited Atg5 transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.

Glyphosate exposure attenuates testosterone synthesis via NR1D1 inhibition of StAR expression in mouse Leydig cells.

Glyphosate is a broad-spectrum herbicide that impairs testosterone synthesis in mammals. Leydig cells (LCs), the primary producers of testosterone, demonstrate rhythmic expression of circadian clock genes both in vivo and in vitro. The nuclear receptor NR1D1 is an important clock component that constitutes the subsidiary transcriptional/translational loop in the circadian clock system. Nr1d1 deficiency resulted in diminished fertility in both male and female mice. However, whether NR1D1 is involved in the glyphosate-mediated inhibition of testosterone synthesis in LCs remains unclear. Here, the involvement of NR1D1 in glyphosate-mediated inhibition of testosterone synthesis was investigated both in vitro and in vivo. Glyphosate exposure of TM3 cells significantly increased Nr1d1 mRNA levels, but decreased Bmal1, Per2, StAR, Cyp11a1, and Cyp17a1 mRNA levels. Western blotting confirmed elevated NR1D1 and reduced StAR protein levels following glyphosate exposure. Glyphosate exposure also reduced testosterone production in TM3 cells. In primary LCs, glyphosate exposure also upregulated Nr1d1 mRNA levels and downregulated the mRNA levels of other clock genes (Bmal1 and Per2) and steroidogenic genes (StAR, Cyp17a1, Cyp11a1, and Hsd3b2), and inhibited testosterone synthesis. Moreover, glyphosate exposure significantly reduced the amplitude and shortened the period of PER2::LUCIFERASE oscillations in primary LCs isolated from mPer2Luciferase knock-in mice. Four weeks of oral glyphosate upregulated NR1D1 at both the mRNA and protein levels in mouse testes, and this was accompanied by a reduction in StAR expression. Notably, serum testosterone levels were also drastically reduced in mice treated with glyphosate. Moreover, dual-luciferase reporter and EMSA assays revealed that in TM3 cells NR1D1 inhibits the expression of StAR by binding to a canonical RORE element present within its promoter. Together, these data demonstrate that glyphosate perturbs testosterone synthesis via NR1D1 mediated inhibition of StAR expression in mouse LCs. These findings extend our understanding of how glyphosate impairs male fertility.

Retracted: Cell Fate and Tissue Remodeling in Canine Urethral Repair Using a Bone Marrow Mesenchymal Stem Cell+Endothelial Progenitor Cell Amniotic Patch.

The Editors of Tissue Engineering: Part A retract the article entitled, "Cell Fate and Tissue Remodeling in Canine Urethral Repair Using a Bone Marrow Mesenchymal Stem Cell+Endothelial Progenitor Cell Amniotic Patch," by Wenxin Zhang, Xin Zhang, Yihua Zhang, Xinke Zhang, Tong Zou, Wen Zhao, Yangou Lv, Jinglu Wang, Pengxiu Dai, Hao Cui, Yi Zhang, Dengke Gao, Chenmei Ruan, and Xia Zhang (epub ahead of print September 21, 2020; DOI: http://doi.org/10.1089/ten.tea.2020.0129). After the online publication of the article, the authors have indicated that they "feel that we have not yet studied our work completely and some new great results are discovered. So after carefully thinking, we are going to rearrange this manuscript and try to give more precise model. [sic]" The authors have not explained what those expected results will be, so it remains unclear the direction their work is headed. The authors also indicated that they plan to submit an updated version of the paper to Tissue Engineering in the future. Upon submission the new manuscript will undergo rigorous peer review, and there is no guarantee of acceptance.

Parathyroid hormone (1-34) promotes the effects of 3D printed scaffold-seeded bone marrow mesenchymal stem cells on meniscus regeneration.

