New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy.

Despite the development of cancer therapies, the success of most treatments has been impeded by drug resistance. The crucial role of tumor cell plasticity has emerged recently in cancer progression, cancer stemness and eventually drug resistance. Cell plasticity drives tumor cells to reversibly convert their cell identity, analogous to differentiation and dedifferentiation, to adapt to drug treatment. This phenotypical switch is driven by alteration of the transcriptome. Several pluripotent factors from the KLF and SOX families are closely associated with cancer pathogenesis and have been revealed to regulate tumor cell plasticity. In this review, we particularly summarize recent studies about KLF4, KLF5 and SOX factors in cancer development and evolution, focusing on their roles in cancer initiation, invasion, tumor hierarchy and heterogeneity, and lineage plasticity. In addition, we discuss the various regulation of these transcription factors and related cutting-edge drug development approaches that could be used to drug "undruggable" transcription factors, such as PROTAC and PPI targeting, for targeted cancer therapy. Advanced knowledge could pave the way for the development of novel drugs that target transcriptional regulation and could improve the outcome of cancer therapy.

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Optimization of 3D Point Clouds of Oilseed Rape Plants Based on Time-of-Flight Cameras.

Three-dimensional (3D) structure is an important morphological trait of plants for describing their growth and biotic/abiotic stress responses. Various methods have been developed for obtaining 3D plant data, but the data quality and equipment costs are the main factors limiting their development. Here, we propose a method to improve the quality of 3D plant data using the time-of-flight (TOF) camera Kinect V2. A K-dimension (k-d) tree was applied to spatial topological relationships for searching points. Background noise points were then removed with a minimum oriented bounding box (MOBB) with a pass-through filter, while outliers and flying pixel points were removed based on viewpoints and surface normals. After being smoothed with the bilateral filter, the 3D plant data were registered and meshed. We adjusted the mesh patches to eliminate layered points. The results showed that the patches were closer. The average distance between the patches was 1.88 × 10-3 m, and the average angle was 17.64°, which were 54.97% and 48.33% of those values before optimization. The proposed method performed better in reducing noise and the local layered-points phenomenon, and it could help to more accurately determine 3D structure parameters from point clouds and mesh models.

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System.

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained.IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Development of food-grade expression system for d-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose.

D-Allulose 3-epimerase (DAE) has been applied to produce D-allulose, a low-calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food-grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harboring three DAE genes in an expression vector exhibited the highest enzyme activity with 22.7 U/mg. Whole-cell transformation of DAE16 produced 225 g/L D-allulose with a volumetric productivity of 353 g·g -1 ·hr -1 . The catalytic efficiency of strain C-DAE9 integrating total 11 DAE genes in chromosome was 16.4-fold higher than strains carrying one DAE. Fed-batch culture of C-DAE9 gave enzyme activity of 44,700 U/L. We also expressed a thermostable invertase in C. glutamicum and obtained enzyme activity of 29 U/mg. Immobilized cells expressing DAE or invertase exhibited 80% of retained activity after 30 cycles of catalytic reactions. Those immobilized cells were coupled to produce 61.2 g/L D-allulose from cane molasses in a two-step reaction process. This study provided an efficient approach for enzyme preparation and allowed access to produce D-allulose from other abundant and low-cost feedstock enriched with sucrose.

Target animal safety testing of an oral salicylanilide suspension, oxyclozanide, for the treatment of fascioliasis in bovine in China.

The aim of this study was to determine the potential toxicity risk of an oxyclozanide suspension to the target animal, bovine. In this experiment, 32 Simmental beef cattle were fattened and fed a full-price diet without antimicrobial agents. The test cattle were divided into 4 groups, which were treated with 0, 1, 3, and 5 times the recommended dosage through continuous intermittent oral administration at intervals of 2 days. The body weight of the cattle was recorded before and after the experiment, and the weight changes were calculated. The safety of the drugs was evaluated by weight gain, observation of clinical toxicity, haematology, clinical chemistry and histopathology. The results showed that the cattle had different degrees of diarrhoea, loss of appetite and depression after administration. The results of clinicopathology had no significant effect. The results of pathological examination showed that there was a certain degree of damage in the 5 times recommended dose group. The recommended dose was safe to use. Thus, the recommended dose should be given by a single oral administration to ensure the safe use of this drug in the clinic.