BACKGROUND: Cell-based tissue engineering represents a promising management for meniscus repair and regeneration. The present study aimed to investigate whether the injection of parathyroid hormone (PTH) (1-34) could promote the regeneration and chondroprotection of 3D printed scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in a canine total meniscal meniscectomy model. METHODS: 3D printed poly(e-caprolactone) scaffold seeded with BMSCs was cultured in vitro, and the effects of in vitro culture time on cell growth and matrix synthesis of the BMSCs-scaffold construct were evaluated by microscopic observation and cartilage matrix content detection at 7, 14, 21, and 28 days. After that, the tissue-engineered meniscus based on BMSCs-scaffold cultured for the appropriate culture time was selected for in vivo implantation. Sixteen dogs were randomly divided into four groups: PTH + BMSCs-scaffold, BMSCs-scaffold, total meniscectomy, and sham operation. The regeneration of the implanted tissue and the degeneration of articular cartilage were assessed by gross, histological, and immunohistochemical analysis at 12 weeks postoperatively. RESULTS: In vitro study showed that the glycosaminoglycan (GAG)/DNA ratio and the expression of collagen type II (Col2) were significantly higher on day 21 as compared to the other time points. In vivo study showed that, compared with the BMSCs-scaffold group, the PTH + BMSCs-scaffold group showed better regeneration of the implanted tissue and greater similarity to native meniscus concerning gross appearance, cell composition, and cartilage extracellular matrix deposition. This group also showed less expression of terminal differentiation markers of BMSC chondrogenesis as well as lower cartilage degeneration with less damage on the knee cartilage surface, higher expression of Col2, and lower expression of degeneration markers. CONCLUSIONS: Our results demonstrated that PTH (1-34) promotes the regenerative and chondroprotective effects of the BMSCs-3D printed meniscal scaffold in a canine model, and thus, their combination could be a promising strategy for meniscus tissue engineering.

Genome-Wide Analysis Reveals Changes in Long Noncoding RNAs in the Differentiation of Canine BMSCs into Insulin-Producing Cells.

Long noncoding RNAs (lncRNAs) have been extensively explored over the past decade, including mice and humans. However, their impact on the transdifferentiation of canine bone marrow mesenchymal stem cells (cBMSCs) into insulin-producing cells (IPCs) is largely unknown. In this study, we used a three-step induction procedure to induce cBMSCs into IPCs, and samples (two biological replicates each) were obtained after each step; the samples consisted of "BMSCs" (B), "stage 1" (S1), "stage 2" (S2), "stage 3" (S3), and "islets" (I). After sequencing, 15,091 lncRNAs were identified, and we screened 110, 41, 23, and 686 differentially expressed lncRNAs (padjusted < 0.05) in B vs. S1, S1 vs. S2, S2 vs. S3, and I vs. S3 pairwise comparisons, respectively. In lncRNA target prediction, there were 166,623 colocalized targets and 2,976,362 correlated targets. Gene Ontology (GO) analysis showed that binding represented the main molecular functions of both the cis- and trans-modes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the insulin signaling pathway, Rap1 signaling pathway, tight junctions, MAPK signaling pathway, and cell cycle were enriched for these relative genes. The expression of lncRNAs was verified using qRT-PCR. This study provides a lncRNA catalog for future research concerning the mechanism of the transdifferentiation of cBMSCs into IPCs.

Biochemical characterization and structural analysis of ulvan lyase from marine Alteromonas sp. reveals the basis for its salt tolerance.

Marine macroalgae have gained considerable attention as renewable biomass sources. Ulvan is a water-soluble anionic polysaccharide, and its depolymerization into fermentable monosaccharides has great potential for the production of bioethanol or high-value food additives. Ulvan lyase from Alteromonas sp. (AsPL) utilizes a ß-elimination mechanism to cleave the glycosidic bond between rhamnose 3-sulfate and glucuronic acid, forming an unsaturated uronic acid at the non-reducing end. AsPL was active in the temperature range of 30-50 °C and pH values ranging from 7.5 to 9.5. Furthermore, AsPL was found to be halophilic, showing high activity and stability in the presence of up to 2.5 M NaCl. The apparent Km and kcat values of AsPL are 3.19 ±â€¯0.37 mg mL-1 and 4.19 ±â€¯0.21 s-1, respectively. Crystal structure analysis revealed that AsPL adopts a ß-propeller fold with four anti-parallel ß-strands in each of the seven propeller blades. The acid residues at the protein surface and two Ca2+ coordination sites contribute to its salt tolerance. The research on ulvan lyase has potential commercial value in the utilization of algal resources for biofuel production to relieve the environmental burden of petrochemicals.