The Characterization and Modification of a Novel Bifunctional and Robust Alginate Lyase Derived from Marinimicrobium sp. H1.

Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0-10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.

Time-Series Chlorophyll Fluorescence Imaging Reveals Dynamic Photosynthetic Fingerprints of sos Mutants to Drought Stress.

Resistance to drought stress is one of the most favorable traits in breeding programs yet drought stress is one of the most poorly addressed biological processes for both phenomics and genetics. In this study, we investigated the potential of using a time-series chlorophyll fluorescence (ChlF) analysis to dissect the ChlF fingerprints of salt overly sensitive (SOS) mutants under drought stress. Principle component analysis (PCA) was used to identify a shifting pattern of different genotypes including sos mutants and wild type (WT) Col-0. A time-series deep-learning algorithm, sparse auto encoders (SAEs) neural network, was applied to extract time-series ChlF features which were used in four classification models including linear discriminant analysis (LDA), k-nearest neighbor classifier (KNN), Gaussian naive Bayes (NB) and support vector machine (SVM). The results showed that the discrimination accuracy of sos mutants SOS1-1, SOS2-3, and wild type Col-0 reached 95% with LDA classification model. Sequential forward selection (SFS) algorithm was used to obtain ChlF fingerprints of the shifting pattern, which could address the response of sos mutants and Col-0 to drought stress over time. Parameters including QY, NPQ and Fm, etc. were significantly different between sos mutants and WT. This research proved the potential of ChlF imaging for gene function analysis and the study of drought stress using ChlF in a time-series manner.

Purification and Characterization of a Novel Alginate Lyase from the Marine Bacterium Bacillus sp. Alg07.

Alginate oligosaccharides with different bioactivities can be prepared through the specific degradation of alginate by alginate lyases. Therefore, alginate lyases that can be used to degrade alginate under mild conditions have recently attracted public attention. Although various types of alginate lyases have been discovered and characterized, few can be used in industrial production. In this study, AlgA, a novel alginate lyase with high specific activity, was purified from the marine bacterium Bacillus sp. Alg07. AlgA had a molecular weight of approximately 60 kDa, an optimal temperature of 40 °C, and an optimal pH of 7.5. The activity of AlgA was dependent on sodium chloride and could be considerably enhanced by Mg2+ or Ca2+. Under optimal conditions, the activity of AlgA reached up to 8306.7 U/mg, which is the highest activity recorded for alginate lyases. Moreover, the enzyme was stable over a broad pH range (5.0-10.0), and its activity negligibly changed after 24 h of incubation at 40 °C. AlgA exhibited high activity and affinity toward poly-ß-d-mannuronate (polyM). These characteristics suggested that AlgA is an endolytic polyM-specific alginate lyase (EC 4.2.2.3). The products of alginate and polyM degradation by AlgA were purified and identified through fast protein liquid chromatography and electrospray ionization mass spectrometry, which revealed that AlgA mainly produced disaccharides, trisaccharides, and tetrasaccharide from alginate and disaccharides and trisaccharides from polyM. Therefore, the novel lysate AlgA has potential applications in the production of mannuronic oligosaccharides and poly-α-l-guluronate blocks from alginate.

Manganese superoxide dismutase (SOD2): is there a center in the universe of mitochondrial redox signaling?

It is becoming increasingly clear that mitochondria drive cellular functions and in vivo phenotypes by directing the production rate and abundance of metabolites that are proposed to function as signaling molecules (Chandel 2015; Selak et al. 2005; Etchegaray and Mostoslavsky 2016). Many of these metabolites are intermediates that make up cellular metabolism, part of which occur in mitochondria (i.e. the TCA and urea cycles), while others are produced "on demand" mainly in response to alterations in the microenvironment in order to participate in the activation of acute adaptive responses (Mills et al. 2016; Go et al. 2010). Reactive oxygen species (ROS) are well suited for the purpose of executing rapid and transient signaling due to their short lived nature (Bae et al. 2011). Hydrogen peroxide (H2O2), in particular, possesses important characteristics including diffusibility and faster reactivity with specific residues such as methionine, cysteine and selenocysteine (Bonini et al. 2014). Therefore, it is reasonable to propose that H2O2 functions as a relatively specific redox signaling molecule. Even though it is now established that mtH2O2 is indispensable, at least for hypoxic adaptation and energetic and/or metabolic homeostasis (Hamanaka et al. 2016; Guzy et al. 2005), the question of how H2O2 is produced and regulated in the mitochondria is only partially answered. In this review, some roles of this indispensable signaling molecule in driving cellular metabolism will be discussed. In addition, we will discuss how H2O2 formation in mitochondria depends on and is controlled by MnSOD. Finally, we will conclude this manuscript by highlighting why a better understanding of redox hubs in the mitochondria will likely lead to new and improved therapeutics of a number of diseases, including cancer.

Construction of engineered Saccharomyces cerevisiae strain to improve that whole-cell biocatalytic production of melibiose from raffinose.

There are excessive by-products in the biocatalysis process of this whole-cell biocatalytic production of melibiose from raffinose with current Saccharomyces cerevisiae strains. To solve this problem, we constructed engineered strains based on a liquor yeast (S. cerevisiae) via gene deletion (mel1 gene), heterologous integration (fsy1 or/and ffzi1 gene from Candida magnoliae), and gene overexpression (gcr1 gene). Functional verification showed that deletion of the mel1 gene led to elimination of the reactions catalyzed by α-galactosidase, as well as elimination of the degradation of melibiose and the formation of galactose by-product. Insertion of the fsy1 or/and ffzi1 gene and overexpression of the gcr1 gene could contribute to fructose transport for enhancing the biopurification rate of the fructose by-product. Compared with the wild-type strain, the optimal engineered strain of MP8 (Δmel1::fsy1 cm ::ffzi1 cm ::gcr1 sc ) had improved about 30% on yield, 31% on productivity, and 36% on purity of the melibiose product.

Oxidation of Cucurbitadienol Catalyzed by CYP87D18 in the Biosynthesis of Mogrosides from Siraitia grosvenorii.

Mogrosides, the principally bioactive compounds extracted from the fruits of Siraitia grosvenorii, are a group of glycosylated cucurbitane-type tetracyclic triterpenoid saponins that exhibit a wide range of notable biological activities and are commercially available worldwide as natural sweeteners. The biosynthesis of mogrosides involves initial cyclization of 2,3-oxidosqualene to the triterpenoid skeleton of cucurbitadienol, followed by a series of oxidation reactions catalyzed by Cyt P450s (P450s) and then glycosylation reactions catalyzed by UDP glycosyltransferases (UGTs). We previously reported the identification of a cucurbitadienol synthase (SgCbQ) and a mogrol C-3 hydroxyl glycosyltransferase (UGT74AC1). However, molecular characterization of further transformation of cucurbitadienol to mogrol by P450s remains unavailable. In this study, we report the successful identification of a multifunctional P450 (CYP87D18) as being involved in C-11 oxidation of cucurbitadienol. In vitro enzymatic activity assays showed that CYP87D18 catalyzed the oxidation of cucurbitadienol at C-11 to produce 11-oxo cucurbitadienol and 11-hydroxy cucurbitadienol. Furthermore, 11-oxo-24,25-epoxy cucurbitadienol as well as 11-oxo cucurbitadienol and 11-hydroxy cucurbitadienol were produced when CYP87D18 was co-expressed with SgCbQ in genetic yeast, and their structures were confirmed by liquid chromatography-solid-phase extraction-nuclear magnetic resonance-mass spectrometry coupling (LC-SPE-NMR-MS). Taken together, these results suggest a role for CYP87D18 as a multifunctional cucurbitadienol oxidase in the mogrosides pathway.

High-level intra- and extra-cellular production of D-psicose 3-epimerase via a modified xylose-inducible expression system in Bacillus subtilis.

D-Psicose 3-epimerase (DPEase) converts D-fructose into D-psicose which exists in nature in limited quantities and has key physiological functions. In this study, RDPE (DPEase from Ruminococcus sp. 5_1_39BFAA) was successfully constitutively expressed in Bacillus subtilis, which is the first report of its kind. Three sugar-inducible promoters were compared, and the xylose-inducible promoter P xylA was proved to be the most efficient for RDPE production. Based on the analysis of the inducer concentration and RDPE expression, we surmised that there was an extremely close correlation between the intracellular RDPE expression and xylose accumulation level. Subsequently, after the metabolic pathway of xylose was blocked by deletion of xylAB, the intra- and extra-cellular RDPE expression was significantly enhanced. Meanwhile, the optimal xylose induction concentration was reduced from 4.0 to 0.5 %. Eventually, the secretion level of RDPE reached 95 U/mL and 2.6 g/L in a 7.5-L fermentor with the fed-batch fermentation, which is the highest production of DPEase by a microbe to date.