Redesign of a novel D-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of D-allulose.

BACKGROUND: A novel D-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a D-allulose 3-epimerase family enzyme based on its high specificity for D-allulose. It usually converts both D-fructose and D-tagatose to respectively D-allulose and D-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. RESULTS: SaDAE showed a high activity on D-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s-1 mM-1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward D-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. CONCLUSIONS: We redesigned SaDAE for thermostability and biocatalytic production of D-allulose. The research will aid the development of industrial biocatalysts for D-allulose.

Biochemical characterization and biocatalytic application of a novel d-tagatose 3-epimerase from Sinorhizobium sp.

Sinorhizobium sp. d-tagatose 3-epimerase (sDTE) catalyzes the conversion of d-tagatose to d-sorbose. It also recognizes d-fructose as a substrate for d-allulose production. The optimal temperature and pH of the purified sDTE was 50 °C and 8.0, respectively. Based on the sDTE homologous model, Glu154, Asp187, Gln213, and Glu248, form a hydrogen bond network with the active-site Mn2+ and constitute the catalytic tetrad. The amino acid residues around O-1, -2, and -3 atoms of the substrates (d-tagatose/d-fructose) are strictly conserved and thus likely regulate the catalytic reaction. However, the residues at O-4, -5, and -6, being responsible for the substrate-binding, are different. In particular, Arg65 and Met9 were found to form a unique interaction with O-4 of d-fructose and d-tagatose. The whole cells with recombinant sDTE showed a higher bioconversion rate of 42.5% in a fed-batch bioconversion using d-fructose as a substrate, corresponding to a production of 476 g L-1d-allulose. These results suggest that sDTE is a potential industrial biocatalyst for the production of d-allulose in fed-batch mode.

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates.

BACKGROUND: Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. RESULTS: Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). CONCLUSIONS: The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

A novel l-leucine 5-hydroxylase from Nostoc piscinale unravels unexpected sulfoxidation activity toward l-methionine.

Hydroxy amino acids are produced by Fe(II)/αKG-dependent dioxygenases and used widely as medicinal intermediates for chemical synthesis. A novel l-leucine 5-hydroxylase gene from Nostoc piscinale (NpLDO) was cloned into pET28a (+), pColdI and pQE-80 L plasmids. Using a two-step purification process (Ni-affinity chromatography and gel filtration), highly purified recombinant NpLDO was obtained. Recombinant NpLDO displayed unexpectedly high sulfoxidation activity toward l-methionine. The reaction products were analyzed by high-performance liquid chromatography. Sequence alignment analysis implied that residues of His150, His236 and Asp152 constitute the catalytic triad of NpLDO, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase superfamily. Biochemical data showed that NpLDO catalyzed regio- and stereoselective hydroxylation of l-leucine and sulfoxidation of l-methionine with Fe(II) and l-ascorbic acid as cofactor, and αKG as cosubstrate, respectively.

Biochemical characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV.

Ulvans, complex polysaccharides found in the ulvales (green seaweed) cell wall, contain predominantly 3-sulfated rhamnose (Rha3S) linked to either d-glucuronic acid, l-iduronic acid or d-xylose. The ulvan lyase endolytically cleaves the glycoside bond between Rha3S and uronic acid via a ß-elimination mechanism. Ulvan lyase has been identified as belonging to the polysaccharide lyase family PL24 or PL25 in the carbohydrate active enzymes database, in which fewer members have been characterized. We present the cloning and characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV (PsPL). The enzymes were heterologously expressed in Escherichia coli BL21 (DE3) and purified as the His-tag fusion protein using affinity chromatography, ion-exchange chromatography and size-exclusion chromatography. The degradation products were determined by thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS) to be mainly disaccharides and tetrasaccharides. Ulvan lyase provides an example of degrading ulvales into oligosaccharides. Arg265, His152 and Tyr249 were considered to serve as catalytic residues based on PsPL structural model analysis.