[Screening, identification and fermentation optimization of a collagenase-producing strain and purification of the collagenase].

Objective: The purpose of this study was to isolate novel strains from the soil nearby meat processing factories to produce collagenase. After the yield of collagenase from the strain improved, the collagenase was purified and used for hydrolyzing collagen. Methods: The strain was identified based on morphological features, physiological and biochemical characteristics and 16S rRNA gene phylogenetic tree analysis. The yield of collagenase was increased by optimizing the fermentation condition, and the collagenase isolated from the fermentation supernatant of the strain was finally purified with strong anion exchange resins. Results: The collagenase-producing strain was identified as Bacillus cereus. The optimized fermentation conditions of the strain were: 2.0% glucose as optimum carbon source, 1.5% tryptone as optimum nitrogen source, 0.005% of Ca2+ as optimum metal ion. The optimum temperature and pH were 37 ℃ and 7.5, respectively. Under the optimum conditions, the enzyme activity of collagenase was (65.81±2.06) U/mL, 1.5-fold increased than that before the optimization. After purified with strong anion exchange resins, a collagenase with the purity higher than 90%, the molecular weight about 100 kDa, and the specific activity of 7615.0±78.7 U/mg was obtained. Conclusion: The activity of Bacillus cereus collagenase was higher than the reported collagenases. Using this novel collagenase, collagen could be degraded into short biological peptides in a short time. Hence, this collagenase has application prospects in many fields, such as food, medical, health care products and cosmetics.

Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii.

Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report successful identification of SgCbQ and UGT74AC1, which were previously predicted via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.

Biosynthesis of l-Sorbose and l-Psicose Based on C-C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain.

The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a "one-pot" reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars.

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars.

Biosynthesis of 2-deoxysugars using whole-cell catalyst expressing 2-deoxy-D-ribose 5-phosphate aldolase.

2-Deoxy-D-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERA(K12) (S238D/F200I/ΔY259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards D-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-D-ribose yield from 0.41 mol/mol D-glyceraldehyde to 0.81 mol/mol D-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-D-robose (DR), with a yield of 0.71 mol/mol D-glyceraldehyde and average productivity of 0.13 mol/L·h (17.94 g/L·h). These results demonstrate the potential for large-scale production of 2-deoxy-D-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-D-altrose from D-erythrose, as well as 2-deoxy-L-xylose and 2-deoxy-L-ribose from L-glyceraldehyde.

Construction of allitol synthesis pathway by multi-enzyme coexpression in Escherichia coli and its application in allitol production.

An engineered strain for the conversion of D-fructose to allitol was developed by constructing a multi-enzyme coupling pathway and cofactor recycling system in Escherichia coli. D-Psicose-3-epimerase from Ruminococcus sp. and ribitol dehydrogenase from Klebsiella oxytoca were coexpressed to form the multi-enzyme coupling pathway for allitol production. The cofactor recycling system was constructed using the formate dehydrogenase gene from Candida methylica for continuous NADH supply. The recombinant strain produced 10.62 g/l allitol from 100 mM D-fructose. To increase the intracellular concentration of the substrate, the glucose/fructose facilitator gene from Zymomonas mobilis was incorporated into the engineered strain. The results showed that the allitol yield was enhanced significantly to 16.53 g/l with a conversion rate of 92 %. Through optimizing conversion conditions, allitol was produced effectively on a large scale by the whole-cell biotransformation system; the yield reached 48.62 g/l when 500 mM D-fructose was used as the substrate.

Production of allitol from D-psicose by a novel isolated strain of Klebsiella oxytoca G4A4.

A novel bacterium capable of producing allitol from D-psicose was isolated from soil and identified as Klebsiella oxytoca G4A4. An efficient method for the transformation of D-psicose to allitol was achieved through the resting cell reaction. Ribitol as an inducer is suitable for cell cultivation, and cells are most active in Tris-HCl buffer (pH 8.0) at 37 °C with a density of 40 (OD600 nm ). After the reaction, the final conversion rates of the washed cells were approximately 87, 83, and 55% at D-psicose concentrations of 0.25, 0.5, and 1%, respectively. The product from D-psicose was purified and determined to be allitol by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy.